InteractiveFly: GeneBrief

Shaker: Biological Overview | References

BIOLOGICAL OVERVIEW

Sleep disconnects animals from the external world, at considerable risks and costs that must be offset by a vital benefit. Insight into this mysterious benefit will come from understanding sleep homeostasis: to monitor sleep need, an internal bookkeeper must track physiological changes that are linked to the core function of sleep. In Drosophila, a crucial component of the machinery for sleep homeostasis is a cluster of neurons innervating the dorsal fan-shaped body (dFB) of the central complex. Artificial activation of these cells induces sleep, whereas reductions in excitability cause insomnia. dFB neurons in sleep-deprived flies tend to be electrically active, with high input resistances and long membrane time constants, while neurons in rested flies tend to be electrically silent. Correlative evidence thus supports the simple view that homeostatic sleep control works by switching sleep-promoting neurons between active and quiescent states. This study demonstrates state switching by dFB neurons, identify dopamine as a neuromodulator that operates the switch, and delineate the switching mechanism. Arousing dopamine caused transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects were transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involved the downregulation of voltage-gated A-type currents carried by Shaker and Shab, and the upregulation of voltage-independent leak currents through a two-pore-domain potassium channel that is termed Sandman. Sandman is encoded by the CG8713 gene and translocates to the plasma membrane in response to dopamine. dFB-restricted interference with the expression of Shaker or Sandman decreased or increased sleep, respectively, by slowing the repetitive discharge of dFB neurons in the ON state or blocking their entry into the OFF state. Biophysical changes in a small population of neurons are thus linked to the control of sleep-wake state (Pimentel, 2016).

Recordings were made from dFB neurons (which were marked by R23E10-GAL4 or R23E10-lexA-driven green fluorescent protein (GFP) expression (Donlea, 2014) while head-fixed flies walked or rested on a spherical treadmill. Because inactivity is a necessary correlate but insufficient proof of sleep, the analysis was restricted to awakening, which is defined as a locomotor bout after ≥5 min of rest, during which the recorded dFB neuron had been persistently spiking. To deliver wake-promoting signals, the optogenetic actuator CsChrimson was expressed under TH-GAL4 control in the majority of dopaminergic neurons, including the PPL1 and PPM3 clusters, whose fan-shaped body (FB)-projecting members have been implicated in sleep control (Liu, 2012; Ueno, 2012). Illumination at 630 nm, sustained for 1.5 s to release a bolus of dopamine, effectively stimulated locomotion. dFB neurons paused in successful (but not in unsuccessful) trials , and their membrane potentials dipped by 2-13 mV below the baseline during tonic activity. When flies bearing an undriven CsChrimson transgene were photostimulated, neither physiological nor behavioural changes were apparent. The tight correlation between the suppression of dFB neuron spiking and the initiation of movement might, however, merely mirror a causal dopamine effect elsewhere, as TH-GAL4 labels dopaminergic neurons throughout the brain. Because localized dopamine applications to dFB neuron dendrites similarly caused awakening, this possibility remote is considered remote (Pimentel, 2016).

Flies with enhanced dopaminergic transmission exhibit a short-sleeping phenotype that requires the presence of a D1-like receptor in dFB neurons (Liu, 2012; Ueno, 2012), suggesting that dopamine acts directly on these cells. dFB-restricted RNA interference (RNAi) confirmed this notion and pinpointed Dop1R2 as the responsible receptor, a conclusion reinforced by analysis of the mutant Dop1R2^MI08664 allele. Previous evidence that Dop1R1, a receptor not involved in regulating baseline sleep, confers responsiveness to dopamine when expressed in the dFB indicates that either D1-like receptor can fulfill the role normally played by Dop1R2. Loss of Dop1R2 increased sleep during the day and the late hours of the night, by prolonging sleep bouts without affecting their frequency. This sleep pattern is consistent with reduced sensitivity to a dopaminergic arousal signal (Pimentel, 2016).

To confirm the identity of the effective transmitter, avoid dopamine release outside the dFB, and reduce the transgene load for subsequent experiments, optogenetic manipulations of the dopaminergic system were induced with pressure ejections of dopamine onto dFB neuron dendrites. Like optogenetically stimulated secretion, focal application of dopamine hyperpolarized the cells and suppressed their spiking. The inhibitory responses could be blocked at several nodes of an intracellular signalling pathway that connects the activation of dopamine receptors to the opening of potassium conductances: by RNAi-mediated knockdown of Dop1R2; by the inclusion in the patch pipette of pertussis toxin (PTX), which inactivates heterotrimeric G proteins of the G_i/o family; and by replacing intracellular potassium with caesium, which obstructs the pores of G-protein-coupled inward-rectifier channels. Elevating the chloride reversal potential above resting potential left the polarity of the responses unchanged, corroborating that potassium conductances mediate the bulk of dopaminergic inhibition (Pimentel, 2016).

Coupling of Dop1R2 to G_i/o, although documented in a heterologous system, represents a sufficiently unusual transduction mechanism for a predicted D1-like receptor to prompt verification of its behavioural relevance. Like the loss of Dop1R2, temperature-inducible expression of PTX in dFB neurons increased overall sleep time by extending sleep bout length (Pimentel, 2016).

While a single pulse of dopamine transiently hyperpolarized dFB neurons and inhibited their spiking, prolonged dopamine applications (50 Discovering the esses tha eep-promoting neurons back ON will hold important c min) switched the cells from electrical excitability (ON) to quiescence (OFF). The switching process required dopamine as well as Dop1R2, but once the switch had been actuated the cells remained in the OFF state - and flies, awake - without a steady supply of transmitter. Input resistances and membrane time constants dropped to 53.3 ± 1.8 and 24.0 ± 1.3% of their initial values (means ±s.e.m., n = 15 cells), and depolarizing currents no longer elicited action potentials (15 out of 15 cells). The biophysical properties of single dFB neurons, recorded in the same individual before and after operating the dopamine switch, varied as widely as those in sleep-deprived and rested flies (Pimentel, 2016).

Dopamine-induced changes in input resistance and membrane time constant occurred from similar baselines in all genotypes and followed single-exponential kinetics with time constants of 1.07-1.10 min. The speed of conversion points to post-translational modification and/or translocation of ion channels between intracellular pools and the plasma membrane as the underlying mechanism(s). In 7 out of 15 cases, recordings were held long enough to observe the spontaneous recommencement of spiking, which was accompanied by a rise to baseline of input resistance and membrane time constant, after 7-60 min of quiescence. The temporary suspension of electrical output is thus part of the normal activity cycle of dFB neurons and not a dead end brought on by the experimental conditions (Pimentel, 2016).

Most people sleep 7-8 h per night, and, if deprived of sleep, performance suffers greatly; however, a few do well with just 3-4 h of sleep -- a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, mutagenesis in Drosophila melanogaster was performed, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, minisleep (mns), a line that sleeps for one-third of the wild-type amount, was found. mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, short-sleeping Shaker flies were shown to have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency (Pimentel, 2016).

dFB neurons in the ON state expressed two types of potassium current: voltage-dependent A-type. The current-voltage (I-V) relation of I_A resembled that of Shaker, the prototypical A-type channel (Timpe, 1988; Iverson, 1988): no current flowed below -50 mV, the approximate voltage threshold of Shaker; above -40 mV, peak currents increased steeply with voltage and inactivated with a time constant of 7.5 ± 2.1 ms. Non-A-type currents showed weak outward rectification with a reversal potential of -80 mV, consistent with potassium as the permeant ion, and no inactivation (Pimentel, 2016).

Switching the neurons OFF changed both types of potassium current. I_A diminished by one-third, whereas I_non-A nearly quadrupled when quantified between resting potential and spike threshold. The weak rectification of I_non-A in the ON state vanished in the OFF state, giving way to the linear I-V relationship of an ideal leak conductance. dFB neurons thus upregulate I_A in the sleep-promoting ON state. When dopamine switches the cells OFF, voltage-dependent currents are attenuated and leak currents augmented. This seesaw form of regulation should be sensitive to perturbations of the neurons' ion channel inventory: depletion of voltage-gated A-type (K_V) channels (which predominate in the ON state) should tip the cells towards the OFF state; conversely, loss of leak channels (which predominate in the OFF state) should favour the ON state. To test these predictions, sleep was examined in flies carrying R23E10-GAL4-driven RNAi transgenes for dFB-restricted interference with individual potassium channel transcripts (Pimentel, 2016).

RNAi-mediated knockdown of two of the five K_V channel types of Drosophila (Shaker and Shab) reduced sleep relative to parental controls, while knockdown of the remaining three types had no effect. Biasing the potassium channel repertoire of dFB neurons against A-type conductances thus tilts the neurons' excitable state towards quiescence, but leaves transient and sustained dopamine responses unaffected. The seemingly counterintuitive conclusion that reducing a potassium current would decrease, not increase, action potential discharge is explained by a requirement for A-type channels in generating repetitive activity of the kind displayed by dFB neurons during sleep. Depleting Shaker from dFB neurons shifted the interspike interval distribution towards longer values, as would be expected if K_V channels with slow inactivation kinetics replaced rapidly inactivating Shaker as the principal force opposing the generation of the next spike. These findings identify a potential mechanism for the short-sleeping phenotypes caused by mutations in Shaker (Cirelli, 2005), its β subunit Hyperkinetic, or its regulator sleepless (Pimentel, 2016).

Leak conductances are typically formed by two-pore-domain potassium (K_2P) channels. dFB-restricted RNAi of one member of the 11-strong family of Drosophila K_2P channels, encoded by the CG8713 gene, increased sleep relative to parental controls; interference with the remaining 10 K_2P channels had no effect. Recordings from dFB neurons after knockdown of the CG8713 gene product, which is termed Sandman, revealed undiminished non-A-type currents in the ON state and intact responses to a single pulse of dopamine but a defective OFF switch: during prolonged dopamine applications, I_non-A failed to rise, input resistances and membrane time constants remained at their elevated levels, and the neurons continued to fire action potentials (7 out of 7 cells). Blocking vesicle exocytosis in the recorded cell with botulinum neurotoxin C (BoNT/C) similarly disabled the OFF switch. This, combined with the absence of detectable Sandman currents in the ON state, suggests that Sandman is internalized in electrically active cells and recycled to the plasma membrane when dopamine switches the neurons OFF (Pimentel, 2016).

Because dFB neurons lacking Sandman spike persistently even after prolonged dopamine exposure, voltage-gated sodium channels remain functional in the OFF state. The difficulty of driving control cells to action potential threshold in this state must therefore be due to a lengthening of electrotonic distance between sites of current injection and spike generation. This lengthening is an expected consequence of a current leak, which may uncouple the axonal spike generator from somatodendritic synaptic inputs or pacemaker currents when sleep need is low (Pimentel, 2016).

The two kinetically and mechanistically distinct actions of dopamine on dFB neurons (instant, but transient, hyperpolarization and a delayed, but lasting, switch in excitable state) ensure that transitions to vigilance can be both immediate and sustained, providing speedy alarm responses and stable homeostatic control. The key to stability lies in the switching behaviour of dFB neurons, which is driven by dopaminergic input accumulated over time. Unlike bistable neurons, in which two activity regimes coexist for the same set of conductances, dFB neurons switch regimes only when their membrane current densities change. The current analysis of how dopamine effects such a change, from activity to silence, has uncovered elements familiar from other modulated systems: simultaneous, antagonistic regulation of multiple conductances; and modulation of leak currents. Currently little is known about the reverse transition, from silence to activity, except that mutating the Rho-GTPase-activating protein Crossveinless-c locks dFB neurons in the OFF state, resulting in severe insomnia and an inability to correct sleep deficits. Discovering the signals and processes that switch sleep-promoting neurons back ON will hold important clues to the vital function of sleep (Pimentel, 2016).

K+ channel reorganization and homeostatic plasticity during postembryonic development: biophysical and genetic analyses in acutely dissociated Drosophila central neurons

Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, this study shows drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK_Ca) K+ currents during postembryonic development. Larval and adult neurons display prominent fast-inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK_Ca is far smaller in amplitude. In contrast, pupal neurons are characterized by large sustained IKv and prominent IK_Ca, encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removes inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents is accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolishes the vast majority of IK_Ca, particularly at the pupal stage. Strikingly, the deficiency of IK_Ca in slo pupae is compensated by the transient component of IKv mediated by Sh channels. Thus, IK_Ca appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survive normally, Sh;;slo double mutants deficient in both fail to survive through pupal metamorphosis. Together, these data highlight significant reorganizations and homeostatic compensations of K+ currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process (Saur, 2016).

The intrinsic membrane properties of a neuron, mediated by various voltage- and ligand-activated ion currents that determine its overall excitability state and firing patterns, give the neuron its functional identity. At the circuit level, intrinsic properties dictate the translation of dendritic input to axonal output, which can guide the establishment and remodeling of connections in neural circuits. Drosophila metamorphosis offers a particularly extreme case of postembryonic development since during pupation, the central nervous system (CNS) undergoes a drastic reconstruction via processes that include elimination of certain larval neurons, modifications of the persisting ones, and proliferation and differentiation of adult-specific neurons to accommodate profound changes in body functions and behaviors. Thus, Drosophila metamorphosis provides a unique system to investigate the genetic control of the transforming physiological properties of CNS neurons during this morphologically defined postembryonic developmental process (Saur, 2016).

K⁺currents are a key element of intrinsic properties regulating excitability, frequency coding and firing patterns of a neuron, and two Ca²⁺-activated (I_KCa) currents, fast I_CF and slow I_CS. Five genes are known to encode these currents in Drosophila: Shaker (Sh, I_A), Shal (Shaker cognate l: I_A), Shab (slowly inactivating I_K), Shaw (Shaker cognate w; non-inactivating I_K), and slowpoke (slo, I_KCa). Previous studies of Sh and Shal mutants suggest that Sh channels mediate I_A in embryonic, larval, and adult muscle (Saur, 2016).

Electrophysiological analyses of cultured neurons from Drosophila embryos , larvae, and adult brains have provided in-depth understandings of neuronal K⁺current properties at the specific stages. However, this approach does not ensure a direct 'profiling' of K⁺currents across distinct metamorphic stages due to the 'extra' in vitro culturing periods and potential complications derived from different culture and growth conditions. Only limited studies have investigated the contribution of Sh to K⁺currents in larval and adult neurons in vivo. Additionally, the molecular identities that mediate neuronal I_KCa at the various stages still remain to be determined (Saur, 2016).

This study addressed these questions using acutely dissociated neurons from Drosophila larval, pupal, and adult brains. This approach allowed direct comparisons of K⁺currents expressed in neurons from these distinct developmental stages. Drastic reorganizations are shown of both I_Kv and I_KCa during metamorphosis. The data indicate that the Sh^M mutation affects a transient I_Kv in most (>60%) of larval and adult neurons, which was accompanied by an upregulation of remaining I_Kv, suggesting an unexpectedly large role for Sh in larval and adult neurons. In contrast, the slo¹ mutation eliminated the vast majority of neuronal I_KCa from all stages, and the near complete loss of I_KCa was nevertheless compensated by increased I_Kv in pupal neurons. These data provide the first 'profile' of neuronal K⁺currents during Drosophila postembryonic development, assign contributions for Sh and slo to larval, pupal and adult K⁺currents, and highlight a form of robust neuronal plasticity that governs K⁺current re-assemblies and compensations during postembryonic development (Saur, 2016).

How individual neurons participate in circuitry functions that mediate behaviors depends, to a great degree, on their intrinsic properties in situ. This study adapted a neuronal preparation that allowed comprehensive and quantitative biophysical investigations of ion currents from soma of freshly isolated neurons from the CNS of Drosophila. These neurons were devoid of long neurites and complex arbors, avoiding interferences from uncontrolled remote processes during voltage-clamp recording. In addition, this acute dissociation approach permitted immediate assessments of neuronal ion channels in their natural settings without further culturing, which may alter channel expression and assembly during prolonged in vitro growth or regeneration. This study provides a complete profile of the various K⁺and Ca²⁺currents during Drosophila postembryonic development. Analyses of mutants that are deficient in Sh- and/or slo-encoded channels uncovered their major contributions to the composition, reorganization, and homeostatic plasticity of neuronal K⁺currents at different postembryonic stages (Saur, 2016).

K⁺currents in Drosophila neurons have been the subject of intense study. However, a detailed understanding of how K⁺currents develop, especially at postembryonic phases, has been hampered due to the electrophysiological inaccessibility to most of postembryonic neurons. Using acutely dissociated neurons from different postembryonic stages, the first comprehensive, quantitative biophysical assessments of K⁺current phenotypes in Drosophila central neurons at larval, pupal, and adult stages are provided. I_A displays a U-shaped developmental profile, minimizing at an early pupal stage, whereas both I_CF and I_CS exhibit inverted U profiles, peaking at the same early/mid pupal stages. In contrast, I_K shows a steady increase as metamorphosis progresses, reaching a plateau beyond the mid-pupal stage. Similar profiles were also observed in morphologically and functionally more homogeneous neurons dissociated from the mushroom bodies. Thus, these profiles likely reflect a global program of regulated expression and/or reassembly of different channel subunits in CNS neurons during postembryonic development (Saur, 2016).

This postembryonic I_A profile extends the sequential and progressive development reported for I_A in embryonic neurons in Drosophila. Such monotonic increase of I_A is found in embryonic neurons in vertebrates as well. However, the onset of metamorphosis, i.e., early pupa, is characterized by a vigorous remodeling of CNS at both the individual cell and the circuit levels, during which larval dendritic and axonal branches prune, followed by the growth of adult-specific processes. Notably, transient I_A has been negatively correlated to axonal arborization and growth. For example, Drosophila hyperexcitability mutants that lack both Sh and ether à go-go (eag) display increased larval motoneuron terminal branching, whereas overexpression of Sh-like channels leads to a reduction in morphologically differentiated neurons in vertebrate cell cultures. In this context, suppression of I_A at early pupal stages may increase nerve excitability and promote axonal arborization, activity-dependent synaptic maturation, and neural circuit reorganization during early metamorphosis (Saur, 2016).

The delayed rectifier I_K contributes to membrane repolarization and the maintenance of repetitive nature of action potential firing. This study demonstrates a progressive increase of I_K during postembryonic development, which further extends the gradual increase of I_K in the development of both Drosophila embryos. A larger I_K is expected to limit the number of spikes. Consistent with this theme, mutation of the Shab channel in Drosophila converts characteristic repetitive firing patterns into run-away spikes and high-frequency oscillations in mutant 'giant' neuron cultures. In an identified motoneuron in Manduca, I_K gradually increases during postembryonic metamorphosis, in parallel to the continuing reduction of membrane excitability during its transformation to adult form (Saur, 2016).

Ca²⁺-dependent I_KCa contributes to action potential repolarization and hyperpolarizing after potentials, and hence neuronal membrane excitability and firing patterns. Therefore, Ca²⁺influx through voltage-gated Ca²⁺channels not only orchestrates complex intracellular Ca²⁺signaling processes, but also triggers I_KCa that directly regulates membrane repolarization and in turn limits further Ca²⁺influx. This study found that these two functionally coupled channel types, voltage-gated Ca²⁺channels and Ca²⁺-activated K⁺channels, also displayed similar developmental profiles, suggesting that their expression is also developmentally coupled during metamorphosis. During insect CNS metamorphosis, extensive axonal and dendritic pruning, synaptogenesis, and synaptic modification occur, all of which may depend on spatially and temporally regulated Ca²⁺signaling. The surges of both I_Ca and I_KCa at the onset of metamorphosis may reflect a drastic change in neuronal Ca²⁺signaling and excitability requirements for the CNS remodeling (Saur, 2016).

This demonstration of a significant contribution of Sh channels to I_A in Drosophila CNS neurons contrasts to the previous genetic and electrophysiological studies of I_Kv in cultured embryonic and larval central neurons that have led to the idea that Shal and Shab mediate nearly all of I_A and I_K, respectively, and that Sh does not make significant contributions to I_A in these neurons. However, prolonged culturing of dissociated neurons in vitro could alter intrinsic gene expression programs in studies employing cultured neurons. It is interesting to speculate that processes of neuronal re-growth in culture might recapitulate the condition of CNS remodeling initiation in early pupal development where IA expression is minimal among the postembryonic stages (Saur, 2016).

This analyses of Sh^M neurons demonstrated that the null mutation drastically reduced incidences of the fast inactivating type B Voltage-Gated Channel (VGCs) in all three developmental stages, including larval (70.2 to 20%), pupal (31.8 to 21.4%), and adult (70.8 to 20.8%), and curtailed the current densities of this VGC type by 49%, and 41%, and 52% in mutant larval, pupal, and adult neurons, respectively. These results demonstrate that Sh makes significantly larger than expected contributions to I_A in a majority of CNS neurons. Sh contributions in different developmental stages are not equal, being more prominent in larval and adult neurons and less in pupal neurons, consistent with the reported two expression peaks of Sh transcripts, at the larval and adult stages, respectively. Together, this study on populations of wild-type and Sh^M neurons from various postembryonic stages uncover surprisingly greater roles of Sh in larval and adult neurons (Saur, 2016).

In Drosophila embryonic, larval, and adult muscle cells, slo eliminates the transient, but not the sustained components, of I_KCa, indicating that slo mediates I_CF in muscles. The molecular identities of I_KCa in neurons are less clear, although a previous study using cultured embryonic Drosophila 'giant' neurons suggests that slo appears to affect the late as well as the transient components of I_KCa. The current study on neurons freshly dissociated from various postembryonic brains demonstrated that both I_CF and I_CS were strikingly reduced by slo¹, suggesting that slo mediates the vast majority of somatic I_CF and I_CS in central neurons during all postembryonic stages. However, slo¹ did not completely eliminate I_CF and I_CS in at least a subset of neurons and non-slo I_KCa channels were present. This is indicated by small populations of remaining type A and D Cacophony (CAC) neurons, and most strikingly during metamorphosis, when a great majority of type A and D CACs was converted into type E CAC of small amplitudes in slo pupal neurons. The possibility that these remaining CACs may be mediated by channels encoded by other genes awaits further investigations. In summary, in the Drosophila nervous system, a single gene, slo, appears to encode the majority of the Ca²⁺-activated K⁺currents across the entire embryonic and postembryonic phases (Saur, 2016).

Homeostatic maintenance of neuronal activity represents a form of plasticity by which neurons re-adjust to maintain their membrane excitability in response to chronic internal (e.g. genetic) or external perturbations. This can be achieved through reorganization by re-adjusting the proportions of different ion channel subtypes. For instance, overexpression of Shal-mediated I_A in lobster neurons induces a compensatory increase of hyperpolarization-activated inward I_h (Saur, 2016).

This study presents two cases of channel homeostatic plasticity in response to genetic perturbations in Drosophila. In Sh^M, there was a specific increase of Sh-independent I_A, but not I_K, that was particularly evident in larval and adult neurons, a compensation that could be due to upregulation of other homologues genes in the Sh family. The properties of the remaining voltage-gated K⁺currents after Sh channel removal in Sh^M neurons reveal some clues to the candidate voltage-gated K⁺channels for the homeostatic compensation. The dominant type A VGC in Sh^M, presumably converted from the type B majority in WT, exhibited a much longer time to peak, indicating slower activation kinetics, and the remaining non-Sh I_A in Type B VGC showed a hyperpolarizing shift in steady-state inactivation (a more negative V_1/2). These findings indicate that the Shal channel could be a good candidate based on the kinetic and voltage-dependent properties. Further studies will be required to determine this conjecture (Saur, 2016).

In contrast, slo¹ displayed a specific upregulation of I_A, but not I_K, mediated largely by an increased Sh expression and most evident in mutant pupal neurons. The contrasting stage-dependent compensations of K⁺currents in Sh^M and slo¹ mutants mirror their more prominent contributions in the corresponding postembryonic stages: Sh for larval and adult and slo for pupal stages (Saur, 2016).

The upregulation of Sh currents in slo¹ pupal neurons provide a unique example of cross-family homeostatic regulation of K⁺currents. This is presumably because I_A and I_CF have similar kinetic and other biophysical properties and can provide some functional redundancies. This notion is consistent with the previous study in larval neuromuscular junctions indicating that Sh K⁺channels are upregulated to maintain synaptic strength homeostasis in slo mutants (Saur, 2016).

Similar cross-family compensation is observed in the Drosophila Ca²⁺channel mutant, cacophony (cac). In cac, the majority of Ca²⁺-activated K⁺currents are lost (resulting from the near complete elimination of Ca²⁺currents in mutant neurons), but an upregulation of the Sh I_A is induced to partially compensate for the reduction of I_KCa. In both cases, interestingly, the homeostatic regulation appeared to be unidirectional since I_A was upregulated in slo and cac but neither Ca²⁺currents nor I_KCa were altered in K⁺channel mutants. The data indicate that Sh can serve as a functional backup for other channels with similar physiological properties, e.g.,slo channels. This novel compensation mechanism is indispensable during pupal development, as Sh;;slo double mutants failed to enclose to become adults. Thus, although Sh mediates only a minor portion of I_A in pupal neurons during metamorphosis, its role as a 'plasticity' gene represents an essential feature in Drosophila postembryonic development (Saur, 2016).

Structural analysis and deletion mutagenesis define regions of Quiver/Sleepless that are responsible for interactions with Shaker-type potassium channels and nicotinic scetylcholine receptors

Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake alpha-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein Quiver/Sleepless (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, this study demonstrated that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively these data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function (Wu, 2016).

Ly6 proteins are endogenous prototoxins found in most animals. They contain three short loops extending away from a hydrophobic scaffold that is anchored together by four conserved disulfide bonds. They belong to a superfamily of so-called 'three-fingered' proteins that includes snake α-neurotoxins, which often interfere with ion channel function and cholinergic signaling pathways. Similarly, Ly6 proteins have also been found to regulate ion channels and nicotinic acetylcholine receptors (nAChRs). One of the best-studied Ly6 proteins is Quiver/Sleepless (QVR/SSS or SSS), found in Drosophila. Originally identified phenotypically in unmapped mutants that 'quiver' in response to anesthesia and that exhibit reduced IA currents, the gene encoding SSS was subsequently shown to be required for normal levels of sleep and mapped to the quiver/sleepless (qvr/sss or sss) locus. As its name suggests, loss of sss causes loss of sleep, much as in animals that have abnormally low levels of one of the protein's targets, the Shaker voltage-gated potassium (K) channel, or high levels of another target, Dα3 nAChRs (Wu, 2014). SSS has four primary effects on Shaker channels. It upregulates their protein levels; it accelerates their activation kinetics; it slows their C-type inactivation; and it speeds their recovery from inactivation. SSS also antagonizes nAChRs by unknown means. The net result is that SSS acts as a suppressor of both excitability and cholinergic synaptic transmission (Wu, 2016).

Like other single domain-containing members of the Ly6 family, SSS is highly processed post-translationally. Its N- and C-termini are both cleaved, and the remaining 98 amino acid protein is tethered to cellular membranes. SSS is also modified by N-linked glycosylation. The resulting sugar moiety is a common feature of many extracellular and intrinsic membrane proteins and is therefore unlikely to contribute to selective interactions between SSS and its target molecules. Instead selectivity is likely to be conferred by the protein-forming portion of SSS. How such selectivity is achieved is unknown, however, and is particularly intriguing considering that the small size of mature SSS limits the surface area through which protein-protein interactions can occur (Wu, 2016).

To determine which protein motifs of SSS are required to form complexes with and regulate K channels and nAChRs, a series of structure-function studies were conducted. Deletion mutants of SSS were generated, expressed heterologously, and assayed for co-immunoprecipitation with Shaker K channels and Dα3 nAChRs. Whether each deletion mutant was able to suppress the activity of α4β2 nAChRs was also tested. The data suggest that only loop 2 (i.e., the middle finger) of SSS is required for interactions with both classes of target molecules and for regulation of nAChRs. It was also demonstrate that SSS normally inhibits nAChR activity by reducing levels of receptor at the cell surface, a process for which loop 2 is also required. These data point for the first time to a structural motif required for a Ly6 protein to modulate effectors of neuronal activity. Such information may be useful in design of Ly6 mimetics or antagonists with pharmacological utility in treating nervous system dysfunction in which cholinergic signaling plays a role (Wu, 2016).

The importance of Ly6 proteins is underscored by their implication in diseases such as Mal de Meleda; in nervous system functions such as synaptic transmission, visual plasticity, anxiety, and sleep; and in immune and stem cell functions. In some of these cases individual Ly6 proteins have been shown to exert their effects through antagonism of nAChRs, but the mechanisms underlying these effects and the structural motifs that are involved have not been thoroughly investigated. This study examined the contributions of each of three loops of the Drosophila Ly6 protein SSS to complex formation with K channel and nAChR effectors, to regulation of nAChR activity, and to trafficking of nAChRs (Wu, 2016 and references therein).

Using a de novo structural model of SSS based on lowest free-energy folding predictions this study identified disulfide bonds that appear to define the first two loops or 'fingers' of SSS. This de novo model fell just short of predicting the formation of a third disulfide bond at the base of the third loop, despite the presence of conserved cysteines at the appropriate locations. This suggests that although the current model is similar to known three-finger structures, some differences do exist, and empirical measurements of the actual structure of SSS will be necessary to validate these predictions (Wu, 2016).

This analysis also predicted an additional disulfide bond in the first loop, thus suggesting that SSS structurally resembles non-conventional three-finger toxins, which do not show strong toxicity. PCR-based mutagenesis was used to delete each loop individually as well as to generate two partial, complementary deletions in loop 1. It was hypothesized that by leaving the disulfide-rich hydrophobic core of the protein intact it might be possible to maintain the stability of each deletion mutant and thereby investigate the contribution of each loop to SSS function. This hypothesis appears to be generally correct. Although the deletion of loop 1 did not form stable protein, stable expression of the remaining mutants was observed, including those with smaller deletions in loop 1, suggesting that most mutants were not grossly misfolded or degraded. This hypothesis was reinforced by the PNGase-sensitive migration speeds of each mutant except for SSS-ΔK2, which lacked the predicted glycosylation site, as well as by the detection of all mutants except SSS-ΔK2 at the cell surface. Collectively these data suggest that most deletions do not impair protein stability or trafficking through the secretory pathway from the ER to Golgi to the plasma membrane. These results also validate the general predictions of the structural model for SSS (Wu, 2016).

Despite the reduction of SSS-ΔK2 expression at the cell surface, this deletion mutant as well as the SSS-ΔK1, and SSS-ΔL3 mutants are still capable of forming stable complexes with both Shaker and Dα3 as well as functionally inhibiting α4β2 nAChR activity. Instead, the data indicates that only loop 2 of SSS is necessary for interactions with both channel types and for regulation of α4β2 activity. Interestingly, loop 2 of α-cobratoxin and α-bungarotoxin have been shown to interact with the ligand-binding pockets of acetylcholine binding protein and α1 nAChRs, respectively. Thus, the data is consistent with the middle 'finger' of three-finger proteins being particularly important for forming complexes with and regulating target molecules. Indeed, molecular modeling and site-directed mutagenesis suggest that the mammalian Ly6 protein Lynx1 interacts with the acetylcholine binding protein via residues at the tip of loop 2. However, it is somewhat surprising that the same structural element of SSS is responsible for complex formation with both nAChRs and K channels, since these target proteins have very different structures. While nAChRs have large extracellular ligand binding domains with which to interact with Ly6 proteins, crystal structures of potassium channels reveal very little protein surface exposed on the outer leaflet of the plasma membran or its topological equivalent in the vesicular sorting pathway. The shared requirement of loop 2 for interactions with multiple effector molecules has interesting implications for neuromodulatory functions of SSS. For example, it might be expected that a single molecule of SSS would be available to interact with a single K channel or nAChR but not with both simultaneously. This mutually exclusive interaction might hint at a mechanism by which SSS could co-regulate Shaker and Dα3, perhaps acting as a feedback 'sensor' of the amount of either channel that is expressed or activated. Alternatively, it is possible that SSS, like some homologous snake α-neurotoxins, might form homodimers capable of interacting with two effector molecules simultaneously in a tetrameric complex. Further studies will be necessary to determine the likelihoods of both possible scenarios (Wu, 2016).

Finally, the data reveal a conserved mechanism by which SSS inhibits nAChR function. As was previously showed for the mammalian Ly6 proteins, Ly6h and Lynx2, SSS appears to alter trafficking of nAChRs to reduce receptor levels at the cell surface, thus making them unavailable for activation by agonist. Interestingly, as has also been shown for Lynx2, this effect supercedes the ability of nicotine to chaperone α4β2 nAChRs. For SSS, however, this study has gone one step further and shown that loop 2 is required to suppress nicotine’s trafficking effects. One possible interpretation of this data is thus that loop 2 outcompetes intracellular nicotine for binding to α4β2 nAChRs. If this were the case then it would also suggest that Ly6 proteins exert some of their regulatory effects by interacting with the agonist-binding site of nAChRs. Such a mechanism would also account for how the SSS-ΔK2 mutant, which does not express well at the cell surface, can still interact with and inhibit α4β2 function, since such effects could occur intracellularly (Wu, 2016).

In conclusion, the data demonstrate both a conserved mechanism for regulation of nAChRs by Ly6 proteins and a structural basis for such modes of regulation. In particular the data suggest a model in which the middle 'finger' of some Ly6 proteins interacts with the agonist-binding site of nAChRs to regulate receptor trafficking and ultimately function. In follow-up studies it will be interesting to determine how modes of nAChR regulation attributed to Ly6 proteins such as trafficking, desensitization kinetics, agonist sensitivity and receptor subunit stoichiometry differ in terms of the underlying structural perturbations. Ultimately, potentially subtle differences may be useful in designing drugs with enhanced selectivity for certain nAChR-Ly6 combinations found in restricted regions of the brain and possibly even for specific receptor conformations (Wu, 2016).

Alternative splicing modulates Kv channel clustering through a molecular ball and chain mechanism

Ion channel clustering at the post-synaptic density serves a fundamental role in action potential generation and transmission. This study shows that interaction between the Shaker Kv channel and the PSD-95 scaffold protein underlying channel clustering is modulated by the length of the intrinsically disordered C terminal channel tail. It was further shown that this tail functions as an entropic clock that times PSD-95 binding. Thus a 'ball and chain' mechanism is proposed to explain Kv channel binding to scaffold proteins, analogous to the mechanism describing channel fast inactivation. The physiological relevance of this mechanism is demonstrated in that alternative splicing of the Shaker channel gene to produce variants of distinct tail lengths resulted in differential channel cell surface expression levels and clustering metrics that correlate with differences in affinity of the variants for PSD-95. It is suggested that modulating channel clustering by specific spatial-temporal spliced variant targeting serves a fundamental role in nervous system development and tuning (Zandany, 2015).

The results argue that the Kv channel-scaffold protein interaction is entropy controlled and further demonstrate that the length of the Kv channel tail times complex formation with the PSD-95 partner. The Kv channel tail thus functions as an entropic clock and encodes a C-terminal 'ball and chain' mechanism for Kv channel binding to scaffold proteins that is analogous to the N-terminal 'ball and chain' mechanism describing channel fast inactivation. Several lines of evidence support the analogy between the two N- and C-terminal 'ball and chain' mechanisms. First, seminal studies on fast channel inactivation clearly showed dependence of the inactivation process on chain length, arguing that this reaction is entropy controlled. This has been demonstrated for the Kv C-terminal channel segment is, as this study shows, length-based modulation of Kv tail-PSD-95 affinity and further showed, in a direct manner, that length-dependent changes in the free energy of Kv channel tail-PDZ association are brought about by changes in entropy alone. Second, the earlier studies showed that the N-terminal channel tail acts as a molecular clock that times channel fast inactivation, with the length of the segment determining the kinetics of entry to inactivation. The current findings that Kv channel tail length only affects the kinetics of PDZ association (ka) but not dissociation (kd) agree with findings that suggest a similar timing function for the C-terminal channel tail. Third, and along the same line, complementary data has been presented showing that mutations in a PDZ-binding motif peptide, all producing variants of identical length, only affected the dissociation rate constant of the peptide from the PSD-95 PDZ domain (kd) without changing the association rate constant (ka). This result is in perfect agreement with a 'ball and chain' mechanism. Fourth, the power law dependence of the normalized association rate constant on tail length is adequately explained by a random walk 'ball and chain' theory, as also found for the dependence of the entry to inactivation rate constant in chain length. Finally, the finding that alternative splicing in Shaker Kv channel mRNA only affects the N- and C-terminal tails to produce chains of different length further strengthens the analogy between the 'ball and chain' mechanisms (Zandany, 2015).

One noteworthy difference exists, however, between the two mechanisms. Although the ball-receptor inactivation binding reaction is an intra-molecular process, the binding of the channel to PSD-95 is intermolecular and diffusion limited. This difference will affect the effective local concentration of the 'ball' near its receptor site and will be reflected in the magnitude and type of the forward binding rate constants, being first order for entry to inactivation and second order for tail-PDZ complex formation. However, considering that in the native context PSD-95 is a priori membrane-associated (because of its interactions with other membrane protein partners and its own plamitoylation and the natural variation in Kv channel fast inactivation where the 'ball and chain' sequence is not carried on the channel itself but rather on the auxiliary β subunit with which it interacts, the resemblance between the two inactivation and clustering 'ball and chain' mechanisms becomes even clearer (Zandany, 2015).

The results presented in this study reporting protein tail length modulation of PDZ binding carry implications for PDZ-based protein-protein interactions, in general. Such interactions are of primary importance in protein interaction networks underlying a variety of cellular signalling processes. It is generally assumed that the affinity of PDZ domains to their cognate proteins is solely determined by a short sequence motif composed of three to eight residues. The observed affinities are typically within the sub-μM range, affinities much higher than those measured in this study (μM range and higher. Such assertions were based on structural and functional studies involving isolated PDZ domains and model terminal PDZ-binding peptides and were aimed at deriving estimates for the affinity of the interaction and to reveal the minimal recognition motif important for binding. However, long protein tails, often presenting an intrinsically disordered nature, usually flank terminal PDZ-binding motifs of membrane and other proteins. To date, no rigorous assessment of protein tail length on interaction affinity has been reported. This paper thus emphasizes that under certain circumstances, chain length plays an important role in mediating PDZ-based protein-protein interaction. In the case of the Kv channel, such tail length modulation considers the entropy contribution of random chains to PDZ domain binding, as predicted from polymer chain chemistry. Indeed, the longest Kv tail truncation, producing a nine amino-acid-long tail, reveals affinity in the sub-μM range, comparable to those values reported using model peptides. It is noted, however, that the reported association and dissociation rate constants for this tail are lower than those typically characterizing PDZ-peptide interactions, a fact that may be related to the particular PDZ-Kv channel peptide interaction examined in this study. Although this cannot be further explained, the important point is that this study focused on relative chain length differences and thus, the observed length-dependent trends of the association rate and equilibrium constants are valid. It is therefore proposed that protein tail length may represent another dimension for controlling PDZ-based protein-protein interactions. Such modulation requires the tail to be a random chain, that is, a pure entropic chain (Zandany, 2015).

The conformity of the natural A and B tail variants with the entropic chain characteristics of the Kv-PSD-95 interaction, as manifested in the binding energy and association kinetics length profiles, points to the physiological relevance of the C-terminal 'ball and chain' mechanism characterizing the Kv channel-PSD-95 interaction. Indeed, when assessed at both the tail alone and whole channel levels, the two variants exhibited distinct cell surface expression and clustering patterns that are coherent with the difference in affinity of the two variants, as rationalized by the 'ball and chain' mechanism. The high-affinity short-tail A variant presents higher cell surface expression level and supports larger channel clusters in Schneider cell membranes than does the low-affinity long-tail B variant. Thus, molecular distinctions reflected in the differential interactions of the tail variants with PSD-95 translate into functional differences in the context of cellular channel clustering (Zandany, 2015).

The proposed 'ball and chain' mechanism for channel clustering sheds new light on the roles of the short A and long B tail variants in electrical signalling. It is suggested that spatial and temporal differences in Shaker A and B expression during development and/or channel variability resulting from hetero-oligomeric A and B subunit assembly would give rise to distinct patterns of PSD-95-mediated A and B channel cell surface expression and clustering, in turn resulting in changes in channel current density at the post-synaptic site of homo- or hethero-oligomeric channel localization. This, subsequently, could lead to changes in action potential propagation and transmission, synaptic growth and plasticity. As such, it is tempting to speculate that a splicing-based developmental switch for the Shaker channel is responsible for modulating channel clustering that may underlie changes in nervous system function and tuning during development. Although this appealing hypothesis requires further investigation, it is evident that the Shaker Kv channel protein system exemplifies how linkage between alternative splicing and intrinsic disorder enables functional diversity (Zandany, 2015).

Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB

Axonal retrograde transport of signalling endosomes from the nerve terminal to the soma underpins survival. As each signalling endosome carries a quantal amount of activated receptors, it was hypothesized that it is the frequency of endosomes reaching the soma that determines the scale of the trophic signal. This study shows that upregulating synaptic activity markedly increased the flux of plasma membrane-derived retrograde endosomes (labelled using cholera toxin subunit-B: CTB) in hippocampal neurons cultured in microfluidic devices, and live Drosophila larval motor neurons. Electron and super-resolution microscopy analyses revealed that the fast-moving sub-diffraction-limited CTB carriers contained the TrkB neurotrophin receptor, transiently activated by synaptic activity in a BDNF-independent manner. Pharmacological and genetic inhibition of TrkB activation selectively prevented the coupling between synaptic activity and the retrograde flux of signalling endosomes. TrkB activity therefore controls the encoding of synaptic activity experienced by nerve terminals, digitalized as the flux of retrogradely transported signalling endosomes (Wang, 2016).

Whether raising synaptic activity also promotes an increase in the flux of CTB retrograde transport was examined in vivo, using live Drosophila melanogaster larval preparations. In addition to allowing measurement. of the effect of activity on retrograde carrier trafficking in a complementary model system, this approach also facilitated tracing the effect of presynaptic activity on the complete transport of CTB from the neuromuscular junction to the soma located in the central nervous system of the larvae (see CTB axonal retrograde carrier flux is controlled by synaptic activity in D. melanogaster motor neurons. In the fibers of neurons from wild-type larvae a low, albeit consistent, rate of CTB-positive retrograde carriers was detected. To analyse the flux of retrograde carriers under conditions of high activity, transgenic larvae were employed expressing dominant-negative forms of Ether-è-go-go (Eag) and Shaker (Sh) voltage-gated potassium channels in motor neurons, upregulating synaptic transmission. Consistent with the data in primary hippocampal neurons, increased presynaptic activity at the neuromuscular junction of the eag, Sh mutant larvae also resulted in an increase in the frequency of retrograde CTB carriers. Furthermore, the net transport of CTB could be quantified by measuring the accumulation of fluorescent CTB in the somata located in the ventral nerve cord. These data confirmed that presynaptic activity controls the transport of CTB retrograde carriers as well as their accumulation in the cell bodies located in the central nervous system (Wang, 2016).

This study demonstrates that the level of activity at the presynapse in hippocampal neurons, whether it be low or high activity, can induce changes in the frequency of CTB-positive retrograde carriers undergoing transport from the nerve terminal to the soma. Greater than 50% of CTB carriers are TrkB-positive signalling endosomes, and there is coupling between the level of presynaptic activity and the flux of signalling endosomes undergoing retrograde axonal transport. Given that the amount of activated TrkB per signalling endosome has been shown to be quantal in nature, it is likely that it is the number of signalling endosomes reaching the cell body that is responsible for monitoring the trophic response of each neuron. Importantly, it was demonstrated that the transient TrkB activation that occurs when presynaptic activity is increased controls the activity-dependent increase in the flux of signalling endosomes in a BDNF-independent manner. This suggests that TrkB activation may play an active role in generating signalling endosomes with a retrograde fate independent of BDNF secretion. Finally, SIM and morphometric analyses reveal a high number of sub-diffraction-limited CTB- and TrkB-positive carriers that may be involved in delivering the survival message (Wang, 2016).

CTB is a widely used neuroanatomical tracer with the ability to undergo retrograde transport in neurons. CTB was found to enter nerve terminals in an activity-dependent manner, and following a 2-4 h chase, is found in retrograde axonal carriers that co-localize extensively with TrkB. Importantly, TrkB staining in resting conditions produced a low and surface-localized pattern, which is in sharp contrast with that observed in the CTB/TrkB vesicular carriers following stimulation. Considering that internalization of activated TrkB is elicited by neuronal activity in hippocampal neurons, this suggests that the retrograde carriers containing both TrkB and CTB are signalling endosomes, destined to deliver a survival signal from the terminal to the soma. The data are also consistent with the previous demonstration that CTB can be co-transported in the same retrograde carriers as the neurotrophin receptor p75NTR and TrkB receptors. It has been speculated that activated neurotrophin receptors internalized into terminals through different pathways may converge in the retrograde traffic and be packaged into 'common' compartments, presumably signalling endosomes, which then undergo microtubule-dependent retrograde trafficking to induce signalling cascades and gene expression in the soma (Wang, 2016).

The signalling endosome model has been proposed to describe the propagation of activated Trk signals from the axon terminal to the neuronal soma. Although the presence of signalling endosomes is widely accepted, the precise nature(s) and regulation of these organelles remain unknown. In particular, it is not known how presynaptic activity translates into an increase in signalling endosome-encoded cell survival. Recent studies using super-resolution microscopy have shown that individual signalling endosomes appear to contain a limited number of signalling receptors, suggesting that retrograde survival signalling could be quantal in nature. Consistent with this idea, recent analysis has demonstrated that signalling endosomes have a fixed number of phosphorylated tyrosine receptor kinases. The current findings suggest that it is the flux of signalling endosomes reaching the cell body that encodes the level of presynaptic activity, and translates this into a survival signal. The data reveal a clear increase in the frequency of retrograde carriers delivering both CTB and TrkB from the nerve terminal to the cell soma following a short, transient burst of activity, whereas BoNT/A pretreatment not only completely blocked this increased flux despite equivalent stimulation but also lowered the basal level of retrograde CTB flux in resting conditions. These results strongly support the hypothesis that the soma of a neuron with lower synaptic activity receives fewer signalling endosomes than that of a cell with high synaptic activity, with ramifications for survival (Wang, 2016).

Previous studies have clearly established that there is constitutive delivery of retrogradely transported neurotrophins and their receptors in different types of primary neurons. This study is the first to reveal a coupling between synaptic activity and the number of retrograde signalling endosomes in both hippocampal neurons and motor neurons from D. melanogaster larvae, suggesting that this coupling represents a conserved regulatory mechanism (Wang, 2016).

Genetic manipulations in Drosophila allowed motor neuron activity to be chronically increase, thereby demonstrating a correlation with increased retrograde signalling endosome transport. Retrograde axonal transport has previously been reported in Drosophila larval motor neurons. However, this process is not regulated by the activation of TrkB receptors in flies, but by neurotrophin-type factors such as BMP (Gbb), p150(Glued) and Toll. Investigating the respective contributions of each of these mechanisms will be required to unravel the mechanism(s) controlling the coupling between synaptic activity and the flux of retrogradely transported signalling endosomes (Wang, 2016).

Clathrin-mediated endocytosis and bulk endocytosis are the two main modes for synaptic vesicle retrieval. On the basis of the current data, internalized CTB is sorted into both synaptic vesicles and larger endosomes following high K+-induced synaptic activation. These are therefore likely to be the source of CTB-positive signalling endosomes. Indeed, it is noted that, following a 4 h chase, the number of CTB-labelled endosomes present in nerve terminals was clearly reduced, suggesting that they could contribute to the generation of retrograde carriers leaving the terminals. More work is needed to pinpoint the actual contribution of the different presynaptic endocytic pathways in the process of generating signalling endosomes and recycling synaptic vesicles. Using electron microscopy and super-resolution SIM on the unidirectional axon bundles, a high proportion was found of CTB-positive retrograde carriers with a diameter <150 nm, indicating that these may constitute a significant source of the signalling endosomes that reach the soma. Interestingly, it was also found that a number of these small CTB retrograde endosomes closely aligned with each other to form consecutive structures. These were reminiscent of single-molecule quantum dot BDNF-labelled compartments, previously described as elongated multiple vesicular bodies. Several different types of organelles contain endocytosed neurotrophin receptors, including electron-lucent endosomes ranging from 50 nm to >200 nm in diameter and multivesicular bodies. The results further confirm this morphological heterogeneity of signalling endosomes. Interestingly, they also show that the sub-diffraction-limited pool of retrograde carriers is increased by a pulse of stimulation. A future challenge will be to fully define these compartments, including their cargo, their regulation and their potential role in neuronal survival (Wang, 2016).

The activity-dependent increase in the flux of CTB retrograde carriers is similar to that observed with autophagosomes undergoing axonal retrograde transport. Genetic and pharmacological inhibition of TrkB prevented the activity-dependent increase in signalling endosomes but did not affect the number of autophagosomes undergoing retrograde transport. This result suggests a limited cross-link between these two pathways, and indicates the existence of a TrkB-dependent sorting mechanism that is selective for the generation of retrograde signalling endosomes and is upregulated by synaptic activity (Wang, 2016).

Although TrkB activation was required to couple synaptic activity with the flux of retrograde CTB carriers, this study found that BDNF collators (anti-BDNF blocking antibody and TrkB-Fc), as well as exogenously applied BDNF, failed to affect this coupling. These results suggest that BDNF may not be required to promote this pathway. First, it can be speculated that increased neuronal activity alone promotes the secretion of alternative TrkB activators, such as NT4. In addition, considering that two different BDNF receptors, p75NTR and TrkB, have been reported to have opposing actions in neurons, the observation that BDNF collators have no effect on retrograde transport or the phosphorylation of TrkB could be explained by the fact that these collators alleviate an inhibitory effect of p75NTR signalling on TrkB activation. Third, it is possible that TrkB receptors could be indirectly transactivated by adenosine or by the MAPK pathway, similar to other tyrosine kinase receptors. However, further experiments are required to pinpoint the precise cascade of molecular events leading to the encoding of the level of synaptic activity by the flux of retrograde signalling endosomes in a BDNF-independent and TrkB activation-dependent pathway (Wang, 2016).

The data show that internalized CTB is sorted into both synaptic vesicles and larger endosomal structures reminiscent of bulk endosomes. Bulk endosomes are therefore likely to contribute to the sorting events that lead to the generation of CTB-positive signalling endosomes. Indeed, the data demonstrate that, in response to stimulation, the number of nerve terminals containing bulk endosomes more than doubles, as does the number of endosomes per terminal. More strikingly, after a chase of 4 h, the number of bulk endosomes returns to control levels, suggesting that sorting has taken place and/or that these large endosomes have undergone retrograde trafficking. In support of this view larger CTB-positive structures inside the axon were observed by electron microscopy. Bulk endocytosis is therefore likely to be a key element of this pathway, not only by budding small synaptic-like vesicles in nerve terminals, but also by generating large endosomes that undergo retrograde trafficking. More work is needed to pinpoint the actual contribution of bulk endocytosis in the process of generating signalling endosomes and recycling synaptic vesicles. The fact that synaptic activity increases the flux of CTB retrograde carriers advocates for the presence of a number of regulatory mechanisms that effectively couple these two pathways. Whether both recycling synaptic vesicles and signalling endosomes bud from the same bulk endosomes will require further investigation (Wang, 2016).

In summary, this study has uncovered a coupling between synaptic activity, trophic signalling and the generation of axonal retrograde signalling endosomes, which contain TrkB receptors and nerve terminal-derived CTB as cargo, and have characterized their kinetic and morphological nature. The data reveal a novel role of presynaptic activity in controlling the flux of retrograde signalling endosomes that will eventually reach the neuronal cell body and deliver cell survival signals. These results suggest that the flux of signalling endosomes undergoing axonal transport constitutes a digital output of the level of synaptic activity experienced by nerve terminals. How the flux of signalling endosomes reaching the cell body is decoded to pursue or halt a survival signal warrants further investigation (Wang, 2016).

The role of cAMP in synaptic homeostasis in response to environmental temperature challenges and hyperexcitability mutations

Homeostasis is the ability of physiological systems to regain functional balance following environment or experimental insults and synaptic homeostasis has been demonstrated in various species following genetic or pharmacological disruptions. Among environmental challenges, homeostatic responses to temperature extremes are critical to animal survival under natural conditions. Axon terminal arborization in Drosophila larval neuromuscular junctions (NMJs) has been shown to be enhanced at elevated temperatures; however, the amplitude of excitatory junctional potentials (EJPs) remains unaltered despite the increase in synaptic bouton numbers. This study determine the cellular basis of this homeostatic adjustment in larvae reared at high temperature (HT, 29 ° C). Synaptic current focally recorded from individual synaptic boutons was unaffected by rearing temperature (<15 degrees C to >30 degrees C). However, HT rearing decreased the quantal size (amplitude of spontaneous miniature EJPs, or mEJPs), which compensates for the increased number of synaptic releasing sites to retain a normal EJP size. The quantal size decrease is accounted for by a decrease in input resistance of the postsynaptic muscle fiber, indicating an increase in membrane area that matches the synaptic growth at HT. Interestingly, a mutation in rutabaga (rut) encoding adenylyl cyclase (AC) exhibited no obvious changes in quantal size or input resistance of postsynaptic muscle cells after HT rearing, suggesting an important role for rut AC in temperature-induced synaptic homeostasis in Drosophila. This extends a previous finding of rut-dependent synaptic homeostasis in hyperexcitable mutants, e.g., slowpoke (slo). In slo larvae, the lack of BK channel function is partially ameliorated by upregulation of presynaptic Shaker (Sh) IA current to limit excessive transmitter release in addition to postsynaptic glutamate receptor recomposition that reduces the quantal size (Ueda, 2015).

The transcription factors islet and lim3 combinatorially regulate ion channel gene expression

Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. This study used the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9/ExEx, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K(+) channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. Body wall muscle was used as a test tissue because in conditions of low Ca(2+), the fast K(+) current is carried solely by Sh channels (unlike neurons in which a second fast K(+) current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression (Wolfram, 2014).

Flight and seizure motor patterns in Drosophila mutants: simultaneous acoustic and electrophysiological recordings of wing beats and flight muscle activity

Tethered flies allow studies of biomechanics and electrophysiology of flight control. Microelectrode recordings were performed of spikes in an indirect flight muscle (the dorsal longitudinal muscle, DLMa) coupled with acoustic analysis of wing beat frequency (WBF) via microphone signals. Simultaneous electrophysiological recording of direct and indirect flight muscles has been technically challenging; however, the WBF is thought to reflect in a one-to-one relationship with spiking activity in a subset of direct flight muscles, including muscle m1b. Therefore, this approach enables systematic mutational analysis for changes in temporal features of electrical activity of motor neurons innervating subsets of direct and indirect flight muscles. This study reports the consequences of specific ion channel disruptions on the spiking activity of myogenic DLMs (firing at approximately 5 Hz) and the corresponding WBF (approximately 200 Hz). Mutants of the genes encoding: 1) voltage-gated Ca(2+) channels (cacophony, cac), 2) Ca(2+)-activated K(+) channels (slowpoke, slo), and 3) voltage-gated K(+) channels (Shaker, Sh) and their auxiliary subunits (Hyperkinetic, Hk and quiver, qvr). Flight initiation in response to an air puff was severely disrupted in both cac and slo mutants. However, once initiated, slo flight was largely unaltered, whereas cac displayed disrupted DLM firing rates and WBF. Sh, Hk, and qvr mutants were able to maintain normal DLM firing rates, despite increased WBF. Notably, defects in the auxiliary subunits encoded by Hk and qvr could lead to distinct consequences, that is, disrupted DLM firing rhythmicity, not observed in Sh. This mutant analysis of direct and indirect flight muscle activities indicates that the two motor activity patterns may be independently modified by specific ion channel mutations, and that this approach can be extended to other dipteran species and additional motor programs, such as electroconvulsive stimulation-induced seizures (Iyengar, 2014).

Flight tones have been used for over a century to analyze dipteran flight. Off-the-shelf microphones could be readily added to monitor flight tones in the tethered preparations previously used to monitor spiking activity in DLMs to analyze properties of the giant-fiber escape reflex as well as seizure discharges in Drosophila. Because the fundamental frequency of the flight tone corresponds to the WBF, this approach enables direct comparisons between indirect muscle firing with wing beats in the same fly. More importantly, analysis of different categories of mutants reveals that mutations in particular genes may affect either WBF (e.g., Sh) or DLM firing (qvr) only, or both (e.g., cac ), suggesting that the firing of direct and indirect flight muscles is driven by distinct motor pattern generators that are genetically separable (Iyengar, 2014).

Among the populations sampled in this study, a span of variability was observed in WBF (range of 170-220 Hz) and DLM firing (range of 2.22-5.98 Hz) in WT and various mutants. While significance of such variability is unclear, previous analyses of WBF across several thousand flies from several isogenized lines of D. melanogaster males report similarly large variability with relatively small contributions due to differences in age, ambient temperature, or flight duration alone. However, it should be noted that WBF and DLM spiking did not co-vary across individuals, that is, for any particular fly, higher WBF did not necessarily couple with higher DLM firing rates during sustained flight. These observations suggest that this holds true in other Drosophila species (Iyengar, 2014).

Significantly, the performance of tethered flies does not reflect all aspects of free-flight behavior. For example, the WBF has been observed to be systematically higher in free flight. It has been suggested that such differences may be introduced by the unnatural posture of horizontally positioned tethered fly, in contrast to a more vertical orientation during free flight resulting in sensory cues not usually experienced by the fly. It is also known that flight power control depends on both wing stroke frequency and amplitude, with a lower stroke amplitude associated with higher stroke frequency, and vice versa. It will be desirable in future studies to include both WBF and amplitude measurements. Further, muscle mechanical power output also depends on temperature, stretch activation, intramuscular Ca2+ levels (partially from influx during muscle firing, and conceivably mechano-sensory feedback. It would be interesting to compare in further studies how the changes in the parameters observed in the current study translate to modified free-flight parameters in the various mutants. (Iyengar, 2014).

The ion-channel mutants that were examined all have previously documented motor coordination deficits. The slo mutants with defective KCa BK channels were first identified on the basis of their uncoordinated, sluggish behavior at high temperature (38°C). Mutant adults display 'poor flight' as measured by a drop-and-flight Flight-Tester assay, and larvae also show abnormal central pattern generation responsible for peristaltic contractions. The altered BK currents in the slo alleles in this study have been described in several tissues, including the DLM, larval muscles, presynaptic terminals, and cultured embryonic neurons. These studies demonstrate the striking consequences of altered BK channels resulting in prolonging Ca2+ influx through Ca2+ channels, broadening action potentials, promoting repetitive firing, and altering synaptic transmission (Iyengar, 2014).

The observations highlight aspects of tethered flight that depend on the functioning of the slo BK channel. Air puff-triggered flight initiation was severely affected, with no success in slo¹ and severe reductions in slo98 in a large number of trials. However, several slo⁹⁸ individuals were able to initiate spontaneous flight, which showed relatively normal WBF and DLM firing patterns. These results suggest several possibilities, including: (1) the slo mutations preferentially affect putative command components of the circuit for flight initiation, (2) potential defects in relevant sensory contributions required for flight, and (3) homeostatic adjustments in neural circuits since slo is known to initiate compensatory up-regulation of Sh channels in cell bodies and presynaptic terminals of neurons (Iyengar, 2014).

The impact of disrupted Sh channels on locomotion has been well studied in a variety of experimental paradigms. The Sh channel complex is composed of the α subunit (KV1) encoded by Sh, the auxiliary β subunit (KVβ1) encoded by Hk, and the extracellular modulatory subunit encoded by qvr. Mutations of these genes display the hallmark ether-induced leg-shaking behavior plus locomotive defects, including larval crawling, and adult walking. Additionally, the roles of Sh and Hk in the giant fiber-mediated escape reflex and its habituation have been documented (Engel, 1998). Previous voltage-clamp experiments have demonstrated the altered biophysical parameters of Sh channels caused by Sh, Hk, and qvr mutant alleles (Iyengar, 2014).

The results reveal previously unnoticed features and relationships among these alleles. Sh^M, a null allele, and Sh¹²⁰, displaying mild effects on muscle currents but severe neurotransmission defects, show similar defects in WBF, among the three parameters analyzed (WBF, DLM firing frequency, CV of DLM inter-spike intervals). However, Sh⁵, a neomorphic allele, produced WT-like flight in all three parameters, even though it is known to have a unique wing-scissoring phenotype in addition to leg-shaking, coupled with a unique I-V curve, requiring stronger depolarization for activation. These observations suggest a potential link between the allele-dependent phenotypic differences and the known extensive post-transcriptional mRNA splice-forms of the Sh gene (Iyengar, 2014).

In comparison with Sh mutants, the null allele, Hk^IE18, produced phenotypes consistent with Sh^M in terms of WBF, whereas Hk¹ displays nearly normal WBF. However, a higher DLM inter-spike-interval CV is observed in both Hk alleles, indicating less regular firing due to this auxiliary β subunit. In contrast, mutations in the qvr subunit can lead to more extreme consequences. Flight in qvr¹ flies with disrupted mRNA splicing displayed WBF changes, similar to Sh^M. However, the null allele, qvr^EY, was clearly more extreme than Sh^M. In fact, qvr^EY's increased DLM inter-spike interval CV was greatest among all mutants examined in this study. This result indicates the possibility that qvr also modulates, in addition to Sh channels, other molecular components regulating membrane excitability. Recently, qvr was found to modify nicotinic acetylcholine receptor channel function, an abundant excitatory neurotransmitter receptor in the Drosophila central nervous system (Iyengar, 2014).

It should be noted that among mutants of the Sh channel complex, the average DLM firing rate was largely preserved across mutant genotypes, similar to the consequences of slo BK mutations. In contrast to K+ channel mutations, severe DLM firing rate modifications were found in cac Ca2+ channel mutations. The gene cac encodes an N-type Ca2+ channel homologous to vertebrate CaV2 channels. These channels are present in motor neuron soma and are also localized in the nerve terminal to mediate Ca2+ influx required for neurotransmitter release. Both cac alleles examined in this study displayed consistent increases in WBF and DLM firing rates. Furthermore, because WBF serves as a proxy for direct flight muscle activity, the results demonstrate that cac mutations disrupt spike pattern generation during flight in the motor neurons driving direct and indirect flight muscle groups. This contrasts with the K+ channel mutations in which WBF increases, but DLM firing rates remain largely unaltered. Importantly, previous studies indicate that cac currents are not prominent in muscles, and thus the observed alterations in both WBF and DLM spiking rate are unlikely to be due to changes in muscle properties, but rather due to changes to the motor neuron and its input (Iyengar, 2014).

Wing movements are known to be involved during a number of behaviors, such as male courtship song production, which has been predominantly studied acoustically. An immediate question with interesting implications is if there is any relationship between wing beats during courtship song and flight. A number of well-studied courtship mutants are available and correlational analysis may produce insight. One such mutant, cac, has well-characterized song defects and this study demonstrates its severe consequences on key parameters involved in flight control. Motor programs involved in these two activities may be correlated in the same fly, using previously devised methods to induce courtship songs in tethered males (Iyengar, 2014).

Another well known, striking behavioral phenotype involving patterned wing beats is seizure, observed in certain bang-sensitive mutants following mechanical shock or in other genotypes induced by ECS. While DLM spiking during ECS-triggered seizures is well described, the activity in the direct flight muscles has not been monitored during these events. This study has demonstrated that the wing buzzing patterns characteristic to individual genotypes during seizures may also be monitored acoustically, enabling correlation between DLM and direct flight muscle activities during another stereotypical behavioral repertoire (Iyengar, 2014).

As such, the current findings may provide an opportunity to extend the scope of analysis across motor programs underlying different categories of behaviors. With the wealth of behavioral mutant collections, it is conceivable that future Drosophila studies will elucidate the common and distinct features of discrete programs driving different muscle groups during behaviors, such as flight, seizure, and other stereotypic motor activities (Iyengar, 2014).

Kruppel mediates the selective rebalancing of ion channel expression

Ion channel gene expression can vary substantially among neurons of a given type, even though neuron-type-specific firing properties remain stable and reproducible. The mechanisms that modulate ion channel gene expression and stabilize neural firing properties are unknown. In Drosophila , this study demonstrates that loss of the Shal potassium channel induces the compensatory rebalancing of ion channel expression including, but not limited to, the enhanced expression and function of Shaker and slowpoke. Using genomic and network modeling approaches combined with genetic and electrophysiological assays, it was demonstrated that the transcription factor Kruppel is necessary for the homeostatic modulation of Shaker and slowpoke expression. Remarkably, Kruppel induction is specific to the loss of Shal, not being observed in five other potassium channel mutants that cause enhanced neuronal excitability. Thus, homeostatic signaling systems responsible for rebalancing ion channel expression can be selectively induced after the loss or impairment of a specific ion channel (Parrish, 2014).

This study provides evidence that the cell fate regulator Kr is a critical player in the compensatory control of potassium channel gene expression. It is speculated that the induction of Kr drives a pattern of gene expression, first used to establish neuronal identity in the embryo and then, postembryonically, to rebalance ion channel expression in the face of persistent or acute perturbation of the Shal channel. Surprisingly, Kr is induced after the loss of Shal, but not other potassium channel gene mutations that have been shown to cause neural hyperexcitability. It is concluded that Shal function is specifically coupled to a homeostatic feedback system that includes the Kr-dependent transcriptional response. As such, these data imply the existence of discoverable 'rules' that define how individual neurons will respond to mutations in ion channel genes. Recent work underscores the possibility that the regulation of ion channel expression can be conserved from Drosophila to mammalian central neurons. In Drosophila, the translational regulator Pumilio was shown to be necessary and sufficient for the modulation of sodium channel transcription after persistent changes to synaptic transmission in the CNS. More recent data indicate that Pumilio-2 regulates NaV1.6 translation in rat visual cortical pyramidal neurons in a manner consistent with that observed in Drosophila. In mammalian neurons, Kr-like genes (KLF) respond to neuronal activity and are studied intensively in the context of axonal regeneration, but a role in ion channel expression or homeostatic rebalancing has yet to be defined (Parrish, 2014).

Kr and its homologs are potent regulators of neuronal cell fate. KLF4 and KLF5, in particular, have been shown to both maintain and reprogram embryonic stem cell fate. This study has provided evidence that Kr protein levels diminish to nearly undetectable levels in the postembryonic CNS. Kr expression is then induced to achieve potassium channel regulation. It is tempting to speculate that the rebalancing of ion channel expression postembryonically is a reinduction of the embryonic mechanisms that initially specify neuronal active properties (Parrish, 2014).

Drosophila QVR/SSS modulates the activation and C-type inactivation kinetics of Shaker K+ channels

The quiver/sleepless (qvr/sss) gene encodes a small, glycosylphosphatidylinositol-anchored protein that plays a critical role in the regulation of sleep in Drosophila. Loss-of-function mutations in qvr/sss severely suppress sleep and effect multiple changes in in situ Shaker K⁺ currents, including decreased magnitude, slower time-to-peak, and cumulative inactivation. Recently, it was demonstrated that Sleepless (Sss) protein modulates Shaker channel activity, possibly through a direct interaction at the plasma membrane. This study shows that Sss accelerates the activation of heterologously expressed Shaker channels with no effect on deactivation or fast N-type inactivation. Furthermore, this Sss-induced acceleration is sensitive to the pharmacological disruption of lipid rafts and sufficiently accounts for the slower time-to-peak of in situ Shaker currents seen in qvr/sss mutants. It was also found that Sss decreases the rate of C-type inactivation of heterologously expressed Shaker channels, providing a potential mechanism for the cumulative inactivation phenotype induced by qvr/sss loss-of-function mutations. Kinetic modeling based on the in vitro results suggests that the Sss-dependent regulation of channel kinetics accounts for nearly 40% of the decrease in Shaker current magnitude in flies lacking Sss. Sleep duration in qvr/sss-null mutants is restored to normal by a qvr/sss transgene that fully rescues the Shaker kinetic phenotypes but only partially rescues the decrease in current magnitude. Together, these results suggest that the role of Sss in the regulation of sleep in Drosophila correlates more strongly with the effects of Sss on Shaker kinetics than current magnitude (Dean, 2011).

In an in vitro system, Sss increased the rate of Shaker channel activation without affecting the rate of deactivation. While Sss also increased the rate of decay of heterologously expressed wild-type Shaker currents at voltages <30 mV, this may be an indirect effect of enhancing the rate of channel opening. Enhancing the rate of activation of inactivating channels not only hastens the time-to-peak, but also increases the rate of current decay. Thus, an increase in activation alone is sufficient to explain the in situ and in vitro effects of Sss on wild-type Shaker channels (Dean, 2011).

Consistent with previous reports (Wang, 2000; Wang, 2010), this study found that repetitive depolarization of qvr/sss mutant larval muscles leads to a significant loss of Shaker current. The cumulatively inactivated component, recently named I_AS (Wang, 2010), is eliminated by muscle-directed restoration of Sss in sss^P1 mutants, suggesting that Sss normally prevents the cumulative inactivation of Shaker currents in wild-type flies. In in vitro studies, Sss did not affect recovery from either N- or C-type inactivation, but it decreased the rate of entry into the C-type inactivated state. Sss may also slow C-type inactivation of Shaker in Drosophila, as the cumulative inactivation phenotype observed in qvr/sss mutants could be caused by an absence of Sss-dependent resistance to C-type inactivation. This hypothesis is supported by the reduction of cumulative inactivation at supraphysiological concentrations of extracellular K⁺. However, direct quantitative comparisons between the in situ Shaker currents and the in vitro currents of ShBδN T449A cannot be made, as the precise Shaker isoforms underlying the former are unknown, and the latter has a fundamentally different rate of C-type inactivation than wild-type channels. Similarly, testing the specific contribution of C-type inactivation to the sss^P1 behavioral phenotypes may also be difficult. It was not possible to rescue the sss^P1 phenotype with a high K⁺ diet, but it is suspected that systemic K⁺ concentration in Drosophila is tightly regulated, as it is in mammals (Dean, 2011).

Mutations in sss profoundly decrease Drosophila I_A magnitude; however, it is unclear whether the loss of Shaker current in qvr/sss mutants is caused by decreases in Shaker expression or changes in Shaker kinetics. Kinetic modeling based on in vitro experiments suggest that kinetic effects of Sss account for nearly 40% of the total loss in I_A magnitude exhibited by sss^P1 mutants; the rest is likely due to a decrease in channel quantity. Interestingly, the cumulative inactivation of I_A comprises a large portion of the in situ current loss. Because the rescue of the cumulative inactivation phenotype nearly doubles the Shaker magnitude after repetitive depolarization, this kinetic rescue could account for the entirety of the previously reported partial rescue of total Shaker current magnitude. Therefore, in sss^P1 mutants, targeted expression of Sss completely rescues altered Shaker gating kinetics, cumulative inactivation, and slower t_Peak, as previously reported (Wu, 2010), while only minimally rescuing total Shaker current magnitude. Nevertheless, Sss expression fully rescues the sss^P1 mutant sleep phenotype when driven broadly throughout the Drosophila brain as well as selectively in cholinergic neurons (Wu, 2010). Thus, the severity of the sleep phenotype in sss^P1 mutants may better correlate with an alteration in Shaker current kinetics than magnitude. For example, Sss overexpression in wild-type flies reduces Shaker current in Drosophila muscles but does not induce a sleep phenotype, even when expressed using drivers that rescued the sleep phenotype in sss^P1 flies. Additionally, Drosophila bearing Shaker-null alleles exhibit a less severe sleep phenotype than sss^P1 mutants despite having no Shaker current. Because organisms can compensate for constitutive changes in ion channel quantity to preserve a neuron's overall excitability, it is speculated that static decreases in Shaker quantity are easier to compensate for than the dynamic, use-dependent loss of Shaker K⁺ current seen in qvr/sss mutants. Future studies of in vivo central neurons in adult Drosophila may better elucidate the neurophysiological significance of the kinetic effects of Sss on Shaker (Dean, 2011).

The Shaker β-subunit Hyperkinetic (Hk) accelerates rate of Shaker activation to a degree similar to that induced by Sss; however, Hk is an intracellular protein whereas Sss is tethered to the extracellular side of the membrane by a GPI-anchor. Although GPI-anchored auxiliary subunits have not yet been proposed for voltage-gated K⁺ channels, a recent study found that the voltage-gated Ca²⁺ channel auxiliary subunit a₂d is indeed GPI-anchored. Just as adequate Ca²⁺ channel expression depends on a₂d, a deficiency of Sss leads to decreases in Shaker protein levels in vivo (Wu, 2010). Additionally, a₂d-containing Ca²⁺ channels concentrate in lipid rafts, much like Sss and Shaker. Furthermore, the Sss-induced increase in activation of heterologously expressed Shaker channels was sensitive to cholesterol depletion, similar to K⁺ currents in ex vivo neurons from the Drosophila mushroom bodies, raising the possibility that the mechanism by which Sss interacts with Shaker involves lipid rafts. Thus, GPI-anchored proteins like Sss and a₂d may be critical regulators of ion channel function and quantity at the cell surface (Dean, 2011).

Sss enhances gating charge movement, but the mechanism and stoichiometry by which Sss affects the charge-bearing S4 transmembrane segment remain unknown. Because Sss and Shaker coimmunoprecipitate when coexpressed in a heterologous system (Wu, 2010), one potential mechanism is a direct protein-protein interaction between Sss and the extracellular loops of Shaker. The S1-S2 linker is a promising candidate, as a threonine to isoleucine substitution in this loop (Sh^mns) also induces a sleep phenotype; however, the functional consequences of that allele have not yet been thoroughly characterized (Cirelli, 2005; Dean, 2011 and references therein).

Increasing the forward rate constant a in the two-step model of Shaker activation was sufficient to reproduce the effects of Sss on the activation of ionic currents and the time course of gating charge movement. A recent structure-function study of Shaker gating by Tao (2010) suggests that activation requires at least four transitions between five discrete states of the S4 voltage sensor. The forward rate constant a in the ZHA model may correspond to the initial transitions of the voltage sensor, and thus Sss may act upon the early intermediate states of S4, including destabilizing the resting state when the first charge of S4 is located below the S2 phenylalanine cap (Dean, 2011).

There are also potential mechanisms for the Sss-dependent modulation of activation and C-type inactivation aside from a direct Sss-Sh interaction. For instance, Sss may indirectly act upon Shaker through another protein, as even in an in vitro system like HEK cells, native proteins found in lipid rafts (e.g., caveolin) can modulate K⁺ channels. Another potential effect of Sss may be to increase local extracellular K⁺ accumulation upon channel opening and K⁺ efflux, therefore indirectly slowing C-type inactivation. Sss may even affect channel kinetics by altering interactions between surrounding lipids and Shaker channels, an important consideration given that membrane phosopholipids play critical roles in ion channel function. A lipid-channel interaction mediated by Sss is consistent with the finding that the sigmoidicity of macroscopic current activation was unaltered by Sss, regardless of the degree of acceleration that Sss induced. Had the interaction between Sss and Shaker followed a strict stoichiometry in which individual subunits within a tetramer were selectively enhanced, then it is unlikely that activation sigmoidicity could have been preserved over the range of activation speeds observed. Because sigmoidicity is preserved over the entire range of activation speeds, it is suspected that every subunit of each channel in a patch is equally affected by the variable expression levels of Sss. The effect may be unique to Shaker, as human Slo1 activation kinetics are not affected by Sss expression despite also trafficking to lipid rafts, suggesting that either Shaker is particularly sensitive to lipid membrane composition or that cholesterol plays a permissive role in the effects of Sss on Shaker kinetics. Nevertheless, effects of Sss on channels other than Shaker cannot be entiredly excluded (Dean, 2011).

In summary, Sss regulates the kinetics of Shaker channels on timescales ranging from milliseconds to hundreds of milliseconds. These kinetic effects, in addition to the Sss-dependent effects on Shaker expression and subcellular localization (Wu, 2010), suggest that Sss is an important regulator of Shaker and that the modulation of Shaker K⁺ conductance is a critical component of the sleep behavior in Drosophila (Dean, 2011).

Modulation of the frequency response of Shaker potassium channels by the Quiver peptide suggesting a novel extracellular interaction mechanism

Recent studies have indicated that the Shaker potassium channel regulates sleep in Drosophila. The Drosophila quiver (qvr) gene encodes a novel potassium channel subunit that modulates the Shaker potassium channel. The Qvr peptide contains a signal sequence for extracellular localization. Qvr may regulate a unique feature of the Shaker I_A current that confers special neuronal excitability patterns. Studies of the Shaker channel properties in the qvr mutation background should provide an opportunity to uncover physiologic modulation of potassium channels. This study investigated the impact of qvr protein on the Shaker channel properties and its implications in synaptic function in vivo. Synaptic transmission was studied at the larval neuromuscular junction, and the transient potassium current I_A was characterized in larval muscles. Two different functional states of I_A were identified in qvr larval muscles, as reflected by two distinct components, I_AF and I_AS, differing in their kinetics of recovery from inactivation and sensitivity to a K⁺ channel blocker. Correspondingly, qvr mutant larvae exhibit multiple synaptic discharges following individual nerve stimuli during repetitive activity (Wang, 2010)).

Most invertebrate muscles, including that of Drosophila, do not express Na⁺ channel, and rely on Ca²⁺-mediated action potentials for muscle contraction. A step depolarizing potential generates five major currents in Drosophila larval muscles. Four outward K⁺ currents including the voltage-gated transient I_A and the delayed rectifier I_K, and the Ca²⁺-dependent fast I_CF and the slow I_CF, plus an inward Ca²⁺ current. Genetic and pharmacological studies have shown that some of these currents consist of distinct components. A previous study showed that the qvr mutations affect only the transient I_A, but not I_K, I_CF, I_CS or the calcium current (Wang, 2000; Wang, 2011 and references therein).

When ten episodes of a depolarizing step to +10 mV were applied to a qvr mutant muscle, the first outward transient current (I_A) was more than twice that of the subsequent nine currents, but not as large as the wild-type current. The subsequent nine currents were almost the same size in amplitude with the same kinetics, suggesting that there were two components of I_A in qvr mutant muscles. This contrasts with the use-dependent inactivation of a homogeneous component, which should display a gradual decay in the current amplitude upon repetitive depolarization. The first and the subsequent I_A currents displayed the same time to peak and the same inactivation kinetics, which could be visualized when the two traces were normalized. The fast- and slow-recovery components are therefore named I_AF and I_AS, respectively. Operationally, the first I_A is assumed to represent the sum of I_AF and I_AS, and the average of subsequent nine I_A currents includes only I_AF. The similarity in I-V curve between qvr¹ and qvr^δ43-1/qvr¹ suggests the observed phenotype is attributable to the qvr locus and not likely an effect of an unidentified second-site mutation in the background (Wang, 2010).

The involvement of the qvr K⁺ channel subunit in sleep regulation and its potential interaction with the Sh K⁺ channel from extracellular domain (Koh, 2008) call for more biophysical studies of the Qvr function. The present study shows that Qvr is important for the maintenance of neuronal excitability. Frequent stimulations of the motor axons in qvr mutant larvae generate discretely increased neuromuscular transmission. In a detailed biophysical characterization of the Sh potassium channel, this study found that the transient I_A current in qvr mutant muscles exhibits two discrete components, I_AF and I_AS, which are not observed in normal muscles. I_AF displays faster kinetics of recovery from inactivation and more sensitivity to 4-aminopyridine (4-AP) than I_AS. Furthermore, analysis of I_AF and I_AS in double mutants, Sh qvr, eag qvr and Hk qvr, identified a potential conformational change in the Sh K⁺ channel conferred by Qvr (Wang, 2010).

Previous study shows that qvr mutations affect only the I_A channel in both conductance and kinetics, without altering I_K, I_CF, I_CS, and I_Ca, suggesting that the Qvr assumes a role in modulating the a-subunit of the I_A channel (Wang, 2000). Its phenotypic similarities to the three known K⁺ channel mutants Sh, Hk and eag provide hints that the qvr gene might code for a distinct K⁺ channel subunit. The Qvr protein is predicted to contain a GPI-attachment site and the GPI anchor can be cleaved by PLC (Koh, 2008). This study has investigated the potential conformational change conferred by the interaction between Qvr and the Sh K⁺ channel. First, a pharmacological approach was used. 4-AP has been well established as a Sh channel blocker that binds to the cytoplasmic pore region of the channel. The sensitivities to 4-AP of I_AF and I_AS showed an IC₅₀ of 60 and 200 microM, respectively, which were much higher than the IC₅₀ of only 7 microM in wild-type muscles. The differential 4-AP sensitivities in I_AF and I_AS imply different conformation in the cytoplasmic pore region. Then several K⁺ channel mutants were used to identify potential site of conformational change. The Sh⁵ mutation, which has an amino acid replacement in the S4-S5 linker of the Sh polypeptide, caused an increase in 4-AP sensitivity. Among several different double mutants, Sh⁵qvr¹ exhibited a special property: a sum amplitude of I_AF and I_AS is on par with the I_A amplitude in Sh⁵ mutant muscles. These results suggest that the interaction between Qvr and the Sh channel confers conformational change at the Sh⁵ mutation site (Wang, 2010).

The excitability control of the motor neurons can be attributed to the function of the Sh channel. It is interesting to note that the 4-AP sensitive I_A current plays an important role in the gating of action potentials in hippocampal CA3 pyramidal neurons. Action potentials in the pyramidal neurons can be blocked by the activation of the I_A current with a brief hyperpolarization a few milliseconds before the induction of an action potential. The discrete increase in EJC at the neuromuscular junction in Drosophila larvae suggests that Qvr is important in the controlling the propagation of action potentials. This result is in accord with previous findings that efficient membrane repolarization is required to suppress supernumerary action potentials in the motor axon (Wang, 2011 and references therein).

Sleep is thought as a physiological state that increases the efficiency of behavior by regulating its timing and energy use (Siegel, 2009). Studies of rat cerebral energy consumption suggest that about half of the brain energy is used to drive signals along axons and across synapse. This study suggests that modulation of the Sh K⁺ channel by Qvr is a potential gating mechanism for the generation of action potentials in the nervous system. One can speculate that physiologic modulation of Qvr function plays an important role in controlling action potentials in relevant neural circuit for sleep. However, qvr expression does not fluctuate with the circadian cycle (Koh, 2008). Therefore, future experiments to demonstrate whether and how wakefulness/sleep states modulate the biophysical properties of the Sh K⁺ channel, reminiscent of the comprehensive studies in the mammalian thalamocortical systems, will be important to provide a mechanistic link between sleep and K⁺ channel function. Alternatively, it is possible that wakefulness/sleep states do not modulate Sh K⁺ channel and the abnormal sleep phenotype in K⁺ channel mutant flies is due to their hypersensitivity to sensory stimulations and a hypersensitive motor system. Future studies of Qvr function in Drosophila central brain with optical imaging may shed light on the role of Qvr in sleep regulation (Wang, 2010).

Sleepless, a Ly-6/Neurotoxin family member, regulates levels, localization, and activity of Shaker

Sleep is a whole-organism phenomenon accompanied by global changes in neural activity. Sleepless (Sss) as a novel glycosylphosphatidyl-inositol-anchored protein required for sleep in Drosophila. This study demonstrates a critical role for Sss in regulating the sleep-modulating potassium channel, Shaker. Sss and Shaker exhibit similar expression patterns in the brain and specifically affect each other's expression levels. sss mutants exhibit altered Shaker localization, reduced Shaker current density, and slower Shaker current kinetics. Transgenic expression of sss in sss mutants rescues defects in Shaker expression and activity cell-autonomously and also suggests that Sss functions in wake-promoting, cholinergic neurons. Importantly, in heterologous cells, Sss accelerates kinetics of Shaker currents and can be co-immunoprecipitated with Shaker, suggesting that Sss interacts with Shaker and modulates its activity. Sss is predicted to belong to the Ly-6/neurotoxin superfamily, suggesting a novel mechanism for regulation of neuronal excitability by endogenous toxin-like molecules (Wu, 2010).

This study presents in vivo and in vitro evidence that Sss is a novel modulator of Shaker expression, subcellular localization, and activity, and thus is an important regulator of nervous system function. While Sss probably modulates neuronal excitability at multiple anatomical loci, dissociation of the neural circuits responsible for sleep and ether-dependent leg-shaking suggests that the role of Sss in sleep regulation is distinct from its effect on motor control. The data suggest that Sss acts on Shaker in a cell-autonomous manner and that expression of Sss in cholinergic neurons restores sleep in sss mutants, although unidentified non-cholinergic neurons included in the Cha-Gal4 expression pattern may also be required. Since upregulation of Shaker by Sss in cholinergic neurons presumably decreases excitability and results in increased sleep, excitation of these cholinergic neurons is likely to promote wakefulness in Drosophila. Recent studies have demonstrated involvement of monoaminergic signaling and GABA-responsive peptidergic cells in regulating wakefulness in Drosophila. Thus, as in mammals40, sleep in Drosophila is controlled by arousal systems that include distinct populations of cholinergic, monoaminergic, and peptidergic neurons (Wu, 2010).

Sss and Shaker are enriched in the same regions of the Drosophila brain, and Sss appeared to affect the subcellular distribution of Shaker. Thus, in sss mutants the distribution of Shaker channels shifts from an enrichment in processes to a predominance in cell bodies in brains and thoracic ganglia. In addition, loss of Sss or Shaker resulted in a reduction of the other protein, without a concomitant reduction in transcript, suggesting that these proteins stabilize each other in a complex. The reduction in brain Shaker expression in sss mutants could be rescued by transgenic expression of Sss. However, only partial rescue of muscle IA amplitude was observed in sss mutants with overexpression of Sss in muscles. Along these lines, it was also found that overexpression of Sss in wild-type muscles reduced I_A amplitude, suggesting that the presence of either too little or too much Sss can impair Shaker function, at least at the larval NMJ (Wu, 2010).

In addition to modulating the level of Shaker, Sss regulates kinetics of Shaker-dependent potassium currents. Kinetics of Shaker-mediated I_A potassium currents in muscle were selectively slower in sss mutants, a phenotype that could be rescued by targeted expression of sss in muscle. In heterologous cells, co-expression of Shaker and Sss accelerated Shaker currents and resulted in detectable complex formation between the two proteins. Taken together, these data suggest that Sss directly interacts with Shaker to regulate its levels, localization, and activity (Wu, 2010).

Properties of voltage-gated potassium channels, such as expression level, subcellular localization, and gating characteristics are influenced by a number of associated regulatory proteins including Kvβ/Hyperkinetic, KCNEs, KChIPs, and KChAP. The in vivo relevance of these regulatory proteins is underscored by the finding that mutations in some of the genes encoding them are associated with human diseases, including Long QT syndromes. Unlike most other known regulators of voltage-gated potassium channels, which generally interact with cytoplasmic domains of channel proteins, Sss, as a GPI-anchored protein tethered to the plasma membrane, probably interacts with an extracellular domain of the Shaker channel. The predicted structure of Sss is also unlike those of other known endogenous regulators of voltage-gated potassium channels. Bioinformatic analysis predicts that Sss contains a compact disulfide-bonded beta-sheet structure (three-finger fold) found in the Ly-6/neurotoxin superfamily of proteins. This diverse family includes proteins involved in the modulation of receptor function and immune complex formation, as well as snake neurotoxins that bind the extracellular domains of various ion channels at the cell surface (Wu, 2010).

Snake neurotoxins do not have GPI anchors like Sss. However, ER-targeted expression of soluble dendrotoxin, a specific blocker of Shaker-type potassium channels, results in increased surface expression of Kv1.1, a mammalian ortholog of Shaker. This finding led to a postulate of the existence of an endogenous toxin-like ER protein that tethers Shaker channels to the ER membrane and with which dendrotoxin competes for binding. Sss may be such an endogenous neurotoxin-like molecule regulating Shaker function and localization. However, rather than retaining Shaker in the ER, Sss appears to increase surface localization of the channel, either through promotion of Shaker trafficking to or retention at the cell surface. Lynx1, another GPI-anchored neurotoxin/Ly-6 family member found in mammals, binds to and modulates the activity of a ligand-gated ion channel (nicotinic acetylcholine receptor). Thus, regulation of various ion channels by toxin-like GPI-anchored proteins may be an evolutionarily conserved mechanism, and Sss and Lynx-1 may be founding members of a family of cell-surface proto-toxins that modulate ion channel properties to control neuronal excitability and signaling. Although BLAST analysis with the primary sequence of Sss does not reveal an obvious mammalian ortholog, there are a number of mammalian proteins with a Ly-6 domain and a GPI anchor, one of which may represent a functional homolog of Sss (Wu, 2010).

In summary, this study has demonstrate that Sss is a novel regulator of Shaker expression, localization, and function in vivo. It is proposed that Sss acts as an endogenous 'proto-toxin' that forms a complex with Shaker and promotes its stability and activity at the cell surface. Since dysregulation of channel function causes a number of inherited human diseases, including migraine, epilepsy, and cardiac arrhythmias, identification and characterization of additional toxin-like regulators of ion channels may prove to be a fruitful approach for discovering novel treatment options for these diseases (Wu, 2010).

A hierarchy of cell intrinsic and target-derived homeostatic signaling

Homeostatic control of neural function can be mediated by the regulation of ion channel expression, neurotransmitter receptor abundance, or modulation of presynaptic release. These processes can be implemented through cell autonomous or intercellular signaling. It remains unknown whether different forms of homeostatic regulation can be coordinated to achieve constant neural function. One way to approach this question is to confront a simple neural system with conflicting perturbations and determine whether the outcome reflects a coordinated, homeostatic response. This study demonstrates that two A-type potassium channel genes, shal and shaker, are reciprocally, transcriptionally coupled to maintain A-type channel expression. This homeostatic control of A-type channel expression was shown to prevent target-dependent, homeostatic modulation of synaptic transmission. Thus, this study uncovered a homeostatic mechanism that reciprocally regulates A-type potassium channels, and a hierarchical relationship was defined between cell-intrinsic control of ion channel expression and target-derived homeostatic control of synaptic transmission (Bergquist, 2010).

An electrophysiology-based forward genetic screen identified three potassium channel mutations, including mutations in shal and Drosophila K_V3.2, that block the expression of synaptic homeostasis following inhibition of postsynaptic glutamate receptor function. This study focused on how mutations in a single potassium channel, shal, lead to a blockade of synaptic homeostasis. It was first demonstrated that loss of shal induces a compensatory increase in shaker expression, and vice versa, suggesting homeostatic maintenance of A-type channel abundance in Drosophila motoneurons. The compensatory increase in shaker expression is remarkable, however, because it does not replace the A-type current recorded at the motoneuron soma. Rather, increased Shaker functions to restrict neurotransmitter release from the motoneuron terminal, decreasing baseline release and blocking any further homeostatic enhancement of presynaptic release. There are several implications. First, the data demonstrate that the unique subcellular localization of each ion channel will determine how any compensatory change in ion channel abundance affects neural activity and synaptic transmission. Second, it appears that cell-autonomous control of intrinsic excitability can occlude the expression of subsequent intercellular homeostatic signaling. This suggests a hierarchical control of cell-intrinsic excitability compared to circuit level homeostatic regulation. This also calls into question the concept of a master, homeostatic sensor of neuronal activity. Finally, a form of compensation was defined that may largely preserve neuronal output properties without restoring cellular excitation at the level of the cell soma (Bergquist, 2010).

This study has demonstrated that compensatory increase in Shaker expression is necessary and sufficient to block the subsequent expression of synaptic homeostasis following postsynaptic GluR inhibition. In a shal mutant, a ~250% increase in shaker expression was detected. If this increase in Shaker expression is presented in any of three different ways, 1) genetically by introducing shaker mutations, 2) transgenically through neuron-specific dsRNA knockdown of shaker, or 3) pharmacologically, then synaptic homeostasis is restored in the shal mutant. Furthermore, acute block of Shaker by 4-AP following PhTx provides evidence that increased Shaker levels block the expression of synaptic homeostasis, not the induction of this form of homeostatic plasticity. Finally, it was demonstrated that exogenous overexpression of a Shaker transgene (EKO) is sufficient to block synaptic homeostasis in an otherwise wild type background. Thus, the compensatory increase in Shaker expression in the shal mutant blocks subsequent expression of synaptic homeostasis (Bergquist, 2010).

Numerous experiments are provided that argue against the possibility that loss of Shaker rescues synaptic homeostasis through a non-specific potentiation of synaptic transmission. First, neuronal expression of shaker RNAi in the shal mutant background reduces shaker transcript (~70% reduction) and restores synaptic homeostasis without potentiating baseline transmission. Second, pharmacological inhibition of Shaker was performed using 4-AP concentrations that have a minimal effect on baseline synaptic transmission (~27% change), yet synaptic homeostasis is restored. Finally, synaptic homeostasis is also blocked in the K_V3.2 mutant, but there is no change in shaker expression nor does presynaptic knockdown of shaker in the K_V3.2 mutant rescue synaptic homeostasis. It is concluded that the increased shaker expression is specific to the shal mutant and that reducing shaker expression or function in the shal mutant is sufficient to reveal the expression of synaptic homeostasis in the shal mutant (Bergquist, 2010).

Why does increased expression of Shaker, at or near the synaptic terminal block the expression of synaptic homeostasis? It is presumed that increased expression of Shaker in the shal mutant causes a decrease in action potential width. Unfortunately, it is not possible to record the presynaptic action potential from the synaptic terminal because the terminal is embedded within the muscle and is otherwise surrounded by the muscle basal lamina. There are several possible ways that a narrower action potential could block expression of synaptic homeostasis. One possibility is that synaptic homeostasis requires an increase in action potential duration and this is prevented by increased Shaker expression. If so, it is unlikely that Shaker is the direct target of this homeostatic signaling system because homeostatic compensation is observed in the shaker mutant background. Alternatively, a narrower action potential could prevent recruitment of newly inserted presynaptic calcium channels. Genetic data indicate that synaptic homeostasis involves a change in calcium influx at a fixed number of active zones and this could be achieved by an increase in the number of presynaptic calcium channels (Bergquist, 2010).

The transcriptional coupling of shaker and shal would seem to be a homeostatic mechanism since both channels encode A-type potassium currents. However, these channels localize to different subcellular compartments. Thus, increased Shaker expression should not homeostatically restore wild-type motoneuron excitability since the somatic A-current remains absent. Rather, increased Shaker seems to inhibit presynaptic neurotransmitter release and may thereby guard against inappropriately enhanced glutamatergic transmission. This effect differs from current homeostatic hypotheses because baseline neural activity is not re-established, but neural output is constrained within reasonable limits (Bergquist, 2010).

The importance of channel localization during homeostatic compensation is also highlighted by recent studies in vertebrate central neurons. It was recently demonstrated that K_V4.2 knockout animals lack dendritically recorded A-type currents in hippocampal neurons. The absence of a dendritic A-type current potentiates back propagating action potentials and enhances LTP. Thus, at the level of the neuronal dendrite, this is an example of failed homeostatic compensation. However, this study also documents a compensatory increase in somatically recorded K_V1-type currents. It seems plausible that the observed compensatory increase in somatic K_V1-type currents could counteract increased dendritic excitability and, thereby, homeostatically restrain neural output. This possibility is supported by data from additional studies examining K_V4.2 knockouts in other neuronal cell types. In these studies, neuronal firing properties measured at the soma are largely normal in the K_V4.2 knockout despite the absence of the dendritic A-type current (Bergquist, 2010).

This study demonstrates that shal and shaker, which encode A-type potassium channels, are reciprocally, homeostatically coupled. What drives the compensatory change in ion channel expression following loss of a given ion channel? One possibility, suggested by prior research in other systems is that the neuron senses a persistent change in cellular activity and initiates a homeostatic response that modulates the expression of other ion channels. The data are consistent with an activity-dependent model. Knockdown of shaker expression (65% of the wild type level) leads to a 223% increase in shal expression. Remarkably, a 1300% increase was observed in shal expression in the shaker¹⁴ mutant, which is a point mutation resulting in a non-functional channel. In the shaker¹⁴ experiment, the mutant shaker transcript continues to be expressed at 80% wild type levels. Thus, the degree to which shal expression is increased correlates with the severity of altered channel function rather than the loss of shaker message. This suggests that altered channel function or altered neural activity could be the trigger for the compensatory response. These data also raise an interesting question. If the expression of one ion channel, such as shal, is specifically coupled to the expression of another channel, such as shaker, how could this be achieved by a general monitor of neural activity (Bergquist, 2010)?

Several studies have now documented that prolonged inhibition of an ion channel, or genetic ablation of an ion channel, can lead to increased expression of a different ion channel with overlapping function, again suggesting coupling between specific pairs of ion channels. For example, loss of Na_V1.6 causes increased expression of Na_V1.1 in purkinje cells and increased expression of Na_V1.2 in retinal ganglion cells. Similarly, loss of A-type potassium currents in K_V4.2 (the vertebrate shal homolog) knockout animals causes a compensatory increase in both I_K and I_SS that preserves action potential shape and neuronal firing properties. In these examples, the compensatory changes in sodium or potassium channel expression seem to homeostatically maintain appropriate neuronal firing properties. These studies support the hypothesis that ion channels are free variables that can be adjusted by a homeostatic monitor of neural activity and that specific pairs of ion channels may be homeostatically coupled (Bergquist, 2010).

An alternate form of regulation has been suggested by work in lobster stomatogastric neurons where there is evidence for an activity-independent mechanism that couples shal and I_h expression. In this system, overexpression of shal leads to increased I_h current (channel expression was not tested). However, overexpression of a pore-blocked shal also leads to increased I_h current. Thus, altered neural function does not appear to be the trigger for a compensatory change in I_h current. Rather, the cell could monitor the level shal message or protein and regulate I_h current accordingly. This mechanism would allow for specific coupling of ion channel pairs, but appears to be different from the phenomenon identified in Drosophila motoneurons (Bergquist, 2010).

One interesting possibility is that the developmental programs that initially specify the active properties of a given neuron could, later, control ion channel expression in a homeostatic manner. Modeling studies suggest that there are large numbers of physiologically plausible combinations of ion channels that could give rise to a cell with a specific firing property. However, if the expression of pairs or combinations of ion channels are somehow coupled, then the parameter space for defining the firing properties of a given cell type would be dramatically simplified. It is interesting, therefore, to speculate that the apparent homeostatic compensation for loss of a given ion channel could represent the re-use of an earlier developmental program that initially served to balance the expression of specific pairs or combinations of ion channels during cell fate specification. It will be important to determine whether there are any general rules by which one might predict how a cell will respond to the altered expression of a specific ion channel or whether all such relationships will be defined in a cell-type specific manner (Bergquist, 2010).

The regulation of A-type currents in Drosophila motoneurons occludes trans-synaptic, homeostatic modulation of neurotransmitter release. The consequence is that the postsynaptic muscle target is unable to restore normal synaptic drive from the motoneuron terminal and remains hypo-excitable. Specifically, EPSP amplitudes are significantly smaller in the shal; GluRIIA double mutant animals compared to either shal or GluRIIA alone. Thus, at the neuromuscular junction, the regulation of motoneuron intrinsic excitability supercedes the homeostatic control of motor unit function (Bergquist, 2010).

The homeostatic modulation of synaptic transmission can be induced in seconds to minutes. By contrast, the compensatory control of ion channel expression clearly involves gene transcription and is likely to be induced more slowly. One question is whether, given enough time, the mechanisms of synaptic homeostasis can adjust to the change in ion channel expression observed in the shal mutant background. This does not appear to be the case. The GluRIIA mutation causes a persistent change in postsynaptic receptor function leading to a persistent homeostatic increase in presynaptic release that is present throughout the four days of larval development. Synaptic homeostasis is still blocked in the GluRIIA; shal double mutant and a statistically similar increase shaker transcription is observed (Bergquist, 2010).

It is worth emphasizing that the homeostatic modulation of presynaptic release appears to have been executed, unaltered in the shal mutant background because acute application of 4-AP reveals normal homeostatic compensation in the shal mutant. These data argue against the possibility that independent homeostatic signaling systems are somehow coordinated at the level of the motor unit, or perhaps neural circuit. Thus, even though an initial homeostatic action is restorative, any change in the balance of ion conductances that control the action potential could dramatically alter how a cell responds to a future perturbation. It has been speculated in systems ranging from crustacean central neurons to the vertebrate cortex, that normal cell-to-cell differences in ionic conductances recorded from an identified cell type might reflect the activity of homeostatic signaling systems. The question remains whether these different cells respond similarly to future homeostatic pressures (Bergquist, 2010).

Pre- and post-synaptic mechanisms of synaptic strength homeostasis revealed by slowpoke and shaker K+ channel mutations in Drosophila

Naturally occurring, systematic variations in synaptic strength have been reported at neuromuscular junctions along the dorsal-ventral (D-V) axis of the Drosophila larval body wall. These gradual changes were correlated with differences in presynaptic neurotransmitter release regulated by nerve terminal excitability and in postsynaptic receptor composition influencing miniature excitatory junctional potential (mEJP) amplitude. Surprisingly, synaptic strength and D-V differentials at physiological Ca(2+) levels were not significantly altered in slowpoke (slo) and Shaker (Sh) mutants, despite their defects in two major repolarizing forces, Ca(2+)-activated Slo (BK) and voltage-activated Sh currents, respectively. However, lowering [Ca(2+)](o) levels revealed greatly altered synaptic mechanisms in these mutants, indicated by drastically enhanced excitatory junctional potentials (EJPs) in Sh but paradoxically reduced EJPs in slo. Removal of Sh current in slo mutants by 4-aminopyridine blockade or by combining slowith Sh mutations led to strikingly increased synaptic transmission, suggesting upregulation of presynaptic Sh current to limit excessive neurotransmitter release in the absence of Slo current. In addition, slo mutants displayed altered immunoreactivity intensity ratio between DGluRIIA and DGluRIIB receptor subunits. This modified receptor composition caused smaller mEJP amplitudes, further preventing excessive transmission in the absence of Slo current. Such compensatory regulations were prevented by rutabaga (rut) adenylyl cyclase mutations in rut slo double mutants, demonstrating a novel role of rut in homeostatic plasticity, in addition to its well-established function in learning behavior (Lee, 2008).

Differential contributions of Shaker and Shab K⁺ currents to neuronal firing patterns in Drosophila

Different K⁺ currents participate in generating neuronal firing patterns. The Drosophila embryonic 'giant' neuron culture system has facilitated current- and voltage-clamp recordings to correlate distinct excitability patterns with the underlying K⁺ currents and to delineate the mutational effects of identified K⁺ channels. Mutations of Sh and Shab K⁺ channels remove part of inactivating I_A and sustained I_K, respectively, and the remaining I_A and I_K reveal the properties of their counterparts, e.g., Shal and Shaw channels. Neuronal subsets displaying the delayed, tonic, adaptive, and damping spike patterns are characterized by different profiles of K⁺ current voltage dependence and kinetics and by differential mutational effects. Shab channels regulate membrane repolarization and repetitive firing over hundreds of milliseconds, and Shab neurons show a gradual decline in repolarization during current injection and their spike activities become limited to high-frequency, damping firing. In contrast, Sh channels acted on events within tens of milliseconds, and Sh mutations broadened spikes and reduced firing rates without eliminating any categories of firing patterns. However, removing both Sh and Shal I_A by 4-aminopyridine (4-AP) converts the delayed to damping firing pattern, demonstrating their actions in regulating spike initiation. Specific blockade of Shab I_K by quinidine mimic the Shab phenotypes and convert tonic firing to a damping pattern. These conversions suggest a hierarchy of complexity in K⁺ current interactions underlying different firing patterns. Different lineage-defined neuronal subsets, identifiable by employing the GAL4-UAS system, display different profiles of spike properties and K⁺ current compositions, providing opportunities for mutational analysis in functionally specialized neurons (Peng, 2007).

Characteristic firing patterns are found among different types of neurons that subserve specific functions in the nervous system. A variety of inward Na⁺ and Ca²⁺ currents and outward K⁺ currents take part in shaping neuronal action potentials and firing patterns. In particular, molecular studies have revealed a much greater diversity of K⁺ channel subtypes than that of Na⁺ and Ca²⁺ channels. Regulation of such molecular diversity facilitates the fine tuning of neuronal excitability and thus enriches spike patterning (Peng, 2007).

K⁺ currents have been classified according to their gating, kinetic, and pharmacological properties. In a variety of excitable cells, voltage-activated outward K⁺ currents are composed of transient, inactivating I_A and sustained, noninactivating I_K. Drosophila Shaker (Sh) mutants, initially isolated on the basis of their abnormal shaking behavior, have made possible the cloning of the first K⁺ channel gene and subsequent identification of three additional Drosophila K⁺ channel genes of the Sh family, Shab, Shaw, and Shal, and their vertebrate counterparts Kv 1, 2, 3, and 4. In heterologous expression systems, Sh and Shal channels mediate I_A-like, fast-inactivating currents, whereas both Shaw and Shab regulate I_K-like, slowly inactivating currents. Drosophila point mutations of Sh and Shab have demonstrated the in vivo roles of I_A and I_K channels at different levels, from cellular physiology to behavior, and can provide information about the regulation of neuronal excitability. Rich repertories of neuronal spike patterns have been described in several Drosophila semi-intact preparations. Nevertheless, it is difficult to delineate the spike patterns generated by synaptic interactions from those reflecting intrinsic neuronal membrane excitability in these preparations. Dissociated cell culture systems provide isolated conditions without cell-cell contacts and controlled ionic environment to eliminate contributions from synaptic interactions (Peng, 2007).

The 'giant' neuron culture system of Drosophila derived from cytokinesis-arrested embryonic neuroblasts displays differentiated morphological and molecular characteristics of different neuronal lineages. These enlarged cells facilitate Ca²⁺ imaging and electrophysiological recordings of Na⁺ and Ca²⁺ action potentials of different firing patterns. To explore the biophysical characteristics and functional roles of Sh and Shab channels, current- and voltage-clamp recordings were performed on the same cells in this culture system. The remaining I_A in Sh and I_K in Shab null mutants could provide opportunities to distinguish properties of Sh versus non-Sh I_A channels (encoded by Shal and possibly other unidentified genes) as well as Shab versus non-Shab I_K channels (encoded by Shaw and other candidate genes). The current results demonstrate the profound effect of Shab mutations on spike firing patterns and the distinctions between Sh channels and their counterpart, such as Shal channels. Advantage was taken of the GAL4-UAS system to demonstrate different profiles of K⁺ current kinetics and firing properties in cell-lineage defined neuronal subsets and their specific alterations by Sh and Shab mutations (Peng, 2007).

This study demonstrates that the Drosophila 'giant' neuron culture system can provide a bridge between heterologous expression systems and in vivo preparations to facilitate the study of how firing patterns are generated by interactions of molecularly identified channel subtypes and controlled by genes of interest. The current-clamp study has shown a diversity of firing patterns in WT cultures. Voltage-clamp recordings on the same cells further revealed the relationship of firing patterns and the compositions of underlying K⁺ currents. In addition, manipulating K⁺ channel compositions by employing mutations and pharmacological agents provided independent lines of evidence for the distinct contributions of each K⁺ current component to the control of membrane excitability (Peng, 2007).

In Drosophila muscles, it has been demonstrated that I_A is eliminated by Sh mutations and I_K is affected by Shab mutations. Mutations of ether a go-go (eag), another gene encoding a K⁺ channel subunit, can also reduce I_A and I_K in muscles. Several studies propose a different K⁺ channel expression pattern in neurons: I_A channels are encoded by both Sh and Shal, but major component of I_K is produced by Shab channels (Tsunoda, 1995). Consistently, in 'giant' neuron cultures, Sh and Shab null mutations only reduce, but did not eliminate I_A or I_K, indicating the presence of a substantial non-Sh component for I_A and a minor non-Shab component for I_K in neurons (Peng, 2007).

The results provide a first description of the striking phenotype of Shab neurons. During sustained current injection, Shab neurons rely on other noninactivating K⁺ currents for action potential repolarization as the transient I_A becomes progressively inactivated. The resultant abnormal damping spike pattern and unusual regenerative activities contrast the roles of Shab and non-Shab channels (including Shaw). In most Shab neurons, spiking or nonspiking, a novel 'repolarization decline' phenotype is observed that reflects a failure in maintaining a steady level of membrane repolarization. Furthermore, the spiking activity in Shab cultures was restricted to the damping firing pattern and was coupled with T3 current kinetics. Consistently, quinidine-treatments, which specifically remove Shab currents in WT neurons, closely mimic the 'repolarization decline' phenotype and converted spiking patterns into damping firing (Peng, 2007).

The mutational effects demonstrate that Shab I_K is more important for signal processing at time scales of hundreds of milliseconds. Shab mutant neurons are capable of generating normal action potentials in response to brief stimuli, but prolonged current injection causes abnormal high-frequency, run-away spikes that degenerate into dwindling oscillations. WT neurons expressing both Shab and non-Shab sustained currents fire full-blown action potentials ~25 Hz on average. In contrast, T3 Shab neurons (I_S/I_P >0.5) that retain substantial non-Shab currents, including Shaw, generate high-frequency firing (up to 60 Hz) either immediately on depolarization or with a gradual development. Interestingly, it has been shown that pharmacological blockade or mutations of Shaw-like Kv3 channels disable the high-frequency firing (often up to 1 kHz) found in certain neuronal types in the hippocampus, basal ganglia, and auditory nuclei. Because the exact dynamics of membrane repolarization can determine the timing of recovery of inward Na⁺ and Ca²⁺ currents from inactivation for resetting another cycle of spike activity, the balancing act between Shab and non-Shab I_K currents can enrich spike frequency control during repetitive firing (Peng, 2007).

An interesting parallel of the striking Shab phenotype during repetitive firing is observed at the larval neuromuscular junction (Ueda, 2006). With single nerve stimuli, mutant Shab synaptic transmission appears normal. During high-frequency nerve stimulation, an explosive neuromuscular transmission (up to 10-fold gain, termed the 'big bang' phenomenon) could suddenly occur when cumulative inactivation of I_A reaches a critical level. The phenomenon can also be induced in WT by repetitive stimulation following quinidine treatment and by single nerve stimuli in quinidine-treated Sh preparations (Ueda, 2006). The generation of a plateau membrane potential in the motor axon terminals, which are enriched with both Na⁺ and Ca²⁺ channels, has been proposed to account for this phenomenon when both I_A and I_K are weakened by mutations, drugs, or activity-dependent inactivation. These observations are reminiscent of the 'repolarization decline' in Shab neurons and quinidine-treated WT neurons during prolonged current injection. Taken together, an intricate interaction between slowly inactivating I_K and fast inactivating I_A is important during the dynamic process of repetitive firing for maintaining the cycles of membrane excitation and repolarization (Peng, 2007).

Notably, simple removal of Shab current by acute quinidine treatment on WT cultures converts firing patterns to damping rather than nonspiking activities. However, in Shab mutant cultures, a drastic increase of nonspiking neurons was observed, contrary to the expectation of increased excitability caused by reduced I_K. A clue to this unexpected finding is provided by the observation that in some nonspiking Shab cells, regenerative oscillations could still be initiated when the transient I_A was suppressed by 4-AP or a depolarizing prepulse. Voltage-clamp measurements yielded direct evidence for an increase in the inactivating I_A in Shab cultures. The peak total current (I_P), which represents the sum of peak I_A and I_K, remained undiminished despite the fact that the sustained current component (I_S) was significantly decreased in mutant Shab neurons. In contrast, pharmacological removal of Shab currents reduced both I_S (~35%) and I_P (~30%). These observations resemble the homeostatic regulation of ion channel previously reported in other preparations. When neuronal spike activity is manipulated, a homeostatic regulation can be initiated to adjust the relative abundance of ion channels and other proteins. Over-expression of Shal I_A in lobster neurons triggers a compensatory increase of hyperpolarization-activated inward I_h. Therefore a compensatory upregulation of the transient K⁺ current component in mutant Shab cultures could account for the lower percentage of spiking cells. Overexpression of transient I_A could prevent spike initiation in the standard current-clamp protocols employed in this study. Additional types of compensatory mechanisms, such as decreased expression of inward Na⁺ or Ca²⁺ currents, might also occur in Shab neurons. However, preliminary results indicate that Ca²⁺ current density in Shab mutant cultures remains unaltered compared with that in WT cells, although potential modification of Na⁺ currents require further investigation (Peng, 2007).

In contrast to Shab channels, Sh channels play a role in regulating rapid events within a millisecond time scale. Broadened action potentials were observed in mutant Sh neurons with delayed and tonic firing patterns. This demonstrates a role of action potential repolarization for Sh channels, consistent with greatly prolonged action potentials documented in the cervical giant fiber of Sh mutants (Peng, 2007).

A well-established function of transient K⁺ currents is pertinent to the control of spike initiation time during excitatory inputs. Mutational and pharmacological analyses confirm the important but overlapping roles of Sh and Shal I_A channels in controlling spike initiation. Neurons exhibiting delayed firing persisted in Sh cultures, supporting the idea that within only a small subset of neurons, I_A channels are exclusively or predominantly encoded by Sh. Furthermore, no disproportional reduction in any of the categories of firing patterns was observed in Sh cultures, indicating that Sh and Shal I_A may serve redundant roles in suppressing transient depolarization, and thus the action of Shal I_A alone could retain the dynamic manifestation of the delayed and other firing patterns (Peng, 2007).

Delayed or tonic firing in WT and Sh cultures could be converted into damping patterns after 4-AP treatment, suggesting that with total elimination of transient I_A, spike repolarization deteriorates during repetitive firing. Nevertheless, such damping firing patterns were distinct from those observed in Shab or quinidine-treated WT neurons, in that the high-frequency oscillations typical of Shab neurons were never observed. In summary, a clear rank of firing rate was observed in the damping patterns of the three genotypes: Shab > WT > Sh (Peng, 2007).

The slower decay of transient I_A in mutant Sh cultures is in agreement with previous reports on pupal and larval cultures, indicating that the remaining Shal channels inactivate more slowly than Sh channels. This is also consistent with the conclusion based on Sh and Shal channel expression experiments in the frog oocyte. The results also suggest that Shal channels follow a slower kinetics of recovery from inactivation, something that awaits confirmation in other preparations. However, steady-state inactivation measurements of I_A in Sh cultures suggest that Shal channels inactivate at voltages more positive than that for Sh channels, contrary to the preceding reports. The reason for the discrepancy is unknown, although expression patterns of the Sh and Shal splice variants as well as their potential association with auxiliary channel subunits could differ among the embryonic, larval and pupal cultures used in these studies. Splice variants of the Sh products are known to mediate K⁺ currents of varying degrees of inactivation when expressed heterologously in Xenopus oocytes. Similarly, subtypes of mammalian Kv1 channels also display different inactivation properties (Peng, 2007).

In some cases, 4-AP did not completely eliminate the transient component in T3 cells in WT and Sh cultures. Such 4-AP insensitive transient component may reflect low sensitivity to 4-AP in certain splicing isoforms of Sh or Shal subunits. Alternatively, inactivating isoforms of Shaw may exist, since a mammalian homologue of Shaw, Kv3.4, mediates inactivating K⁺ currents (Peng, 2007).

Although the tonic firing pattern is insensitive to 4-AP in WT neurons, 4-AP blockade of the remaining Shal currents in Sh neurons converts the tonic firing pattern to adaptive or damping in two out of three cases. Apparently, the currents contributing to the tonic firing pattern have been reconfigured in some Sh neurons relative to WT neurons. In addition, the population of nonspiking neurons is also increased in Sh cultures, contrary to the expectation that removal of Sh currents leads to hyperexcitability. Again, the possibility of downregulation of inward Na⁺ and Ca²⁺ currents in Sh neurons must be considered, similar to the case for Shab mutant neurons. Furthermore, upregulation of Shal or other transient currents in Sh mutant neurons is possible. However, over-expression of non-Sh transient currents would be rather limited because a significant reduction of I_P was still evident in Sh cultures. Taken together, these observations suggest diverse and cell-specific compensatory mechanisms still await further exploration in the heterogeneous population of the nervous system (Peng, 2007).

Both Sh and Shab mutations alter the abundance of the individual kinetic categories of total voltage-activated K⁺ currents among cultured neurons. This redistribution conceivably leads to a population conversion of neuronal types in mutant cultures. For example, part of the more populated T1-like neurons in Shab cultures reflects a conversion from T2 and T3 neurons on removal of Shab I_K as indicated by the unusually fast decay kinetics in some Shab T1 cells that resemble the decay time course of WT T2 and T3 neurons. Similarly, the overpopulated T3 cells in Sh cultures might be converted from T1 and T2 neurons, reflecting enhanced representation of Shal currents. The assumption of population conversion on removal of Sh and Shal channels is corroborated by the results of drug treatments in both voltage- and current-clamp experiments that reveal differential expression of K⁺ channels required for generation of different firing patterns. Notably, neurons with delayed firing patterns were converted by 4-AP to damping patterns but were not affected by quinidine, suggesting an abundance of transient I_A coupled with a deficiency of Shab I_K in this cell category. In contrast, quinidine converted tonic to damping firing patterns (cf. Zhao, 1997), indicating a strong dependence on Shab I_K during tonic firing. These observations suggest that the damping firing pattern requires actions of fewer K⁺ channel subtypes. In other words, an apparent order of complexity exists for K⁺ current interactions that underlie different firing patterns, suggesting an interesting scheme, in which differential expression or modulation of K⁺ channels (Jonas, 1996; Yao, 2001) may generate a diversity of neuronal firing patterns to fulfill the specific tasks and functional plasticity of neuronal circuits (Peng, 2007).

Several Drosophila Gal4 lines have been used to drive targeted expression of UAS-GFP to identify specific neuronal subsets in larval or pupal culture systems. Using this approach, it was demonstrated that in embryonic 'giant' neuron cultures, neurons of different cell lineages displayed characteristic excitability properties and K⁺ current kinetics. This study observed a substantial inactivating K⁺ current component in 201Y(+) cells in embryonic 'giant' neuron cultures, similar to that reported for cultured neurons dissociated from larval mushroom body (Peng, 2007).

It should be noted that channel distribution among different neuronal compartments in dissociated neuron cultures might not exactly match that of native neurons with their interacting partners in vivo and thus may modify the firing pattern of particular neuronal subpopulations. Moreover, in cell division-arrested embryonic neurons, the channel expression profile may reflect a combination of expression patterns of the neuronal descendants. It is known that the progeny of one insect neuroblast can develop different excitability properties (Peng, 2007).

Despite these caveats, electrophysiological and Ca²⁺ imaging studies on the same preparation have indicated a disparity between soma and neurites in K⁺ channel distribution similar to the pattern found in neurons of other in vivo preparations. In addition, the present physiological results of different K⁺ currents are directly comparable to the measurements from acutely dissociated CNS neurons of Drosophila larvae, pupae and adults in terms of kinetic categories, current compositions and mutational effects. Therefore the 'giant' neuron culture system can serve the purpose to link data of different levels that obtained from heterologous expression systems and in vivo preparations. In this system, results from electrophysiological analyses can be integrated with Ca²⁺ imaging to reveal functional specialization in different neuronal lineages. Combining the baseline information obtained in neuronal cultures with in vivo studies will help elucidate the cellular mechanisms that enable the neuronal circuits of interest to perform particular functional tasks in Drosophila (Peng, 2007).

Distinct frequency-dependent regulation of nerve terminal excitability and synaptic transmission by I_A and I_K potassium channels revealed by Drosophila Shaker and Shab mutations

Regulation of synaptic efficacy by nerve terminal excitability has not been extensively studied. Genetic and pharmacological dissections were performed for presynaptic actions of K⁺ channels in Drosophila neuromuscular transmission by using electrophysiological and optical imaging techniques. Current understanding of the roles of the Shab I_K channel and its mammalian Kv2 counterparts is relatively poor, as compared with that for Shaker I_A channels and their Kv1 homologues. The results revealed the striking effect of Shab mutations during high-frequency synaptic activity, as well as a functional division in synaptic regulation between the Shaker and Shab channels. Shaker channels control the basal level of release, indicated by a response to single nerve stimulation, whereas Shab channels regulate repetitive synaptic activities. These observations highlight the crucial control of nerve terminal excitability by Shaker and Shab channels to confer temporal patterns of synaptic transmission and suggest the potential participation of these channels, along with the transmitter release machinery, in activity-dependent synaptic plasticity (Ueda, 2006).

Modulation of synaptic efficacy during patterned activities of presynaptic neurons is important for motor control and for other neuronal network functions. In principle, synaptic efficacy can be modulated by regulating membrane excitability and transmitter release in the presynaptic terminals, although modulation of the transmitter release mechanisms has been more extensively studied. Potassium currents play crucial roles in regulating membrane excitability and could thus contribute to the control of synaptic efficacy. Both rapidly inactivating A-type (I_A) and delayed rectifier (I_K) K⁺ currents have been documented in presynaptic terminals of different species. In some preparations, including the vertebrate calyx of Held, and the squid giant synapse, delayed rectifier is the predominant K⁺ current, whereas in the hippocampal mossy fiber and pituitary hypophysis nerve terminals, I_A is prominent. However, the biological regulation and functional significance of such differential K⁺-channel expression in the presynaptic terminal still await elucidation (Ueda, 2006 and references therein).

The Drosophila Shaker (Sh) gene and its vertebrate homologs (Kv1 members) are the best studied among the diverse K⁺-channel subunit genes thus far identified. Drosophila Sh channels mediate inactivating I_A in both in vivo and heterologous expression preparations. Several vertebrate Kv1 channels also mediate inactivating I_A. Loss of Kv1.1 function in gene-knockout mice leads to epileptiform bursting discharge and seizure. Drosophila Sh mutants also display seizure-like leg shaking under ether anesthesia, abnormal spike bursting in motor circuits, and greatly enhanced neurotransmission at neuromuscular junctions (Ueda, 2006).

Although the Shab gene is homologous to Sh, the in vivo functions of Shab and its vertebrate homologs, the Kv2 members, are much less known. Heterologous expression of Shab produces sustained delayed rectifier K⁺ currents, consistent with the phenotypes of Shab mutants (i.e., reduced I_K in neurons) and muscle cells. Additionally, dominant-negative Kv2 expression reduces I_K in vertebrate cultured neurons. However, the role of Shab and Kv2 channels in the control of presynaptic terminal excitability and neurotransmission has not been established (Ueda, 2006).

This study reports strikingly different phenotypes of Shab and Sh mutations in neurotransmission, coupled with altered nerve terminal excitability and central pattern generation in Drosophila larvae. Sh I_A is responsible for regulating basal-release levels, whereas Shab I_K plays a more important role in transmission-level control during high-frequency activity. These results demonstrate a functional division between Sh I_A and Shab I_K in regulation of synaptic transmission within different temporal domains and frequency ranges (Ueda, 2006).

Both Sh and Shab subunits, as well as their vertebrate homologues Kv1 and Kv2 , are widely expressed in both the central and peripheral nervous systems. However, the role of Shab and Kv2 channels in synaptic function is less well understood than that of Sh and Kv1 channels. This study provides a first demonstration of the involvement of the Shab channel, and its interaction with the Sh channel, in the regulation of synaptic transmission. Severe deficiencies in motor-pattern generation indicate that these channels regulate membrane excitability and synaptic transmission in many central neurons, in addition to the neuromuscular junction. The results from mutational and pharmacological analyses illustrate the salient temporal characteristics of synaptic transmission that are regulated by Sh I_A and Shab I_K channels. Their complementary properties in voltage dependence and gating kinetics enable a functional division between these two channels. Sh I_A undergoes rapid inactivation with a slower recovery time course and could be progressively removed during rapid repetitive depolarizing pulses. In contrast, inactivation of Shab I_K is limited and slow, with more rapid recovery kinetics. The data indicate that Sh I_A undergoes cumulative inactivation during repetitive depolarization, leading to frequency-dependent facilitation of synaptic transmission. Under this condition, further removal of Shab I_K results in misregulation of nerve terminal excitability and uncontrolled, explosive transmission, such as the big-bang phenomenon observed at Shab neuromuscular junctions. In conclusion, Sh channels effectively control the basal level release triggered by single nerve stimuli, whereas Shab channels are more important in the regulation of repetitive synaptic activities. These distinctions highlight the crucial cooperation between Sh and Shab channels in nerve terminal excitability regulation to confer the patterns of synaptic transmission (Ueda, 2006).

Results from pharmacological experiments demonstrate an effective phenocopy of the Sh and Shab phenotypes by the I_A and I_K blockers 4-AP and quinidine, respectively. This suggests that the elevated basal transmission levels in Sh and the big-bang phenomena in Shab reflect direct effects of defective I_A and I_K channels, rather than the consequence of developmental regulation in response to a chronic deficiency in I_A or I_K (Ueda, 2006).

These long-lasting giant and excitatory junctional potentials (ejps) are consistent with the previous report of plateaued ejps in WT neuromuscular junctions treated with quinidine and dendrotoxin, a Kv1 and Sh channel blocker. With K⁺ channels compromised by mutations or drug treatments, the enhanced Na⁺ influx on the arrival of an orthodromic motor neuron spike may be sufficient to elicit a regenerative Ca²⁺ spike in the nerve terminal that is enriched in Ca²⁺ channels. The slower inactivation of Ca²⁺ channels can outlast the refractory period of Na⁺ spikes and can thus trigger another Na⁺ spike once the Na⁺ channels in the adjacent axonal patch recover from inactivation. These supernumerary Na⁺ spikes, thus, propagate antidromically but each contributes to the maintenance of the regenerative Ca²⁺ potentials for another round of interaction between Ca²⁺ and Na⁺ currents (cf. Ganetzky, 1982). With insufficient I_K in Shab mutants, cumulative inactivation of the Sh channel during repetitive stimulation may well decrease the repolarizing force sufficiently to trigger supernumerary spikes in the nerve terminal, leading to sustained transmitter release. Similar explosive events of prolonged neurotransmission have been described in the squid giant synapse and the frog neuromuscular junction after treatment with the K⁺ channel blocker tetraethylammonium (Ueda, 2006).

Both rapidly inactivating I_A (presumably mediated by Kv1.1/1.4) and noninactivating delayed rectifier I_K (presumably Kv1.2) have recently been detected in presynaptic terminals of vertebrate species and their functional significance requires further exploration. The proportion of these two types of K⁺ currents varies among different preparations and such variability may contribute to the distinct temporal characteristics of synaptic transmission typical of individual preparations. Synapses predominantly expressing I_A over I_K (e.g., hippocampal mossy fibers and pituitary neurohypophysis) are known to be modulated by repetitive input for cumulatively enhanced transmission. Such synapses may be more susceptible to high-frequency excitation and could produce explosive output under some diseased conditions. However, synapses predominantly expressing I_K over I_A (e.g., the calyx of Held in the auditory pathway) (presumably mediated by Kv1.2), could display a high following frequency, as required for high-frequency transmission. In contrast, synapses known to express both I_A and I_K (e.g., the lobster neuromuscular junction), can exhibit several patterns of transmission, as a substrate for both short-term and long-term synaptic plasticity (Ueda, 2006).

The major focus in the study of activity-dependent synaptic plasticity has been directed to the regulation of transmitter release machinery, including the studies using the Drosophila neuromuscular junction preparation. Less attention has been directed to the control of presynaptic terminal excitability in this process. In principle, modulation of inactivating I_A and sustained I_K may lead to important consequences on synaptic efficacy. In Drosophila, differential regulation of membrane repolarization and, thus, neuronal firing properties by Sh and Shab currents have been documented in cultured Drosophila central neurons. Sh current is important for the regularity of spike firing (Yao, 2001) and Shab is important for maintaining spike repolarization during repetitive firing. Further, both I_A and I_K channels are the known targets for cAMP- and cGMP-dependent modulation in Drosophila muscle fibers. In cultured neurons, Sh I_A is modulated by cAMP-and cGMP-dependent pathways and Shab I_K by CaM-dependent kinases. Therefore, modulation of K⁺ currents in nerve terminals may play an essential role in the activity-dependent plasticity of synaptic transmission. The Drosophila neuromuscular junction expresses both I_A and I_K with a number of known structural genes as well as auxiliary proteins that modify the channel properties. This model system thus provides a rich source for genetic interaction analysis and easy manipulation of genes and proteins important in the control of presynaptic terminal excitability and activity-dependent modification of synaptic efficacy (Ueda, 2006).

Dissection of synaptic excitability phenotypes by using a dominant-negative Shaker K+ channel subunit

During nervous system development, synapses undergo morphological change as a function of electrical activity. In Drosophila, enhanced activity results in the expansion of larval neuromuscular junctions. This study examined whether these structural changes involve the pre- or postsynaptic partner by selectively enhancing electrical excitability with a Shaker dominant-negative (SDN) potassium channel subunit. The SDN was found to enhance neurotransmitter release when expressed in motoneurons, postsynaptic potential broadening when expressed in muscles and neurons, and selectively suppresses fast-inactivating, Shaker-mediated IA currents in muscles. SDN expression also phenocopies the canonical behavioral phenotypes of the Sh mutation. At the neuromuscular junction, this study found that activity-dependent changes in arbor size occur only when SDN is expressed presynaptically. This finding indicates that elevated postsynaptic membrane excitability is by itself insufficient to enhance presynaptic arbor growth. Such changes must minimally involve increased neuronal excitability (Mosca, 2005).

Reduced sleep in Drosophila Shaker mutants

Most people sleep 7-8 h per night, and if deprived of sleep performance suffers greatly; however, a few do well with just 3-4 h of sleep-a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, mutagenesis was performed in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, minisleep (mns), a line that sleeps for one-third of the wild-type amount, was found. mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, short-sleeping Shaker flies were found to have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency (Cirelli, 2005).

Search PubMed for articles about Drosophila Shaker

Bergquist, S., Dickman, D. K. and Davis, G. W. (2010). A hierarchy of cell intrinsic and target-derived homeostatic signaling. Neuron 66(2): 220-234. PubMed ID: 20434999

Cirelli, C., Bushey, D., Hill, S., Huber, R., Kreber, R., Ganetzky, B. and Tononi, G. (2005). Reduced sleep in Drosophila Shaker mutants. Nature 434(7037): 1087-1092. PubMed ID: 15858564

Dean, T., Xu, R., Joiner, W., Sehgal, A. and Hoshi, T. (2011). Drosophila QVR/Sss modulates the activation and C-type inactivation kinetics of Shaker K(+) channels. J. Neurosci. 31(31): 11387-95. PubMed ID: 21813698

Donlea, J. M., Pimentel, D. and Miesenbock, G. (2014). Neuronal machinery of sleep homeostasis in Drosophila. Neuron 81(4): 860-872. PubMed ID: 24559676

Iverson, L. E., Tanouye, M. A., Lester, H. A., Davidson, N. and Rudy, B. (1988). A-type potassium channels expressed from Shaker locus cDNA. Proc Natl Acad Sci U S A 85(15): 5723-5727. PubMed ID: 2456579

Iyengar, A. and Wu, C. F. (2014). Flight and seizure motor patterns in Drosophila mutants: simultaneous acoustic and electrophysiological recordings of wing beats and flight muscle activity. J Neurogenet 28: 316-328. PubMed ID: 25159538

Koh, K., Joiner, W. J., Wu, M. N., Yue, Z., Smith, C. J. and Sehgal, A. (2008). Identification of Sleepless, a sleep-promoting factor. Science. 321(5887): 372-6. PubMed ID: 18635795

Lee, J., Ueda, A. and Wu, C. F. (2008). Pre- and post-synaptic mechanisms of synaptic strength homeostasis revealed by slowpoke and shaker K+ channel mutations in Drosophila. Neuroscience 154(4): 1283-1296. PubMed ID: 18539401

Liu, Q., Liu, S., Kodama, L., Driscoll, M. R. and Wu, M. N. (2012). Two dopaminergic neurons signal to the dorsal fan-shaped body to promote wakefulness in Drosophila. Curr Biol 22(22): 2114-2123. PubMed ID: 23022067

Mosca, T. J., Carrillo, R. A., White, B. H. and Keshishian, H. (2005). Dissection of synaptic excitability phenotypes by using a dominant-negative Shaker K+ channel subunit. Proc Natl Acad Sci U S A 102(9): 3477-3482. PubMed ID: 15728380

Parrish, J. Z., Kim, C. C., Tang, L., Bergquist, S., Wang, T., Derisi, J. L., Jan, L. Y., Jan, Y. N. and Davis, G. W. (2014). Kruppel mediates the selective rebalancing of ion channel expression. Neuron 82(3): 537-544. PubMed ID: 24811378

Peng, I. F. and Wu, C. F. (2007). Differential contributions of Shaker and Shab K+ currents to neuronal firing patterns in Drosophila. J. Neurophysiol. 97(1): 780-94. PubMed ID: 17079336

Pimentel, D., Donlea, J. M., Talbot, C. B., Song, S. M., Thurston, A. J. and Miesenbock, G. (2016). Operation of a homeostatic sleep switch. Nature 536: 333-337. PubMed ID: 27487216

Saur, T., Peng, I.F., Jiang, P., Gong, N., Yao, W.D., Xu, T.L. and Wu, C.F. (2016). K+ channel reorganization and homeostatic plasticity during postembryonic development: biophysical and genetic analyses in acutely dissociated Drosophila central neurons. J Neurogenet [Epub ahead of print]. PubMed ID: 27868467

Tao, X., Lee, A., Limapichat, W., Dougherty, D. A. and MacKinnon, R. (2010). A gating charge transfer center in voltage sensors. Science 328(5974): 67-73. PubMed ID: 20360102

Timpe, L. C., Schwarz, T. L., Tempel, B. L., Papazian, D. M., Jan, Y. N. and Jan, L. Y. (1988). Expression of functional potassium channels from Shaker cDNA in Xenopus oocytes. Nature 331(6152): 143-145. PubMed ID: 2448636

Ueda, A. and Wu, C.-F. (2006). Distinct frequency-dependent regulation of nerve terminal excitability and synaptic transmission by I_A and I_K potassium channels revealed by Drosophila Shaker and Shab mutations. J. Neurosci. 26(23): 6238-6248. PubMed ID: 16763031

Ueda, A. and Wu, C. F. (2015). The role of cAMP in synaptic homeostasis in response to environmental temperature challenges and hyperexcitability mutations. Front Cell Neurosci 9: 10. PubMed ID: 25698925

Ueno, T., Tomita, J., Tanimoto, H., Endo, K., Ito, K., Kume, S. and Kume, K. (2012). Identification of a dopamine pathway that regulates sleep and arousal in Drosophila. Nat Neurosci 15(11): 1516-1523. PubMed ID: 23064381

Wang, J. W., Humphreys, J. M., Phillips, J. P., Hilliker, A. J. and Wu, C. F. (2000). A novel leg-shaking Drosophila mutant defective in a voltage-gated K(+)current and hypersensitive to reactive oxygen species. J Neurosci 20(16): 5958-5964. PubMed ID: 10934243

Wang, J. W. and Wu, C. F. (2010). Modulation of the frequency response of Shaker potassium channels by the quiver peptide suggesting a novel extracellular interaction mechanism. J. Neurogenet. 24(2): 67-74. PubMed ID: 20429677

Wang, T., Martin, S., Nguyen, T. H., Harper, C. B., Gormal, R. S., Martinez-Marmol, R., Karunanithi, S., Coulson, E. J., Glass, N. R., Cooper-White, J. J., van Swinderen, B. and Meunier, F. A. (2016). Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB. Nat Commun 7: 12976. PubMed ID: 27687129

Wolfram, V., Southall, T. D., Gunay, C., Prinz, A. A., Brand, A. H. and Baines, R. A. (2014). The transcription factors islet and lim3 combinatorially regulate ion channel gene expression. J Neurosci 34: 2538-2543. PubMed ID: 24523544

Wu, M. N., et al. (2010). SLEEPLESS, a Ly-6/neurotoxin family member, regulates the levels, localization and activity of Shaker. Nat. Neurosci. 13(1):69-75. PubMed ID: 20010822

Wu, M., Robinson, J. E. and Joiner, W. J. (2014). SLEEPLESS is a bifunctional regulator of excitability and cholinergic synaptic transmission. Curr Biol 24(6): 621-629. PubMed ID: 24613312

Wu, M., Liu, C. Z. and Joiner, W. J. (2016). Structural analysis and deletion mutagenesis define regions of Quiver/Sleepless that are responsible for interactions with Shaker-type potassium channels and nicotinic scetylcholine receptors. PLoS One 11(2): e0148215. PubMed ID: 26828958

Zandany, N., Marciano, S., Magidovich, E., Frimerman, T., Yehezkel, R., Shem-Ad, T., Lewin, L., Abdu, U., Orr, I. and Yifrach, O. (2015). Alternative splicing modulates Kv channel clustering through a molecular ball and chain mechanism. Nat Commun 6: 6488. PubMed ID: 25813388

Biological Overview

date revised: 1 January 2024

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.