We have presented 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. Our 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 Drosophila21,23–25,30,37–39
. Thus, as in mammals40
, sleep in Drosophila
is controlled by arousal systems that include distinct populations of cholinergic, monoaminergic, and peptidergic neurons.
We found that SSS and Shaker were enriched in the same regions of the Drosophila brain and that 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, we only observed partial rescue of muscle IA amplitude in sss mutants with overexpression of SSS in muscles. Along these lines, we 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.
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.
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 Kvß/Hyperkinetic, KCNEs, KChIPs, and KChAP41–43
. 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 syndromes41,43
. 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 surface12,13,15,44
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 Kv1.117
, a mammalian ortholog of Shaker. This finding led Vacher et al. (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)34,45
. 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 ortholog11
, 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.
In summary, we demonstrate that SSS is a novel regulator of Shaker expression, localization, and function in vivo
. We propose 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 arrhythmias46,47
, 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.