InteractiveFly: GeneBrief

quiver: Biological Overview | References

Gene name - quiver

Synonyms - sleepless (sss)

Cytological map position - 47F13-47F13

Function - channel-associated protein

Keywords - sleep, brain, muscle, modifier of the activity of Shaker K+ channel

Symbol - qvr

FlyBase ID: FBgn0260499

Genetic map position - chr2R:7330031-7339109

Classification - possible a toxin-like GPI-anchored protein

Cellular location - glycosyl-phosphatidylinositol (GPI)-anchored membrane protein

NCBI links: Precomputed BLAST | EntrezGene


Sleep is an essential process conserved from flies to humans. The importance of sleep is underscored by its tight homeostatic control. In this study, through a forward-genetic screen, a novel gene, sleepless, was identified that is required for sleep in Drosophila. sleepless encodes a brain-enriched, glycosyl-phosphatidylinositol-anchored protein. Loss of Sleepless protein causes an extreme (>80%) reduction in sleep. Furthermore, a moderate reduction in Sleepless protein has minimal effects on baseline sleep, but markedly reduces recovery sleep following sleep deprivation. Genetic and molecular analyses reveal that quiver, a mutation that impairs Shaker-dependent K+ current, is an allele of sleepless. Consistent with this finding, Shaker protein level is reduced in sleepless mutants. It is proposed that Sleepless is a signaling molecule that connects sleep drive to lowered membrane excitability (Koh, 2008).

Insufficient and poor quality sleep is an increasing problem in industrialized nations. Chronic sleep problems diminish quality of life, reduce workplace productivity, and contribute to fatal accidents. Although the biological needs fulfilled by sleep are unclear, they are likely to be important because sleep is conserved from flies to humans, and prolonged sleep deprivation can lead to lethality. Identifying mechanisms that control sleep may lead to novel approaches for improving sleep quality (Koh, 2008).

Sleep is regulated by two main processes: circadian and homeostatic. The circadian clock regulates the timing of sleep, whereas the homeostatic mechanism controls sleep need. Homeostatic pressure to sleep increases with time spent awake and decreases with time spent asleep. Homeostatic control is thought to influence sleep under normal (baseline) conditions as well as recovery (rebound) sleep following deprivation. However, the molecular mechanisms underlying homeostatic regulation of sleep remain unclear (Koh, 2008).

A powerful approach to unraveling a poorly understood biological process is to conduct unbiased genetic screens to identify novel molecules required for that process. The Drosophila model for sleep is well-suited for such an approach, which proved invaluable for elucidation of the molecular basis of the circadian clock. Although several Drosophila genes have been implicated in sleep regulation, only one of these, the gene encoding the Shaker (Sh) K+ channel, was isolated as a result of a genetic screen (Cirelli, 2005). A mutation in this gene causes one of the shortest-sleeping phenotypes known, validating the use of screens and suggesting that control of membrane excitability is a critical requirement for sleep. However, the mechanisms by which sleep homeostatic inputs regulate neuronal excitability remain unknown (Koh, 2008).

In this study, using a large-scale, unbiased genetic screen, a novel gene, sleepless (sss), was identified that is required in Drosophila for both normal baseline sleep and rebound sleep following deprivation. sss encodes a brain-enriched, glycosyl-phosphatidylinositol (GPI)-anchored membrane protein. quiver (qvr), a mutation causing impaired Sh-dependent K+ current (Humphreys, 1966; Wang, 2000), is an allele of sss. Sh protein level is reduced in sss mutant flies. It is proposed that Sss protein signals homeostatic sleep drive by enhancing K+ channel activity and thus reducing neuronal excitability (Koh, 2008).

It is worth noting that sssP2 animals show a moderate reduction in SSS protein and a minimal reduction in baseline sleep, but have severely reduced sleep rebound. The differential requirement for SSS protein in normal versus rebound sleep may be explained in the context of the two-process model of sleep regulation, where sleep is postulated to be controlled by the opposing influences of circadian waking drive and homeostatic sleep drive. In this context, for early-morning rebound sleep to occur, a strong homeostatic signal promoting sleep would be required to counteract a strong circadian input keeping the flies awake. At night when circadian waking drive is weaker or absent, a relatively low level of homeostatic input may suffice to allow flies to sleep. The moderate level of SSS protein in sssP2 mutants may be within the range where sleep is possible when a wake-promoting circadian signal is low (at night), but not when it is high (in the early morning). In contrast, sssP1 and sssΔ40 mutants, which have undetectable levels of SSS expression, display severe reductions in both baseline and rebound sleep. In these mutants, the sleep-promoting signal may be too low to allow flies to sleep even when the circadian waking drive is weak at night (Koh, 2008).

Clues to the role of SSS at the cellular level come from the biochemical characterization of this molecule. The SSS protein is a GPI-anchored membrane protein enriched in the brain. GPI-anchored proteins can function as ligands or co-receptors and can also act as diffusible signals following cleavage of the GPI anchor. Although no circadian or homeostatic regulation of the total levels of SSS protein was detected, such regulation may occur at the level of cleavage of the GPI anchor. Regulation of release is known to be controlled by time of day for other proteins that do not cycle in overall levels, such as pigment-dispersing factor, a molecular output of clock neurons. Alternatively, SSS may be regulated in a subset of cells that express it, which would be undetectable on western blots (Koh, 2008).

A potential mechanism by which SSS regulates sleep is suggested by the finding that qvr is an allele of sss and that the Sh protein level is reduced in sss mutants. Furthermore, qvr mutants exhibit markedly impaired Sh-dependent K+ current at the larval neuromuscular junction (Wang, 2000). Thus it is proposed that SSS lowers membrane excitability by modulating K+ channel expression and activity. It is striking that among thousands of mutants screened in Drosophila, two with the strongest sleep phenotypes affect the Sh K+ channel and its putative regulator, sss. Reduced membrane excitability may thus be a central feature of sleep. Collectively, these data suggest that SSS is a signaling molecule that links homeostatic sleep drive to neuronal excitability (Koh, 2008).

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

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

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

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

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

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

Snake neurotoxins do not have GPI anchors like Sss. However, ER-targeted expression of soluble dendrotoxin, a specific blocker of Shaker-type potassium channels, results in increased surface expression of Kv1.1, a mammalian ortholog of Shaker. This finding led Vacher (2007) to postulate the existence of an endogenous toxin-like ER protein that tethers Shaker channels to the ER membrane and with which dendrotoxin competes for binding. Sss may be such an endogenous neurotoxin-like molecule regulating Shaker function and localization. However, rather than retaining Shaker in the ER, Sss appears to increase surface localization of the channel, either through promotion of Shaker trafficking to or retention at the cell surface. Lynx1, another GPI-anchored neurotoxin/Ly-6 family member found in mammals, binds to and modulates the activity of a ligand-gated ion channel (nicotinic acetylcholine receptor). Thus, regulation of various ion channels by toxin-like GPI-anchored proteins may be an evolutionarily conserved mechanism, and Sss and Lynx-1 may be founding members of a family of cell-surface proto-toxins that modulate ion channel properties to control neuronal excitability and signaling. Although BLAST analysis with the primary sequence of Sss does not reveal an obvious mammalian ortholog, there are a number of mammalian proteins with a Ly-6 domain and a GPI anchor, one of which may represent a functional homolog of Sss (Wu, 2010).

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

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

Recent studies have indicated that the Shaker potassium channel regulates sleep in Drosophila. The Drosophila quiver (qvr) gene encodes a novel potassium channel subunit that modulates the Shaker potassium channel. The Qvr peptide contains a signal sequence for extracellular localization. Qvr may regulate a unique feature of the Shaker IA current that confers special neuronal excitability patterns. Studies of the Shaker channel properties in the qvr mutation background should provide an opportunity to uncover physiologic modulation of potassium channels. This study investigated the impact of qvr protein on the Shaker channel properties and its implications in synaptic function in vivo. Synaptic transmission was studied at the larval neuromuscular junction, and the transient potassium current IA was characterized in larval muscles. Two different functional states of IA were identified in qvr larval muscles, as reflected by two distinct components, IAF and IAS, differing in their kinetics of recovery from inactivation and sensitivity to a K+ channel blocker. Correspondingly, qvr mutant larvae exhibit multiple synaptic discharges following individual nerve stimuli during repetitive activity (Wang, 2011).

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

When ten episodes of a depolarizing step to +10 mV were applied to a qvr mutant muscle, the first outward transient current (IA) was more than twice that of the subsequent nine currents, but not as large as the wild-type current. The subsequent nine currents were almost the same size in amplitude with the same kinetics, suggesting that there were two components of IA in qvr mutant muscles. This contrasts with the use-dependent inactivation of a homogeneous component, which should display a gradual decay in the current amplitude upon repetitive depolarization. The first and the subsequent IA currents displayed the same time to peak and the same inactivation kinetics, which could be visualized when the two traces were normalized. The fast- and slow-recovery components are therefore named IAF and IAS, respectively. Operationally, the first IA is assumed to represent the sum of IAF and IAS, and the average of subsequent nine IA currents includes only IAF. The similarity in I-V curve between qvr1 and qvrδ43-1/qvr1 suggests the observed phenotype is attributable to the qvr locus and not likely an effect of an unidentified second-site mutation in the background (Wang, 2011).

The involvement of the qvr K+ channel subunit in sleep regulation and its potential interaction with the Sh K+ channel from extracellular domain (Koh, 2008) call for more biophysical studies of the Qvr function. The present study shows that Qvr is important for the maintenance of neuronal excitability. Frequent stimulations of the motor axons in qvr mutant larvae generate discretely increased neuromuscular transmission. In a detailed biophysical characterization of the Sh potassium channel, this study found that the transient IA current in qvr mutant muscles exhibits two discrete components, IAF and IAS, which are not observed in normal muscles. IAF displays faster kinetics of recovery from inactivation and more sensitivity to 4-aminopyridine (4-AP) than IAS. Furthermore, analysis of IAF and IAS in double mutants, Sh qvr, eag qvr and Hk qvr, identified a potential conformational change in the Sh K+ channel conferred by Qvr (Wang, 2011).

Previous study shows that qvr mutations affect only the IA channel in both conductance and kinetics, without altering IK, ICF, ICS, and ICa, suggesting that the Qvr assumes a role in modulating the a-subunit of the IA channel (Wang, 2000). Its phenotypic similarities to the three known K+ channel mutants Sh, Hk and eag provide hints that the qvr gene might code for a distinct K+ channel subunit. The Qvr protein is predicted to contain a GPI-attachment site and the GPI anchor can be cleaved by PLC (Koh, 2008). This study has investigated the potential conformational change conferred by the interaction between Qvr and the Sh K+ channel. First, a pharmacological approach was used. 4-AP has been well established as a Sh channel blocker that binds to the cytoplasmic pore region of the channel. The sensitivities to 4-AP of IAF and IAS showed an IC50 of 60 and 200 microM, respectively, which were much higher than the IC50 of only 7 microM in wild-type muscles. The differential 4-AP sensitivities in IAF and IAS imply different conformation in the cytoplasmic pore region. Then several K+ channel mutants were used to identify potential site of conformational change. The Sh5 mutation, which has an amino acid replacement in the S4-S5 linker of the Sh polypeptide, caused an increase in 4-AP sensitivity. Among several different double mutants, Sh5qvr1 exhibited a special property: a sum amplitude of IAF and IAS is on par with the IA amplitude in Sh5 mutant muscles. These results suggest that the interaction between Qvr and the Sh channel confers conformational change at the Sh5 mutation site (Wang, 2011).

The excitability control of the motor neurons can be attributed to the function of the Sh channel. It is interesting to note that the 4-AP sensitive IA current plays an important role in the gating of action potentials in hippocampal CA3 pyramidal neurons. Action potentials in the pyramidal neurons can be blocked by the activation of the IA current with a brief hyperpolarization a few milliseconds before the induction of an action potential. The discrete increase in EJC at the neuromuscular junction in Drosophila larvae suggests that Qvr is important in the controlling the propagation of action potentials. This result is in accord with previous findings that efficient membrane repolarization is required to suppress supernumerary action potentials in the motor axon (Ueda, 2006; Wang, 2011 and references therein).

Sleep is thought as a physiological state that increases the efficiency of behavior by regulating its timing and energy use (Siegel, 2009). Studies of rat cerebral energy consumption suggest that about half of the brain energy is used to drive signals along axons and across synapse. This study suggests that modulation of the Sh K+ channel by Qvr is a potential gating mechanism for the generation of action potentials in the nervous system. One can speculate that physiologic modulation of Qvr function plays an important role in controlling action potentials in relevant neural circuit for sleep. However, qvr expression does not fluctuate with the circadian cycle (Koh, 2008). Therefore, future experiments to demonstrate whether and how wakefulness/sleep states modulate the biophysical properties of the Sh K+ channel, reminiscent of the comprehensive studies in the mammalian thalamocortical systems (McCormick, 1997), will be important to provide a mechanistic link between sleep and K+ channel function. Alternatively, it is possible that wakefulness/sleep states do not modulate Sh K+ channel and the abnormal sleep phenotype in K+ channel mutant flies is due to their hypersensitivity to sensory stimulations and a hypersensitive motor system. Future studies of Qvr function in Drosophila central brain with optical imaging may shed light on the role of Qvr in sleep regulation (Wang, 2011).

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

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

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

Consistent with previous reports (Wang, 2000; Wang, 2010), this study found that repetitive depolarization of qvr/sss mutant larval muscles leads to a significant loss of Shaker current. The cumulatively inactivated component, recently named IAS (Wang, 2010), is eliminated by muscle-directed restoration of Sss in sssP1 mutants, suggesting that Sss normally prevents the cumulative inactivation of Shaker currents in wild-type flies. In in vitro studies, Sss did not affect recovery from either N- or C-type inactivation, but it decreased the rate of entry into the C-type inactivated state. Sss may also slow C-type inactivation of Shaker in Drosophila, as the cumulative inactivation phenotype observed in qvr/sss mutants could be caused by an absence of Sss-dependent resistance to C-type inactivation. This hypothesis is supported by the reduction of cumulative inactivation at supraphysiological concentrations of extracellular K+. However, direct quantitative comparisons between the in situ Shaker currents and the in vitro currents of ShBδN T449A cannot be made, as the precise Shaker isoforms underlying the former are unknown, and the latter has a fundamentally different rate of C-type inactivation than wild-type channels. Similarly, testing the specific contribution of C-type inactivation to the sssP1 behavioral phenotypes may also be difficult. It was not possible to rescue the sssP1 phenotype with a high K+ diet, but it is suspected that systemic K+ concentration in Drosophila is tightly regulated, as it is in mammals (Dean, 2011).

Mutations in sss profoundly decrease Drosophila IA magnitude; however, it is unclear whether the loss of Shaker current in qvr/sss mutants is caused by decreases in Shaker expression or changes in Shaker kinetics. Kinetic modeling based on in vitro experiments suggest that kinetic effects of Sss account for nearly 40% of the total loss in IA magnitude exhibited by sssP1 mutants; the rest is likely due to a decrease in channel quantity. Interestingly, the cumulative inactivation of IA comprises a large portion of the in situ current loss. Because the rescue of the cumulative inactivation phenotype nearly doubles the Shaker magnitude after repetitive depolarization, this kinetic rescue could account for the entirety of the previously reported partial rescue of total Shaker current magnitude. Therefore, in sssP1 mutants, targeted expression of Sss completely rescues altered Shaker gating kinetics, cumulative inactivation, and slower tPeak, as previously reported (Wu, 2010), while only minimally rescuing total Shaker current magnitude. Nevertheless, Sss expression fully rescues the sssP1 mutant sleep phenotype when driven broadly throughout the Drosophila brain as well as selectively in cholinergic neurons (Wu, 2010). Thus, the severity of the sleep phenotype in sssP1 mutants may better correlate with an alteration in Shaker current kinetics than magnitude. For example, Sss overexpression in wild-type flies reduces Shaker current in Drosophila muscles but does not induce a sleep phenotype, even when expressed using drivers that rescued the sleep phenotype in sssP1 flies. Additionally, Drosophila bearing Shaker-null alleles exhibit a less severe sleep phenotype than sssP1 mutants despite having no Shaker current. Because organisms can compensate for constitutive changes in ion channel quantity to preserve a neuron's overall excitability, it is speculated that static decreases in Shaker quantity are easier to compensate for than the dynamic, use-dependent loss of Shaker K+ current seen in qvr/sss mutants. Future studies of in vivo central neurons in adult Drosophila may better elucidate the neurophysiological significance of the kinetic effects of Sss on Shaker (Dean, 2011).

The Shaker β-subunit Hyperkinetic (Hk) accelerates rate of Shaker activation (Chouinard, 1995) to a degree similar to that induced by Sss; however, Hk is an intracellular protein whereas Sss is tethered to the extracellular side of the membrane by a GPI-anchor. Although GPI-anchored auxiliary subunits have not yet been proposed for voltage-gated K+ channels, a recent study found that the voltage-gated Ca2+ channel auxiliary subunit a2d is indeed GPI-anchored. Just as adequate Ca2+ channel expression depends on a2d, a deficiency of Sss leads to decreases in Shaker protein levels in vivo (Wu, 2010). Additionally, a2d-containing Ca2+ channels concentrate in lipid rafts, much like Sss and Shaker. Furthermore, the Sss-induced increase in activation of heterologously expressed Shaker channels was sensitive to cholesterol depletion, similar to K+ currents in ex vivo neurons from the Drosophila mushroom bodies, raising the possibility that the mechanism by which Sss interacts with Shaker involves lipid rafts. Thus, GPI-anchored proteins like Sss and a2d may be critical regulators of ion channel function and quantity at the cell surface (Dean, 2011).

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

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

There are also potential mechanisms for the Sss-dependent modulation of activation and C-type inactivation aside from a direct Sss-Sh interaction. For instance, Sss may indirectly act upon Shaker through another protein, as even in an in vitro system like HEK cells, native proteins found in lipid rafts (e.g., caveolin) can modulate K+ channels. Another potential effect of Sss may be to increase local extracellular K+ accumulation upon channel opening and K+ efflux, therefore indirectly slowing C-type inactivation. Sss may even affect channel kinetics by altering interactions between surrounding lipids and Shaker channels, an important consideration given that membrane phosopholipids play critical roles in ion channel function. A lipid-channel interaction mediated by Sss is consistent with the finding that the sigmoidicity of macroscopic current activation was unaltered by Sss, regardless of the degree of acceleration that Sss induced. Had the interaction between Sss and Shaker followed a strict stoichiometry in which individual subunits within a tetramer were selectively enhanced, then it is unlikely that activation sigmoidicity could have been preserved over the range of activation speeds observed. Because sigmoidicity is preserved over the entire range of activation speeds, it is suspected that every subunit of each channel in a patch is equally affected by the variable expression levels of Sss. The effect may be unique to Shaker, as human Slo1 activation kinetics are not affected by Sss expression despite also trafficking to lipid rafts (Weaver, 2007), suggesting that either Shaker is particularly sensitive to lipid membrane composition or that cholesterol plays a permissive role in the effects of Sss on Shaker kinetics. Nevertheless, effects of Sss on channels other than Shaker cannot be entiredly excluded (Dean, 2011).

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

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 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 paper reports the consequences of specific ion channel disruptions on the spiking activity of myogenic DLMs (firing at approximately 5 Hz) and the corresponding wing beat frequency (approximately 200 Hz). Mutants were examined of: 1) voltage-gated Ca2+ channels (cacophony, cac), 2) Ca2+-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 Hk and qvr could lead to distinct consequences, i.e. 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).

A novel leg-shaking Drosophila mutant defective in a voltage-gated K(+)current and hypersensitive to reactive oxygen species

1,1'-Dimethyl-4,4'-bipyridinium dichloride (methyl viologen; paraquat), an herbicide that causes depletion of NADPH and generates excessive reactive oxygen species (ROS) in vivo, has been used to screen for ROS-sensitive Drosophila mutants. One mutant so isolated, named quiver1 (qvr1), has a leg-shaking phenotype. Mutants of the Shaker (Sh), Hyperkinetic (Hk), and ether a go-go (eag) genes, which encode different K+ channel subunits that regulate the A-type K+ current (IA) in different ways, exhibit leg shaking under ether anesthesia and have heightened metabolic rates and shortened life spans. This study found that Sh, Hk, and eag mutant flies were all hypersensitive to paraquat. Double-mutant combinations among the three channel mutations and qvr1 had drastically enhanced sensitivity to paraquat. Synaptic transmission at the larval neuromuscular junction was increased in the qvr1 mutant to the level of Sh mutants. Similar to eag Sh double mutants, double mutants of eag and qvr1 showed striking enhancement in synaptic transmission and a wings-down phenotype, the hallmarks of extreme hyperexcitability. Voltage-clamp experiments demonstrated that the qvr1 mutation specifically disrupted the Sh-dependent IA current without altering the other currents. Several deficiency strains of the qvr locus failed to complement qvr1 and confirmed that ether-induced leg shaking, reduced IA current, and paraquat hypersensitivity map to the same locus. The results suggest that the qvr gene may encode a novel K+ channel-related polypeptide and indicate a strong link between a voltage-activated K+ current and vulnerability to ROS (Wang, 2000).


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Biological Overview

date revised: 28 February 2012

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