InteractiveFly: GeneBrief

Shaker cognate b: Biological Overview | References

BIOLOGICAL OVERVIEW

The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K⁺ (Kv) channels (see a review of voltage gated K⁺ by Yi and Jan, 2000). In whole-cell recordings of isolated photoreceptors, this study shows that light selectively modulates the delayed rectifier (Shab) current. Shab currents are increased by light with similar kinetics to the light-induced current itself (latency ~20 ms), recovering to control values with a t_1/2 of ~60 s in darkness. Genetic disruption of PLCβ4, responsible for light-induced PIP₂ hydrolysis, abolishes this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds¹), required for PIP₂ resynthesis, the modulation becomes irreversible, but exogenously applied PIP₂ restores reversibility. The modulation is accurately and reversibly mimicked by application of PIP₂ to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP₂ in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade (Krause, 2008).

In Drosophila photoreceptors, light-dependent activation of TRP and TRPL channels produces an inward current, carried mainly by calcium and sodium ions, creating a light-dependent depolarization. This voltage-change is opposed by opening of at least four types of voltage-activated K⁺ channels (Kv channels): a rapidly inactivating A current mediated by Shaker channels, a slow delayed rectifier (Shab), and two others responsible for a fast delayed rectifier and a slow, noninactivating current. Shaker and Shab are the prototypical members of the Kv1 and Kv2 voltage-activated K⁺ channel subfamilies, respectively. The functional significance of these K⁺ channels in the photoreceptors has turned out to be rather complicated. Currents mediated by Shaker and Shab have been proposed to play various roles in adaptation, regulation of gain and frequency response, and in optimizing the response voltage range (Krause, 2008 and referneces therein).

Many K⁺ channels, including those in Drosophila neurons, have been reported to be targets of modulation by intracellular signaling molecules. The transduction cascade, with its various enzymatic and nonenzymatic components, could be a potential modulator of the K⁺ channels. This has not been investigated systematically in Drosophila photoreceptors, although one report suggested that the delayed rectifier was modulated by CaMKII, while the fast Shaker current can be modulated by serotonin. Phototransduction in Drosophila photoreceptors is mediated by a canonical phospholipase C (PLC) signaling cascade located in the microvillar part of the photoreceptors (rhabdomeres), where temporal fluctuations in the number of absorbed photons are transformed into modulation of membrane voltage. In the cascade, light triggers (via rhodopsin isomerization and G proteins) the activation of the phospholipase C isoform β4 (PLCβ4, encoded by the norpA gene). This catalyzes hydrolysis of the membrane phospholipid phosphatidylinositol-4,5-biphosphate (PIP₂) into soluble inositol-1,4,5,-triphosphate (InsP₃) and membrane-bound diacylglycerol (DAG). Of these, DAG or one of its metabolites leads eventually to opening of the Ca²⁺- and Na⁺-permeable TRP and TRPL channels (encoded by the trp and trpl genes), while most available evidence suggests that InsP₃ plays no role in the photoreceptor's light response. The continuous functionality of the photoreceptors is maintained by regeneration of PIP₂ in a cyclic, enzymatic pathway (the PI cycle). This means that illumination of the photoreceptor activates turnover of its membrane lipids and dynamically modulates their composition (Krause, 2008).

The present work shows that the delayed rectifier K⁺ channel (encoded by shab) is selectively, rapidly, and reversibly upregulated by light stimulation. This analysis indicates that this is due to the depletion of PIP₂ by PLC. Because this modulation is coupled to the activation of the phototransduction cascade, it has the potential to form an automated light adaptation mechanism (Krause, 2008).

This study has discovered a regulatory mechanism of a delayed rectifier type Kv channel in Drosophila photoreceptors and has used genetic and electrophysiological approaches to reveal its molecular mechanism. Upon illumination, the delayed rectifier current mediated by Shab channels (Vähäsöyrinki, 2006) is specifically increased. Genetic and pharmacological dissection of the underlying pathway indicated that the light-dependent modulation (LDM) of Shab current is mediated by PLC and the resulting reduction in PIP₂. Finally, direct application of PIP₂ to inside-out patches containing heterologously expressed Shab channels precisely mimics the modulation, suggesting that PIP₂ may interact directly with the channels. The LDM can be argued to be an economical adaptation mechanism, because it is tightly linked to the phototransduction cascade that uses the same cycle (Krause, 2008).

Disruption of PLCβ4 function completely blocks LDM, but neither Ca²⁺ influx nor Ca²⁺ release is required for LDM induction, while a null mutation in the InsP₃ receptor has no effect. Although this does not completely exclude the possibility of other InsP₃-mediated processes, at this stage it could be concluded that the substrate (PIP₂) or one of the lipid products of PLCβ4 activity is the relevant mediator (Krause, 2008).

Strikingly, LDM of Shab becomes irreversible in mutants of the cds gene encoding CDP-DAG synthase, a vital enzyme of the PI cycle required for PIP₂ synthesis. This suggests that LDM is initiated by a reduction in PIP₂, since PIP₂ has been shown to be irreversibly depleted by light in cds¹ mutants. This is strongly supported by finding that exogenous di-C₈ PIP₂ in the intracellular medium can restore the recovery from LDM in cds¹ mutants. The proposal that LDM is initiated by PIP₂ depletion is also fully consistent with its intensity dependence, which closely matches the intensity dependence of PIP₂ depletion, while the time course of recovery of LDM also closely matches the time course of PIP₂ resynthesis (Hardie, 2004) monitored using PIP₂ biosensors (Krause, 2008).

Strikingly, it was found that the LDM could be accurately recapitulated by applying exogenous PIP₂ to the cytosolic surface of excised patches containing recombinant Shab channels expressed in Drosophila S2 cells. While the possibility of additional factors in excised patches cannot be excluded, the robust, rapid, and reversible effects using ATP-free solutions suggest that the effects of PIP₂ may be mediated by direct interactions with the Shab channels themselves. The effective PIP₂ dose-response function shows significant inhibition only with quite high concentrations -- i.e., Shab would appear to have rather low affinity for PIP₂ -- but this is exactly what is required to generate a sensitive and rapid response to PIP₂ depletion (Krause, 2008).

In summary, five independent lines of positive evidence (mutant analysis, intensity dependence, recovery time course, application of exogenous di-C₈ PIP₂ to photoreceptors, modulation of the activation curve of Shab channels in patches), along with evidence against alternative possibilities, lead to the conclusion that LDM of Shab is mediated by a reduction of membrane PIP₂, quite possibly by direct interaction with the channel itself (Krause, 2008).

This study shows that the light-dependent increase of the Shab current is caused by selective increase in amplitude of its slowly inactivating component. This, in turn, originates from a change of voltage dependence of its open probability rather than a change of the open channel conductance, and manifests in a leftward shift of the voltage dependence of activation (Krause, 2008).

It has been shown previously that currents mediated by Shab and its mammalian homolog, Kv2.1, decay with two time constants (Hardie, 1991; Immke, 1999). Immke attributed these two different kinetic components to two separately functioning conformations. The current results now suggest that one of these components can be specifically and independently regulated. Thus, in Drosophila photoreceptors, shab codes for one channel protein, which presumably depending on its pore occupancy, operates in two different kinetic modes (Krause, 2008).

The increase in Shab conductance observed upon PIP₂ depletion is in direct contrast to the only previous reports of modulation of Kv channels by PIP₂, namely members of the rapidly inactivating mammalian Kv1 and Kv3 families, which were reported to increase their open probability upon application of PIP₂ (Oliver, 2004). The mechanism proposed by Oliver was a specific interaction of the negatively charged head group of PIP₂ with the positively charged N terminus, the so-called ball-and-chain motif. Since the slow inactivating Shab channel lacks this motif, it is not surprising that no such positive modulation by PIP₂ was observed. The upregulation of Shab observed upon PIP₂ depletion indicates that members of the Kv2 channel subfamily may instead be inhibited by PIP₂ (Krause, 2008).

Interestingly, the LDM is eliminated in shab¹ mutants, although these mutants still generate a considerable Shab current. The R435Q point mutation in shab¹ is at the border of the NH₂ terminus and the first membrane segment, suggesting that PIP₂ evokes LDM via the NH₂ terminus. This region of the N terminus is generally believed to be involved in heteromultimerization within subfamilies in 6TM K⁺ channels (Choe, 2002), which might suggest that subunit interactions are involved in LDM. In addition, the point mutation of the shab¹ mutant is located within a region rich in basic residues. Similar arginine- and lysine-rich motifs are known in several inward rectifier channels to mediate interaction with PIP₂, and neutralization of just one basic residue can disrupt PIP₂ binding. It will be interesting to see whether PIP₂ interacts directly with this region in Shab and, if so, whether neutralization of Arg⁴³⁵ disrupts the interaction. This region is highly conserved with mammalian Kv2.1 channels; it will also be interesting to see whether this proposed mode of Kv channel modulation by PIP₂ is more general (Krause, 2008).

The onset of LDM is remarkably fast, taking place within a few tens of milliseconds after the light stimulus, and is at least as rapid as the phototransduction current itself when recorded under Ca²⁺-free conditions. This fast onset would be consistent with a direct effect of PIP₂ on the channel and in addition argues for a close spatial proximity to PLCβ4, which itself is localized to the microvilli (Schneuwly, 1991). Given the relatively slow diffusion coefficient of PIP₂ (Golebiewska, 2008) and the fact that phototransduction in flies is already generally recognized as the fastest known G protein-coupled signaling cascade, this speed of response would be difficult to understand unless the Shab channels were localized to the microvillar rhabdomere where the phototransduction machinery is located (for review see Hardie, 2001). Such a localization would also explain an earlier puzzling finding, namely that, in marked contrast to Shaker channels, Shab channels were almost never found in excised patches from the exposed, basal membrane of the photoreceptor (Hardie, 1991). Location of the Shab channels in the rhabdomeral microvilli or base rather than in the basal plasma membrane (where Shaker channels are densely expressed) would also support the idea that LDM plays a relevant role in vivo (Krause, 2008).

The modulation of Shab current (LDM) is tightly linked to the phototransduction cascade that uses the same cycle. Thus, whenever the phototransduction cascade is activated by light, Shab channels are upregulated without requirement of additional signal transduction machinery. At the same time, it automatically confers a dependence on light intensity, thus matching the upregulation of the delayed rectifier conductance to the level of activation of phototransduction cascade (Krause, 2008).

In intracellular recordings of the intact retina, sustained stimuli of ~30,000 effective photons per second are sufficient to depolarize the photoreceptor membrane to a maintained plateau potential of about 30 mV above resting potential. In the experiments reported in this study, similar light intensities resulted in an ~3-fold increase in the Shab conductance at these potentials. This may, however, overestimate the degree of modulation in vivo, since it is likely that the isolated photoreceptors in whole-cell patch-clamp are more sensitive to PIP₂ depletion than cells in the intact retina. With this caveat, it can be expected that under daylight conditions in vivo, the available Shab conductance that can be activated by voltage excursions from the plateau potential should be considerably larger than in the dark and would be expected to decrease in a graded fashion as the ambient intensity decreases. Therefore, adaptive mechanisms attributed to delayed rectifier in photoreceptors should be increased during progressive light adaptation. These include, for example, the speeding up of responses due to the widened bandwidth of the filter properties of the membrane and the less depolarized membrane voltage upon illumination. In this context, the LDM can be viewed as a feed-forward mechanism promoting adaptation (i.e., reduction of depolarization caused by light), because it tends to increase the activation of Shab channels that are already being activated by depolarization (Krause, 2008).

In conclusion This study shows that in Drosophila photoreceptors the slow inactivating component of Shab channel, responsible for the main delayed rectifier current, is increased selectively and independently of other Kv channels upon illumination. The modulation is dependent on light intensity, and it can be evoked even by relatively dim light. The regulation of Shab is initiated by the same PLCβ4 responsible for phototransduction itself. The evidence suggests that a reduction in the membrane lipid PIP₂ is the key regulating factor and is consistent with a direct action of PIP₂ on Shab channels. It is further proposed that light-dependent Shab regulation enhances mid- to long-term light adaptation in fly vision (Krause, 2008).

Operation of a homeostatic sleep switch

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. 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. This study demonstrates state switching by dFB neurons, identifies dopamine as a neuromodulator that operates the switch, and delineates the switching mechanism. Arousing dopamine causes transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects are transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involves 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 was 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 decreases or increases 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) 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. 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 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, 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 replaced 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 Gi/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 Gi/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 ms pulses at 10 Hz, or 20 Hz optogenetic stimulation, both sustained for 2-10 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.), 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 (mean ± s.e.m. = 25.86 ± 7.61 min). 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).

dFB neurons in the ON state expressed two types of potassium current: voltage-dependent A-type (rapidly inactivating) and voltage-independent non-A-type currents. The current-voltage (I-V) relation of iA resembled that of Shaker, the prototypical A-type channel: 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 (mean ± s.e.m.). 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. iA diminished by one-third, whereas inon-A nearly quadrupled when quantified between resting potential and spike threshold. The weak rectification of inon-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 iA 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 (KV) 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 KV 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, causing insomnia, 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 KV 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, its β subunit Hyperkinetic, or its regulator sleepless (Pimentel, 2016).

Leak conductances are typically formed by two-pore-domain potassium (K2P) channels. dFB-restricted RNAi of one member of the 11-strong family of Drosophila K2P channels, encoded by the CG8713 gene, increased sleep relative to parental controls; interference with the remaining 10 K2P channels had no effect. Recordings from dFB neurons after knockdown of the CG8713 gene product, which this study 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, inon-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. This 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; reduction of iA; 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).

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 (Coetzee, 1999; Jan, 1997). 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 (Hille 2001). 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 (Singh, 1999). 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) (cf. Hodge, 2005). 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 (Hille 2001). Mutational and pharmacological analyses confirm the important but overlapping roles of Sh and Shal I_A channels in controlling spike initiation (cf. Choi, 2004; Zhao, 1997). 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).

Genetic modifications of seizure susceptibility and expression by altered excitability in Drosophila Na₊ and K₊ channel mutants

A seizure-paralysis repertoire characteristic of Drosophila 'bang-sensitive' mutants can be evoked electroconvulsively in tethered flies, in which behavioral episodes are associated with synchronized spike discharges in different body parts. Flight muscle DLMs (dorsal longitudinal muscles) display a stereotypic sequence of initial and delayed bouts of discharges (ID and DD), interposed with giant fiber (GF) pathway failure and followed by a refractory period. This study examined how seizure susceptibility and discharge patterns are modified in various K⁺ and Na⁺ channel mutants. Decreased numbers of Na⁺ channels in nap^ts flies (Drosophila mutant with a temperature-sensitive block in nerve conduction) drastically reduced susceptibility to seizure induction, eliminated ID, and depressed DD spike generation. Mutations of different K⁺ channels led to differential modifications of the various components in the repertoire. Altered transient K⁺ currents in Sh¹³³ and Hyperkinetic (Hk) mutants promoted ID induction. However, only Sh¹³³ but not Hk mutations increased DD seizure and GF pathway failure durations. Surprisingly, modifications in sustained K⁺ currents in eag and Shab mutants increased thresholds for DD induction and GF pathway failure. Nevertheless, both eag and Shab, like Sh¹³³, increased DD spike generation and recovery time from GF pathway failure. Interactions between channel mutations with the bang-sensitive mutation Shaker bss demonstrated the role of membrane excitability in stress-induced seizure-paralysis behavior. Seizure induction and discharges were suppressed by nap^ts in bss nap double mutants, whereas Sh heightened seizure susceptibility in bss Sh¹³³ and bss Sh^M double mutants. The results suggest that individual seizure repertoire components reflect different neural network activities that could be differentially altered by mutations of specific ion channel subunits (Lee, 2006: Full text of article).

Sub-cellular Ca²⁺ dynamics affected by voltage- and Ca2+-gated K⁺ channels: Regulation of the soma-growth cone disparity and the quiescent state in Drosophila neurons

Using Drosophila mutants and pharmacological blockers, evidence is provided that distinct types of K⁺ channels differentially influence sub-cellular Ca²⁺ regulation and growth cone morphology during neuronal development. Fura-2-based imaging revealed in cultured embryonic neurons that the loss of either voltage-gated, inactivating Shaker channels or Ca²⁺-gated Slowpoke BK channels led to robust spontaneous Ca²⁺ transients that preferentially occurred within the growth cone. In contrast, loss of voltage-gated, non-inactivating Shab channels did not show such a disparity and sometimes produced soma-specific Ca²⁺ transients. The fast spontaneous transients in both the soma and growth cone were suppressed by the Na⁺ channel blocker tetrodotoxin, indicating that these Ca²⁺ fluctuations stemmed from increases in membrane excitability. Similar differences in regional Ca²⁺ regulation were observed upon membrane depolarization by high K⁺-containing saline. In particular, Shaker and slowpoke mutations enhanced the size and dynamics of the depolarization-induced Ca²⁺ increase in the growth cone. In contrast, Shab mutations greatly prolonged the Ca²⁺ increase in the soma. Differential effects of these excitability mutations on neuronal development were indicated by their distinct alterations in growth cone morphology. Loss of Shaker currents increased the size of lamellipodia and the number of filopodia, structures associated with the actin cytoskeleton. Interestingly, loss of Slowpoke currents strongly influenced tubulin regulation, enhancing the number of microtubule loop structures per growth cone. Together, these findings support the idea that individual K⁺ channel subunits differentially regulate spontaneous sub-cellular Ca²⁺ fluctuations in growing neurons that may influence activity-dependent growth cone formation (Berke, 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

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 (Coetzee, 1999). 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 (Butler, 1989), 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 (Wei, 1990), consistent with the phenotypes of Shab mutants (i.e., reduced I_K in neurons) and muscle cells (Singh, 1999; Chopra, 2000). Additionally, dominant-negative Kv2 expression reduces I_K in vertebrate cultured neurons (Blaine, 2001; Malin, 2002). 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 (Schwarz, 1990; Tejedor, 1997), as well as their vertebrate homologues Kv1 and Kv2 (Trimmer, 1991; Sheng, 1992; Coetzee, 1999), 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 (Baukrowitz, 1995). In contrast, inactivation of Shab I_K is limited and slow, with more rapid recovery kinetics (Wu, 1985; Tsunoda, 1995; Singh, 1999). 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 (Wu, 1989). 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 (Dodson, 2004). 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) (Dodson, 2003), 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) (French, 2004), 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 (Zhong, 1993). In cultured neurons, Sh I_A is modulated by cAMP-and cGMP-dependent pathways (Renger, 1999; Yao, 2001) and Shab I_K by CaM-dependent kinases (Yao, 2001). 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).

Robustness of neural coding in Drosophila photoreceptors in the absence of slow delayed rectifier K⁺ channels

Determining the contribution of a single type of ion channel to information processing within a neuron requires not only knowledge of the properties of the channel but also understanding of its function within a complex system. This paper studied the contribution of slow delayed rectifier K⁺ channels to neural coding in Drosophila photoreceptors by combining genetic and electrophysiological approaches with biophysical modeling. The Shab gene encodes the slow delayed rectifier K⁺ channel, and a novel voltage-gated K+ conductance has been identified. Analysis of the in vivo recorded voltage responses together with their computer-simulated counterparts demonstrates that Shab channels in Drosophila photoreceptors attenuate the light-induced depolarization and prevent response saturation in bright light. Reduction of the Shab conductance in mutant photoreceptors is accompanied by a proportional drop in their input resistance. This reduction in input resistance partially restores the signaling range, sensitivity, and dynamic coding of light intensities of mutant Shab photoreceptors to those of the wild-type counterparts. However, loss of the Shab channels may affect both the energy efficiency of coding and the processing of natural stimuli. These results highlight the role of different types of voltage-gated K+ channels in the performance of the photoreceptors and provide insight into functional robustness against the perturbation of specific ion channel composition (Vähäsöyrinki, 2006).

This study has shown that the Shab gene encodes the slow delayed rectifier voltage-gated K⁺ channels in Drosophila photoreceptors. The functional role of this channel was investigated by using a combination of in vivo and in vitro recordings and model simulations. Three alleles of the Shab gene (Hegde, 1999) (Shab¹, Shab², and Shab³) were used to eliminate ~75, 71, and 100% of the Shab conductance, respectively. Using Hodgkin-Huxley-type modeling, the effect of these alleles on the photo-insensitive membrane of Drosophila photoreceptors was predicted: a dramatic increase in steady-state depolarization and gain at a particular light background but no change in input resistance in the dark. However, all of these alleles produced an unexpected result: they showed little or no change in steady-state depolarization or gain in the light. This appears to be attributable to decreased input resistance, coupled to the decrease in Shab conductance, which restores their light–voltage relationships and sensitivity to resemble those of the WT photoreceptors over the physiological voltage range (Vähäsöyrinki, 2006).

The decreased input resistance associated with deletion of the Shab channels is similar to that found in Shaker mutant photoreceptors (Niven, 2003a), although it is unclear whether the underlying mechanisms are the same. Despite similarities, however, these two types of photoreceptor show robustness in different aspects of their function. In Shaker mutant photoreceptors, the decrease in input resistance partially restores the efficient use of the operating voltage range (Niven, 2003a). Shab mutant photoreceptors, conversely, show remarkable robustness in their light-voltage relationships, sensitivities (light contrast gains), and reliability of dynamic coding (information capacities). The decreased input resistance in the Shab mutant photoreceptors significantly improved these aspects of their performance, but clear disadvantages may also follow from the deletion of the voltage-gated channels (Vähäsöyrinki, 2006).

Drosophila photoreceptors, like those of other insects, generate a maintained depolarization in response to increasing light intensity. Because of their voltage dependency, Shab channels open at high light intensities, thereby attenuating the light-induced depolarization and preventing response saturation. In addition, the activation of Shab channels at high light intensities decreases the membrane time constant, allowing photoreceptors to encode faster events (Weckström, 1995). Although these general effects of the K⁺ channels were reproduced by the increased nonspecific background conductance, a significant decrease in the coding efficiency of the photoreceptors is to be expected. Modeling has provided evidence that the maintenance of the resting potential when photoreceptors are in the dark is energetically expensive. Few Shab channels will be active in the dark, contributing relatively little to the input resistance. In contrast to the Shab channels, which are activated only in response to relatively large depolarizing events, the decreased input resistance in Shab mutants imposes a constant, high-energy cost to the photoreceptors. Therefore, Shab mutant photoreceptors are likely to consume significantly more energy in the dark or at low light levels than the WT photoreceptors (Vähäsöyrinki, 2006).

In addition to reducing energetic expenditure, the kinetics of Shab channels also contribute to the nonlinear processing of natural stimuli, which contain strong temporal fluctuations, large changes in intensity, and a higher proportion of low-frequency signals. It has been shown previously that the effects of dynamic nonlinearities generated by voltage-gated Shaker channels are important for the performance of Drosophila photoreceptors in coding naturalistic stimuli (Niven, 2004). Such nonlinear effects of voltage-gated K⁺ channels cannot be reproduced by changes in input resistance, which is close to a linear effect, or by an increase in the magnitude of other voltage-gated conductances, whose kinetic properties are different. It is suggested that Shab channels are essential for tuning the membrane properties of the photoreceptors to their input statistics and dynamics. Despite a reduction in the amount of information at low frequencies, the information capacities of Shab³ photoreceptors exposed to white-noise stimuli were close to those of WT Drosophila photoreceptors because of an increase in the information at high frequencies. However, when processing natural images with a high proportion of low-frequency signal content, Shab mutant photoreceptors may have significantly lower information capacities than their WT counterparts (Vähäsöyrinki, 2006).

Additional insights into the robustness of neural processing in Drosophila photoreceptors may come from the identification of the genes encoding other voltage-gated K⁺ channels. The results suggest that at least two more voltage-gated K⁺ channels remain to be identified genetically: the novel non-inactivating channel and the fast delayed rectifier channel, which has been characterized previously. These conductances are not encoded by either the Shab or Shaker genes and are likely to be formed from separate gene products, not heteromultimers. The Shal gene would seem the most likely candidate for the fast-inactivating voltage-gated K⁺ channels because its properties are similar to those reported for Shal channels identified in other tissues and when heterogously expressed. The identity of the novel non-inactivating channel is less clear. In oocyte expression studies, both Shaw, the remaining member of the Shaker-like gene family in Drosophila, and eag (ether-a-go-go) are reported to encode non-inactivating K⁺ conductances, but both have rapid activation kinetics inconsistent with the slow activation observed for the residual current in Shaker¹⁴;Shab³ double mutants. However, mutant and/or overexpression studies of these candidates in the photoreceptors are needed before ruling them out with certainty (Vähäsöyrinki, 2006).

What could be the possible neuronal mechanism behind the decrease in the input resistance? This decrease was reproduced in the models by increasing the leak conductances. In Hodgkin-Huxley-type models, the leak conductances are routinely used to cover all of the undefined processes required to fit the models to the experimental data. Thus, leak conductances can be considered as a descriptive representation of possibly several neuronal mechanisms. A recent study has demonstrated that electrical properties may be restored by the compensatory increase in K⁺ leak conductance in cerebellar granule cells in which a tonic synaptic conductance was eliminated. Whether the leak conductances have cellular counterparts or whether they describe overall effect of more complicated processes remains to be determined in future work (Vähäsöyrinki, 2006).

The results show that regulation of the electrical properties maintains the light-;voltage relationship in Drosophila photoreceptors after imposed changes in their ion channel composition. This relates closely to recent studies in which robustness of electrical properties is shown to optimize information processing by maintaining the dynamic range of signaling within useful limits. However, it is also clear that certain features of voltage-gated ion channels cannot be fully restored by concurrent changes in the passive membrane properties or by upregulation or downregulation of other ion channels (Niven, 2003a; Niven, 2003b; Niven, 2004). Understanding the mechanisms and limitations of robustness of neural coding could have important implications for the evolution of nervous systems, especially when addressing why specific ion channels are expressed in specific neurons (Vähäsöyrinki, 2006).

Search PubMed for articles about Drosophila Shab

Baukrowitz, T. and Yellen, G. (1995). Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron 15: 951-960. PubMed ID: 7576643

Berke, B. A., Lee, J., Peng, I. F. and Wu, C. F. (2006). Sub-cellular Ca²⁺ dynamics affected by voltage- and Ca2+-gated K⁺ channels: Regulation of the soma-growth cone disparity and the quiescent state in Drosophila neurons. Neuroscience 142(3): 629-44. PubMed ID: 16919393

Blaine, J. T. and Ribera, A. B. (2001). Kv2 channels form delayed-rectifier potassium channels in situ. J. Neurosci. 21: 1473-1480. PubMed ID: 11222637

Butler, A., Wei, A. G., Baker, K. and Salkoff, L. (1989). A family of putative potassium channel genes in Drosophila. Science 243: 943-947. PubMed ID: 2493160

Choe, S. (2002). Potassium channel structures. Nat. Rev. Neurosci. 3: 115-121. PubMed ID: 11836519

Choi, J. C., Park, D. and Griffith, L. C. (2003). Electrophysiological and morphological characterization of identified motor neurons in the Drosophila third instar larva central nervous system. J. Neurophysiol. 91(5): 2353-65. PubMed ID: 14695352

Chopra, M., Gu, G. G. and Singh, S. (2000). Mutations affecting the delayed rectifier potassium current in Drosophila. J. Neurogenet. 14: 107-123. PubMed ID: 10992164

Coetzee, W. A., et al. (1999). Molecular diversity of K+ channels. Ann. N. Y. Acad. Sci. 868: 233-285. PubMed ID: 10414301

Dodson, P. D., et al. (2003). Presynaptic rat Kv1.2 channels suppress synaptic terminal hyperexcitability following action potential invasion. J. Physiol. 550: 27-33. PubMed ID: 12777451

Dodson, P. D. and Forsythe, I. D. (2004). Presynaptic K+ channels: electrifying regulators of synaptic terminal excitability. Trends Neurosci. 27: 210-217. PubMed ID: 15046880

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date revised: 25 February 2009

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