InteractiveFly: GeneBrief

Hyperkinetic: Biological Overview | References

Voltage-gated potassium (Kv) channels selectively catalyze the transport of K⁺ across the plasma membrane. The channels are multisubunit complexes, being composed of membrane-integrated Kvα-subunits and of accessory subunits. Kvα-subunits form a tetrameric protein complex that assembles the core of a Kv channel consisting of a pore domain with activation (and inactivation) gate(s) and selectivity filter linked to peripheral voltage-sensor domains. In in vitro expression systems, most Kvα-subunits form functional Kv channels, reproducing basic Kv channel properties such as opening and closing (gating) of the pore in response to a change in the membrane electric field. The activation process is well described by a sequential gating model, in which voltage-sensor movements in each Kvα-subunit are usually followed by a concerted pore opening step to permit passage of K⁺. The details of conformational changes associated with the electromechanical coupling-mechanism between voltage sensor and activation gate are presently under intense investigation (Pongs, 2010 and references therein).

In vivo, Kv channels appear as heteromultimeric complexes coassembled with accessory subunits. Accessory subunits influence a wide range of Kv channel properties, which play important roles during Kv channel ontogeny and function, respectively. The importance of the role of accessory subunits is emphasized by the number of mutations that are associated in both humans and animals with diseases like hypertension, epilepsy, arrhythmogenesis, hypothyroidism, and periodic paralysis. Notably, two of the early described excitability mutants were described in Drosophila melanogaster, with a leg shaking phenotype resulting from mutation in a Kvα-subunit (Shaker) and from mutation in a Kvβ-subunit (hyperkinetic; Chouinard, 1995). What are the exact biological roles of the accessory subunits has been a matter of intense research. To determine cellular function(s), two major avenues were followed. The first one investigates modulatory influences of accessory subunits on various ion channel gating parameters including the aim to closely reproduce in heterologous expression systems electrophysiological and pharmacological Kv channel properties observed in vivo, e.g., in primary neurons in culture or in acute brain slices. The second avenue studies potential roles of accessory subunits in Kv channel assembly and exit from the endoplasmic reticulum (ER), Kv channel trafficking to and from the plasma membrane, Kv channel sorting, and regulation of Kv channel activity by posttranslational modifications. The results unveil common themes, although accessory subunits come in different flavors. One important theme is that the ancillary subunits link Kv channel activity both with extra- and intracellular signaling pathways and protein networks, respectively (Pongs, 2010 and references therein).

Accessory Kv channel subunits exhibit different protein structures reflecting their divergent biological roles. Some accessory subunits, e.g., β-subunits of both BK and Kv7 (KCNQ) channels, are integral membrane proteins with additional NH₂- and/or COOH-terminal sequences extending into the extra- and/or intracellular space. Other accessory subunits are cytosolic proteins and bind to cytoplasmic domains of Kv channel α-subunits, e.g., Kvβ-subunits of Shaker Kv channels and KChIPs of Kv4 channels. To date, no extracellular accessory β-subunits have been found binding to Kv channels from an extracellular site. The first accessory Kv channel subunit cloned and functionally characterized was a Kvβ-(Kvβ1.1) subunit. Studies on the biological function of this subunit class have been very influential for the present understanding of heteromultimeric Kv channel complexes, yet results on the cellular function of Kvβ-subunits are inconclusive and major questions still remain unresolved. An important evolving concept is that auxiliary subunits have, in addition to their influence on Kv channel gating parameters, important roles in Kv channel sorting and trafficking to distinct cellular localizations (Pongs, 2010 and references therein).

Since the discovery of the auxiliary Kvβ-subunits, impressive progress has been made. Many more accessory subunits were discovered, and their properties were studied. Stable association between Kvα and accessory subunits made it possible to obtain crystal structures, for example, the auxiliary Kvβ-subunit in association with the entire Kv1.2 channel, and the ancillary subunit KChIP2 in association with a cytoplasmic Kv4α tetramerization domain. The structural models have many important implications for understanding about mechanisms of interaction between Kvα-subunits and accessory subunits. For functional studies, mostly in vitro expression systems were used. They have been instrumental to analyze influences of accessory subunits on Kv channel gating. Observed influences are often quite remarkable, e.g., members of the Kvβ-subunit family confer rapid inactivation to delayed rectifier-type Kv channels, which otherwise do not inactivate; the ancillary BKβ-subunits of BK channels dramatically increase Ca²⁺ sensitivity of BK channel gating and also markedly affect their pharmacological properties. However, transient heterologous expression systems are potentially controversial and prone to artifacts. Not surprisingly, in vitro studies have frequently yielded conflicting results on possible cellular functions of accessory subunits. This seems particularly true for studies on specificity and stoichiometry of subunit assembly and its impact on Kv channel trafficking (Pongs, 2010 and references therein).

Despite the critical importance for understanding biological functions of Kvβ as well as other accessory subunits for human physiology, detailed structure-function studies on the role of Kvβ-subunits in genetically modified mice are relatively scarce. Furthermore, a clear-cut structure-function relationship between mutated accessory subunit gene and mutant phenotype is difficult to reach in correlating genotype and phenotype of mutant mice. Thus so far limited insights have been gained into disease mechanisms and the underlying pathophysiology of mutations in ancillary subunit genes. More studies of this kind will certainly be helpful to sharpen understanding of the biophysical and cellular basis of disease correlated with mutations in accessory Kv channel subunit genes (Pongs, 2010 and references therein).

Cryptochrome-mediated phototransduction by modulation of the potassium ion channel β-subunit redox sensor

Blue light activation of the photoreceptor Cryptochrome (Cry) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster. This study shows that acute arousal behavioral responses to blue light significantly differ in mutants lacking Cry, as well as mutants with disrupted opsin-based phototransduction. Light-activated Cry couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K+) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk-/- mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K+ channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go-related gene, are ion conducting channels for Cry/Hk-coupled light response. Light activation of Cry is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor (Fogle, 2015).

Cryptochrome (Cry) is a photoreceptor that mediates rapid membrane depolarization and increased spontaneous action potential firing rate in response to blue light in arousal and circadian neurons in Drosophila melanogaster. Cry regulates circadian entrainment by targeting circadian clock proteins to proteasomal degradation in response to light. Cry is expressed in a small subset of central brain circadian, arousal, and photoreceptor neurons in D. melanogaster and other insects, including the large lateral ventral neuron (LNv; l-LNv) subset. The l-LNvs are light-activated arousal neurons, whereas the small lateral ventral neurons (s-LNvs) are critical for circadian function. Previous results suggest that light activated arousal is likely attenuated in cry-null mutants. In addition to modulating light-activated firing rate, membrane excitability in the LNv neurons helps maintain circadian rhythms, and LNv firing rate is circadian regulated (Fogle, 2015).

Based on previous work suggesting that l-LNv electrophysiological light response requires a flavin-specific redox reaction and modulation of membrane K+ channels, the molecular mechanism for Cry phototransduction was examined to determine how light-activated Cry is coupled to rapid membrane electrical changes. Sequence and structural data suggest that the cytoplasmic Kvβs are redox sensors based on a highly conserved aldo-keto-reductase domain (AKR). Although no functional role for redox sensing by Kvβ subunits has been established yet in vivo, studies with heterologously expressed WT and mutant Kvβ subunits show that they confer modulatory sensitivity to coexpressed K+ channels in response to oxidizing and reducing chemical agents. Mammals express six Kvβ genes, whereas Drosophila expresses a single Kvβ designated Hyperkinetic. This study finds that the light-activated redox reaction of the flavin adenine dinucleotide (FAD) chromophore in Cry has a distinct phototransduction mechanism that evokes membrane electrical responses via the Kvβ subunit Hk, which is shown to be a functional redox sensor in vivo (Fogle, 2015).

Acute behavioral arousal to blue light is significantly attenuated in Cry mutants. This study identified a redox signaling couple between blue light-activated Cry and rapid membrane depolarization via the redox sensor of Kvβ channel subunits coassembled with Kvα channel subunits. Additional unknown factors may act as intermediates between Cry and Hk. This finding provides in vivo validation of a very longstanding hypothesis that the highly conserved redox sensor of Kvβ subunit functionally senses cellular redox events to physiological changes in membrane electrical potential. Genetic loss of any single component functionally disrupts the Cry-mediated blue light response, which is functionally rescued by LNv restricted expression of their WT genes in the null backgrounds. Although little is known about the structural contacts between Kvβ and EAG subunits, Kvβ subunits make extensive physical contacts in a fourfold symmetric fashion in 1:1 stoichiometry with the T1 assembly domain of other coassembled tetrameric Kvα subunits that form the complete functional channel. Application of Kvβ redox chemical substrate modulates voltage-evoked channel peak current, steady-state current, and inactivation in heterologously expressed α-β channels, which are reversed by fresh NADPH. These results indicate that measurements of channel biophysical properties can reflect the redox enzymatic cycle of Kvβ as these channel modulatory effects are absent in preparations that lack the expression of WT Kvβ subunits or express redox sensor mutant Kvβ subunits. Whether direct chemical redox reactions occur between Cry and Hk is unclear. For Cry, light or chemical reduction induces one-electron reduction of the FAD cofactor of Cry, whereas the reductive catalytic mechanism of AKRs (such as Hk) requires a hydride ion transferred from NADPH to a substrate carbonyl, then a solvent-donated proton reduces the substrate carbonyl to an alcohol. These differences in redox chemistry between Cry and Hk suggest that other intermediates, such as oxygen, are possibly required for redox coupling (Fogle, 2015).

Spectroscopic analysis of animal and plant Crys suggest that light activation causes reduction of the FAD oxidized base state. Light activation of Drosophila Cry also evokes conformational changes in the C terminus of Cry that clearly promotes Cry C-terminal access to proteolytic degradation and subsequent interactions with the Timeless clock protein, thus signaling degradation and circadian entrainment. However, all existing evidence suggests that light activated Cry-mediated circadian entrainment and membrane electrical phototransduction operate under different mechanisms, including their different activation thresholds and relative dependence on the C terminus of Cry. Further distinguishing the distinct mechanisms of the downstream effects of light-activated Cry, the light-induced conformational changes that couple Cry to ubiquitin ligase binding (thus causing circadian entrainment) occur in oxidized and reduced states of Cry and are unaffected in Cry tryptophan mutants that presumably are responsible for intraprotein electron transfer reactions following light-evoked reduction of the FAD cofactor. Another recent study shows that light- or chemical-evoked reduction of Drosophila Cry FAD is coupled to conformational changes of the Cry C terminus, along with reporting a surprising negative result that DPI has no effect on the reoxidation of the reduced anionic semiquinone of purified Drosophila Cry. DPI could hypothetically influence the electrophysiological light response by blocking the pentose phosphate pathway which produces the Hk redox cofactor NADPH, but this does not explain the light dependence for DPI blocking the electrophysiological light response herein. The available evidence indicates that Cry-mediated light evoked membrane depolarization occurs independently of conformational changes in the Cry C-terminal domain but depends on redox changes in Cry, whereas Cry-mediated light evoked circadian entrainment depends on conformational changes in the Cry C-terminal domain and may or may not depend on Cry redox state (Fogle, 2015).

Light-activated Cry evokes rapid membrane depolarization through the redox sensor of the Kvβ subunit Hk. A general role for circadian regulation of redox state coupled to membrane excitability has been described recently in mammalian suprachiasmatic neurons. Redox modulation of circadian neural excitability may be a well-conserved feature (Fogle, 2015).

Effects of hyperkinetic, a β subunit of Shaker voltage-dependent K⁺ channels, on the oxidation state of presynaptic nerve terminals

The Drosophila Hyperkinetic (Hk) gene encodes a beta subunit of Shaker (Sh) K+ channels and shows high sequence homology to aldoketoreductase. Hk mutations are known to modify the voltage dependence and kinetics of Sh currents, which are also influenced by the oxidative state of the N-terminus region of the Sh channel, as demonstrated in heterologous expression experiments in frog oocytes. However, an in vivo role of Hk in cellular reduction/oxidation (redox) has not been demonstrated. By using a fluorescent indicator of reactive oxygen species (ROS), dihydrorhodamine-123 (DHR), this study shows that the presynaptic nerve terminal of larval motor axons is metabolically active, with more rapid accumulation of ROS in comparison with muscle cells. In Hk terminals, DHR fluorescence was greatly enhanced, indicating increased ROS levels. This observation implicates a role of the Hk beta subunit in redox regulation in presynaptic terminals. This phenomenon was paralleled by the expected effects of the mutations affecting glutathione S-transferase S1 as well as applying H₂O₂ to wild-type synaptic terminals. Thus, the results also establish DHR as a useful tool for detecting ROS levels in the Drosophila neuromuscular junction (Ueda, 2008).

A functional role of the β subunit Kvβ and Hk in the regulation of Kv1 and Sh channels has been well established. Although the sequence homology to aldoketoreductase is a common feature among different Kvβ and Hk varieties, the functional significance of this well conserved sequence has not been fully explored. The current results establish DHR as a useful tool for probing the cellular oxidation status, enabling the demonstration of an in vivo role for the β subunit in the regulation of the cytosolic redox state in the presynaptic nerve terminals. Apparently, a general increase in neuronal excitability is not sufficient to alter ROS metabolism in presynaptic terminals. Examinations of a different hyperexcitable mutant, quiver, that also modulates the Sh I_A properties and causes ether-induced leg shaking, did not indicate altered rates of DHR fluorescence accumulation. Therefore, it will be important in future rescue experiment to compare the efficiency between WT and modified Hk constructs in the putative reductase domain to establish the molecular basis of Hk enzyme activity. Appropriate GAL4 and UAS lines may be constructive for this purpose (Ueda, 2008).

It is known that both oxidating and reducing agents exert clear effects on synaptic physiology. Oxidizing agents such as diamide and 5,5’-dithiobis(2-nitrobenzoic acid)(3,3’-6) suppress activity-dependent facilitation of synaptic transmission in the lobster neuromuscular junction and a form of long-term potentiation in rat hippocampus slices. In contrast, reducing agents can cause hyperexcitability in hippocampal neurons, as indicated by increased synaptic facilitation. In Drosophila Hk mutants, repetitive nerve stimulation leads to striking enhancement of activity-dependent facilitation of transmitter release, associated with supernumerary firing of presynaptic motor axons. Although this aberrant repetitive firing could be attributed to β subunit malfunction in modulation of the Sh I_A channels, additional mechanisms must be considered as well (Ueda, 2008).

First, direct interacting partners of Hk may not be limited to Sh I_A channels. Hk polypeptide is known to bind Eag, another voltage-dependent K⁺ channel α subunit. Coexpression of Eag with Hk subunit greatly enhances Eag-mediated K⁺ current in Xenopus oocytes (Wilson, 1998). Furthermore, additional K⁺ channels have been shown to be modulated by ROS, including a channel encoded by the human eag-related gene (herg), and Ca²⁺-activated BK channels encoded by slowpoke (slo). Since the Eag subunit has been shown to interact with several K⁺ channel subunits including Sh (Ganetzky, 1983), Hk may affect the various K⁺ currents through modulation of the Eag subunit (Ueda, 2008).

Certain polypeptides that modulate downstream targets in the signaling pathway are known to be sensitive to cellular redox states. These include protein tyrosine kinases and phophatases, phospholipases, and transcription factors. Redox regulation of these proteins may affect a variety of cellular processes, including synaptic plasticity, cell proliferation, and neuronal degeneration. Considering that β subunits are widely expressed in the CNS, the β subunit polypeptide may contribute to the modulation of behaviors through cytosolic redox regulation. Hk and Kvβ mutants display several characteristic behavioral changes. Drosophila Hk mutants show ether-induced leg shaking and hypersensitivity to visual stimuli in an escape reflex. Kvβ knockout mice exhibit cold swim-induced tremors and alterations of learning in a water maze paradigm. It will be interesting to determine how direct interaction between Sh and Hk and indirect effects through Hk redox regulation of other targets contribute to different components of the behavior of interest (Ueda, 2008).

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 (Haugland & Wu, 1990), 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).

Effects of social isolation on neuromuscular excitability and aggressive behaviors in Drosophila: altered responses by Hk and gs^ts1, two mutations implicated in redox regulation

Social deprivation is known to trigger a variety of behavioral and physiological modifications in animal species, but the underlying genetic and cellular mechanisms are not fully understood. It has been shown that adult female flies reared in isolation show increased frequency of aggressive behaviors than those reared in a group. This study reports that isolated rearing also caused significantly altered nerve and muscle excitability and enhanced synaptic transmission at larval neuromuscular junctions (NMJs). Mutations of two genes, Hyperkinetic (Hk) and glutathione S-transferase-S1 (gs^ts1), alter the response to social isolation in Drosophila. Hk and gs^ts1 mutations increased adult female aggression and larval neuromuscular hyperexcitability, even when reared in a group. Unlike wild type, these behavioral and electrophysiological phenotypes were not further enhanced in these mutants by isolated rearing. Products of these two genes have been implicated in reactive oxygen species (ROS) metabolism. These mutants have been shown to increase signals from an ROS probe at larval NMJs, and this study revealed distinct effects of isolation rearing on these mutants, compared to the control larvae in ROS-probe signals. The data further demonstrated modified nerve and muscle excitability by a reducing agent, dithiothreitol. These results suggest that altered cellular ROS regulation can exert pleiotropic effects on nerve, synapse, and muscle functions and may involve different redox mechanisms in different cell types to modify behavioral expressions. Therefore, ROS regulation may take part in the cellular responses to social isolation stress, underlying an important form of neural and behavioral plasticity (Ueda, 2009).

Effects of mutant Drosophila K⁺ channel subunits on habituation of the olfactory jump response

The olfactory-jump response assay was used to analyze habituation in Drosophila mutants of potassium (K⁺) channel subunits. As with physiological assays of the giant fiber-mediated escape reflex, mutations at loci that encode K⁺ channel subunits have distinct effects on habituating the olfactory-jump response. The data for slowpoke and ether a go-go indicate similar effects on habituation of the olfactory-jump response and the giant fiber-mediated escape. Habituation in the olfactory jump assay in Hyperkinetic and Shaker mutants was drastically different from the degree of defect in the giant fiber-mediated escape reflex, indicating differential control mechanisms underlying the two forms of non-associative conditioning (Joiner, 2007).

Drosophila Hyperkinetic mutants have reduced sleep and impaired memory

In mammals, sleep is thought to be important for health, cognition, and memory. Fruit flies share most features of mammalian sleep, and a recent study found that Drosophila lines carrying loss-of-function mutations in Shaker (Sh) are short sleeping, suggesting that the Sh current plays a major role in regulating daily sleep amount. The Sh current is potentiated by a beta modulatory subunit coded by Hyperkinetic (Hk). This study demonstrates that severe loss-of-function mutations of Hk reduce sleep and do so primarily by affecting the Sh current. Moreover, using a transgenic approach, this study proved that a wild-type copy of Hk is sufficient to restore normal sleep. Furthermore, short-sleeping Hk mutant lines have a memory deficit, whereas flies carrying a weaker hypomorphic Hk allele have normal sleep and normal memory. By comparing six short-sleeping Sh lines with two normal sleeping ones, it was also found that only allele that reduced sleep also impaired memory. These data identify a gene, Hk, that is necessary to maintain normal sleep, and genetic evidence is provided that short sleep and poor memory are linked (Bushey, 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).

Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons

Molecular analysis and heterologous expression have shown that K+ channel β subunits regulate the properties of the pore-forming alpha subunits, although how they influence neuronal K+ currents and excitability remains to be explored. Cultured Drosophila "giant" neurons derived from mutants of the Hyperkinetic (Hk) gene, which codes for a K+ channel beta subunit, were studied. Whole cell patch-clamp recording revealed broadened action potentials and, more strikingly, persistent rhythmic spontaneous activities in a portion of mutant neurons. Voltage-clamp analysis demonstrated extensive alterations in the kinetics and voltage dependence of K+ current activation and inactivation, especially at subthreshold membrane potentials, suggesting a role in regulating the quiescent state of neurons that are capable of tonic firing. Altered sensitivity of Hk currents to classical K+ channel blockers (4-aminopyridine, alpha-dendrotoxin, and TEA) indicated that Hk mutations modify interactions between voltage-activated K+ channels and these pharmacological probes, apparently by changing both the intra- and extracellular regions of the channel pore. Correlation of voltage- and current-clamp data from the same cells indicated that Hk mutations affect not only the persistently active neurons, but also other neuronal categories. Shaker (Sh) mutations, which alter K+ channel alpha subunits, increased neuronal excitability but did not cause the robust spontaneous activity characteristic of some Hk neurons. Significantly, Hk Sh double mutants are indistinguishable from Sh single mutants, implying that the rhythmic Hk firing pattern is conferred by intact Shα subunits in a distinct neuronal subpopulation. The results suggest that alterations in β subunit regulation, rather than elimination or addition of α subunits, may cause striking modifications in the excitability state of neurons, which may be important for complex neuronal function and plasticity (Yao, 1999).

Interaction of the K channel β subunit, Hyperkinetic, with Eag family members

Assembly of K channel α subunits of the Shaker (Sh) family occurs in a subfamily specific manner. It has been suggested that subfamily specificity also applies in the association of β subunits with Sh channels. This study shows that the Drosophila β subunit homologue Hyperkinetic (Hk) associates with members of the Ether go-go (Eag), as well as Sh, families. Anti-EAG antibody coprecipitates EAG and HK indicating a physical association between proteins. Heterologously expressed Hk dramatically increases the amplitudes of Eag currents and also affects gating and modulation by progesterone. Through their ability to interact with a range of α subunits, the β subunits of voltage-gated K channels are likely to have a much broader impact on the signaling properties of neurons and muscle fibers than previously suggested (Wilson, 1998).

Genetic dissection of functional contributions of specific potassium channel subunits in habituation of an escape circuit in Drosophila

Potassium channels have been implicated in central roles in activity-dependent neural plasticity. The giant fiber escape pathway of Drosophila has been established as a model for analyzing habituation and its modification by memory mutations in an identified circuit. Several genes in Drosophila encoding K+ channel subunits have been characterized, permitting examination of the contributions of specific channel subunits to simple conditioning in an identified circuit that is amenable to genetic analysis. The results show that mutations altering each of four K+ channel subunits (Sh, slo, eag, and Hk) have distinct effects on habituation at least as strong as those of dunce and rutabaga, memory mutants with defective cAMP metabolism. Habituation, spontaneous recovery, and dishabituation of the electrically stimulated long-latency giant fiber pathway response were shown in each mutant type. Mutations of Sh (voltage-gated) and slo (Ca2+-gated) subunits enhanced and slowed habituation, respectively. However, mutations of eag and Hk subunits, which confer K+-current modulation, had even more extreme phenotypes, again enhancing and slowing habituation, respectively. In double mutants, Sh mutations moderated the strong phenotypes of eag and Hk, suggesting that their modulatory functions are best expressed in the presence of intact Sh subunits. Nonactivity-dependent responses (refractory period and latency) at two stages of the circuit were altered only in some mutants and do not account for modifications of habituation. Furthermore, failures of the long-latency response during habituation, which normally occur in labile connections in the brain, could be induced in the thoracic circuit stage in Hk mutants. This work indicates that different K+ channel subunits play distinct roles in activity-dependent neural plasticity and thus can be incorporated along with second messenger 'memory' loci to enrich the genetic analysis of learning and memory (Engel, 1998).

A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus

Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant revealed defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. The Hk polypeptide, determined from analysis of cDNA clones, is a homologue of mammalian K+ channel β subunits (Kv β). Coexpression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence alignments show that Hk, together with mammalian Kv β, represents an additional branch of the aldo-keto reductase superfamily. These results are relevant to understanding the function and evolutionary origin of Kv β (Chouinard, 1995).

Search PubMed for articles about Drosophila Hyperkinetic

Bushey, D., Huber, R., Tononi, G. and Cirelli, C. (2007). Drosophila Hyperkinetic mutants have reduced sleep and impaired memory. J Neurosci 27: 5384-5393. PubMed ID: 17507560

Chouinard, S. W., Wilson, G. F., Schlimgen, A. K. and Ganetzky, B. (1995). A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus. Proc Natl Acad Sci U S A 92: 6763-6767. PubMed ID: 7542775

Engel, J. E. and Wu, C. F. (1998). Genetic dissection of functional contributions of specific potassium channel subunits in habituation of an escape circuit in Drosophila. J Neurosci 18: 2254-2267. PubMed ID: 9482810

Fogle, K. J., Baik, L. S., Houl, J. H., Tran, T. T., Roberts, L., Dahm, N. A., Cao, Y., Zhou, M. and Holmes, T. C. (2015) CRYPTOCHROME-mediated phototransduction by modulation of the potassium ion channel β-subunit redox sensor. Proc Natl Acad Sci U S A 112(7):2245-50. PubMed ID: 25646452

Ganetzky, B. and Wu, C. F. (1983). Neurogenetic analysis of potassium currents in Drosophila: synergistic effects on neuromuscular transmission in double mutants. J Neurogenet 1: 17-28. PubMed ID: 6100303

Haugland, F. N. and Wu, C. F. (1990). A voltage-clamp analysis of gene-dosage effects of the Shaker locus on larval muscle potassium currents in Drosophila. J Neurosci 10: 1357-1371. PubMed ID: 2109786

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

Joiner, M. A., Asztalos, Z., Jones, C. J., Tully, T. and Wu, C. F. (2007). Effects of mutant Drosophila K⁺ channel subunits on habituation of the olfactory jump response. J Neurogenet 21: 45-58. PubMed ID: 17464797

Lee, and Wu, C.-F. (2006). Genetic modifications of seizure susceptibility and expression by altered excitability in Drosophila Na₊ and K₊ channel mutants. J Neurophysiol. 96: 2465-2478. PubMed ID: 17041230

Pongs, O. and Schwarz, J. R. (2010). Ancillary subunits associated with voltage-dependent K+ channels. Physiol Rev 90: 755-796. PubMed ID: 20393197

Ueda, A. and Wu, C. F. (2008). Effects of hyperkinetic, a β subunit of Shaker voltage-dependent K⁺ channels, on the oxidation state of presynaptic nerve terminals. J Neurogenet 22: 1-13. PubMed ID: 18428031

Ueda, A. and Wu, C. F. (2009). Effects of social isolation on neuromuscular excitability and aggressive behaviors in Drosophila: altered responses by Hk and gsts1, two mutations implicated in redox regulation. J Neurogenet 23: 378-394. PubMed ID: 19863269

Wilson, G. F., Wang, Z., Chouinard, S. W., Griffith, L. C. and Ganetzky, B. (1998). Interaction of the K channel beta subunit, Hyperkinetic, with eag family members. J Biol Chem 273: 6389-6394. PubMed ID: 9497369

Yao, W. D. and Wu, C. F. (1999). Auxiliary Hyperkinetic β subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons. J Neurophysiol 81: 2472-2484. PubMed ID: 10322082 Biological Overview

date revised: 25 June 2016

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