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SLEEPLESS, a Ly-6/neurotoxin family member, regulates the levels, localization and activity of Shaker

Abstract

Sleep is a whole-organism phenomenon accompanied by global changes in neural activity. We previously identified SLEEPLESS (SSS) as a glycosylphosphatidyl inositol–anchored protein required for sleep in Drosophila. Here we found that SSS is critical for regulating the sleep-modulating potassium channel Shaker. SSS and Shaker shared similar expression patterns in the brain and specifically affected each other's expression levels. sleepless (sss) loss-of-function mutants exhibited altered Shaker localization, reduced Shaker current density and slower Shaker current kinetics. Transgenic expression of sss in sss mutants rescued defects in Shaker expression and activity cell-autonomously and suggested that SSS functions in wake-promoting, cholinergic neurons. In heterologous cells, SSS accelerated the kinetics of Shaker currents and was co-immunoprecipitated with Shaker, suggesting that SSS modulates Shaker activity via a direct interaction. SSS is predicted to belong to the Ly-6/neurotoxin superfamily, suggesting a mechanism for regulation of neuronal excitability by endogenous toxin-like molecules.

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Figure 1: Rescue of the sleep phenotype of sss mutants with a UAS-sss transgene.
Figure 2: Distribution of SSS and Shaker immunoreactivity in the adult fly brain.
Figure 3: Shaker and SSS specifically affect each other's expression.
Figure 4: Altered Shaker expression and localization in sss mutants.
Figure 5: Rescue of Shaker expression in sss mutants by transgenic expression of sss.
Figure 6: Cell-autonomous rescue of the sss phenotypes at the Drosophila larval NMJ.
Figure 7: SSS modulates Shaker function.

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Acknowledgements

We thank I. Levitan, J. Simpson, D. Bushey, B. Ganetzky, E. Rulifson, K. Kume, G. Korge and the Bloomington Stock Center for providing antibodies and fly stocks. We are grateful to T. Ferguson for help with oocyte preparation and M. Sowcik and R. Xu for technical assistance. This work was funded by a Burroughs-Wellcome Fund Career Award for Medical Scientists (M.N.W.), grants from the US National Institutes of Health (K08NS059671 to M.N.W., T32HL007953 to A. Pack, who supported T.D., R01GM057654 and R01GM078579 to T.H., P01AG017628 to A.S. and K.K., and R01GM088221 to K.K.), and a University Research Foundation Award from the University of Pennsylvania (K.K.). A.S. is an Investigator of the Howard Hughes Medical Institute.

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M.N.W., W.J.J. and K.K. conceived the study, in close consultation with A.S. M.N.W., W.J.J., T.D. and K.K. planned and performed the experiments, and analyzed the data with assistance from Z.Y., C.J.S. and D.C. T.H. provided supervision and advice for electrophysiological experiments. The manuscript was written principally by M.N.W. and K.K. with specific sections written by W.J.J. and T.D. and editorial changes made by A.S. and T.H.

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Correspondence to Kyunghee Koh.

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Wu, M., Joiner, W., Dean, T. et al. SLEEPLESS, a Ly-6/neurotoxin family member, regulates the levels, localization and activity of Shaker. Nat Neurosci 13, 69–75 (2010). https://doi.org/10.1038/nn.2454

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