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

Article metrics


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Hendricks, J.C. et al. Rest in Drosophila is a sleep-like state. Neuron 25, 129–138 (2000).

  2. 2

    Shaw, P.J., Cirelli, C., Greenspan, R.J. & Tononi, G. Correlates of sleep and waking in Drosophila melanogaster. Science 287, 1834–1837 (2000).

  3. 3

    Nitz, D.A., van Swinderen, B., Tononi, G. & Greenspan, R.J. Electrophysiological correlates of rest and activity in Drosophila melanogaster. Curr. Biol. 12, 1934–1940 (2002).

  4. 4

    Anderson, M.P. et al. Thalamic Cav3.1 T-type Ca2+ channel plays a crucial role in stabilizing sleep. Proc. Natl. Acad. Sci. USA 102, 1743–1748 (2005).

  5. 5

    Bushey, D., Huber, R., Tononi, G. & Cirelli, C. Drosophila Hyperkinetic mutants have reduced sleep and impaired memory. J. Neurosci. 27, 5384–5393 (2007).

  6. 6

    Cueni, L. et al. T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites. Nat. Neurosci. 11, 683–692 (2008).

  7. 7

    Espinosa, F., Marks, G., Heintz, N. & Joho, R.H. Increased motor drive and sleep loss in mice lacking Kv3-type potassium channels. Genes Brain Behav. 3, 90–100 (2004).

  8. 8

    Espinosa, F., Torres-Vega, M.A., Marks, G.A. & Joho, R.H. Ablation of Kv3.1 and Kv3.3 potassium channels disrupts thalamocortical oscillations in vitro and in vivo. J. Neurosci. 28, 5570–5581 (2008).

  9. 9

    Cirelli, C. et al. Reduced sleep in Drosophila Shaker mutants. Nature 434, 1087–1092 (2005).

  10. 10

    Douglas, C.L. et al. Sleep in Kcna2 knockout mice. BMC Biol. 5, 42 (2007).

  11. 11

    Koh, K. et al. Identification of SLEEPLESS, a sleep-promoting factor. Science 321, 372–376 (2008).

  12. 12

    Tsetlin, V. Snake venom alpha-neurotoxins and other 'three-finger' proteins. Eur. J. Biochem. 264, 281–286 (1999).

  13. 13

    Greenwald, J., Fischer, W.H., Vale, W.W. & Choe, S. Three-finger toxin fold for the extracellular ligand-binding domain of the type II activin receptor serine kinase. Nat. Struct. Biol. 6, 18–22 (1999).

  14. 14

    Huai, Q. et al. Structure of human urokinase plasminogen activator in complex with its receptor. Science 311, 656–659 (2006).

  15. 15

    Klein, D.E., Stayrook, S.E., Shi, F., Narayan, K. & Lemmon, M.A. Structural basis for EGFR ligand sequestration by Argos. Nature 453, 1271–1275 (2008).

  16. 16

    Albrand, J.P., Blackledge, M.J., Pascaud, F., Hollecker, M. & Marion, D. NMR and restrained molecular dynamics study of the three-dimensional solution structure of toxin FS2, a specific blocker of the L-type calcium channel, isolated from black mamba venom. Biochemistry 34, 5923–5937 (1995).

  17. 17

    Vacher, H., Mohapatra, D.P., Misonou, H. & Trimmer, J.S. Regulation of Kv1 channel trafficking by the mamba snake neurotoxin dendrotoxin K. FASEB J. 21, 906–914 (2007).

  18. 18

    Elliott, D.A. & Brand, A.H. The GAL4 system: a versatile system for the expression of genes. Methods Mol. Biol. 420, 79–95 (2008).

  19. 19

    Joiner, W.J., Crocker, A., White, B.H. & Sehgal, A. Sleep in Drosophila is regulated by adult mushroom bodies. Nature 441, 757–760 (2006).

  20. 20

    Pitman, J.L., McGill, J.J., Keegan, K.P. & Allada, R. A dynamic role for the mushroom bodies in promoting sleep in Drosophila. Nature 441, 753–756 (2006).

  21. 21

    Chung, B.Y., Kilman, V.L., Keath, J.R., Pitman, J.L. & Allada, R. The GABA(A) receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila. Curr. Biol. 19, 386–390 (2009).

  22. 22

    Foltenyi, K., Greenspan, R.J. & Newport, J.W. Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila. Nat. Neurosci. 10, 1160–1167 (2007).

  23. 23

    Parisky, K.M. et al. PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit. Neuron 60, 672–682 (2008).

  24. 24

    Shang, Y., Griffith, L.C. & Rosbash, M. Light-arousal and circadian photoreception circuits intersect at the large PDF cells of the Drosophila brain. Proc. Natl. Acad. Sci. USA 105, 19587–19594 (2008).

  25. 25

    Sheeba, V. et al. Large ventral lateral neurons modulate arousal and sleep in Drosophila. Curr. Biol. 18, 1537–1545 (2008).

  26. 26

    Kaplan, W.D. & Trout, W.E. III. The behavior of four neurological mutants of Drosophila. Genetics 61, 399–409 (1969).

  27. 27

    Tanouye, M.A., Ferrus, A. & Fujita, S.C. Abnormal action potentials associated with the Shaker complex locus of Drosophila. Proc. Natl. Acad. Sci. USA 78, 6548–6552 (1981).

  28. 28

    Rogero, O., Hammerle, B. & Tejedor, F.J. Diverse expression and distribution of Shaker potassium channels during the development of the Drosophila nervous system. J. Neurosci. 17, 5108–5118 (1997).

  29. 29

    Hendricks, J.C. et al. Gender dimorphism in the role of cycle (BMAL1) in rest, rest regulation and longevity in Drosophila melanogaster. J. Biol. Rhythms 18, 12–25 (2003).

  30. 30

    Kume, K., Kume, S., Park, S.K., Hirsh, J. & Jackson, F.R. Dopamine is a regulator of arousal in the fruit fly. J. Neurosci. 25, 7377–7384 (2005).

  31. 31

    Wu, M.N., Koh, K., Yue, Z., Joiner, W.J. & Sehgal, A. A genetic screen for sleep and circadian mutants reveals mechanisms underlying regulation of sleep in Drosophila. Sleep 31, 465–472 (2008).

  32. 32

    Wang, J.W., Humphreys, J.M., Phillips, J.P., Hilliker, A.J. & Wu, C.F. A novel leg-shaking Drosophila mutant defective in a voltage-gated K+ current and hypersensitive to reactive oxygen species. J. Neurosci. 20, 5958–5964 (2000).

  33. 33

    Kelley, L.A. & Sternberg, M.J. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363–371 (2009).

  34. 34

    Miwa, J.M. et al. lynx1, an endogenous toxin-like modulator of nicotinic acetylcholine receptors in the mammalian CNS. Neuron 23, 105–114 (1999).

  35. 35

    Eriksson, M.A. & Roux, B. Modeling the structure of agitoxin in complex with the Shaker K+ channel: a computational approach based on experimental distance restraints extracted from thermodynamic mutant cycles. Biophys. J. 83, 2595–2609 (2002).

  36. 36

    Harvey, A.L. Twenty years of dendrotoxins. Toxicon 39, 15–26 (2001).

  37. 37

    Andretic, R., van Swinderen, B. & Greenspan, R.J. Dopaminergic modulation of arousal in Drosophila. Curr. Biol. 15, 1165–1175 (2005).

  38. 38

    Crocker, A. & Sehgal, A. Octopamine regulates sleep in Drosophila through protein kinase A–dependent mechanisms. J. Neurosci. 28, 9377–9385 (2008).

  39. 39

    Yuan, Q., Joiner, W.J. & Sehgal, A. A sleep-promoting role for the Drosophila serotonin receptor 1A. Curr. Biol. 16, 1051–1062 (2006).

  40. 40

    Saper, C.B., Scammell, T.E. & Lu, J. Hypothalamic regulation of sleep and circadian rhythms. Nature 437, 1257–1263 (2005).

  41. 41

    Li, Y., Um, S.Y. & McDonald, T.V. Voltage-gated potassium channels: regulation by accessory subunits. Neuroscientist 12, 199–210 (2006).

  42. 42

    Misonou, H. & Trimmer, J.S. Determinants of voltage-gated potassium channel surface expression and localization in mammalian neurons. Crit. Rev. Biochem. Mol. Biol. 39, 125–145 (2004).

  43. 43

    Abbott, G.W. & Goldstein, S.A. Potassium channel subunits encoded by the KCNE gene family: physiology and pathophysiology of the MinK-related peptides (MiRPs). Mol. Interv. 1, 95–107 (2001).

  44. 44

    Gumley, T.P., McKenzie, I.F. & Sandrin, M.S. Tissue expression, structure and function of the murine Ly-6 family of molecules. Immunol. Cell Biol. 73, 277–296 (1995).

  45. 45

    Ibañez-Tallon, I. et al. Novel modulation of neuronal nicotinic acetylcholine receptors by association with the endogenous prototoxin lynx1. Neuron 33, 893–903 (2002).

  46. 46

    Boussy, T. et al. Genetic basis of ventricular arrhythmias. Cardiol. Clin. 26, 335–353 (2008).

  47. 47

    Catterall, W.A., Dib-Hajj, S., Meisler, M.H. & Pietrobon, D. Inherited neuronal ion channelopathies: new windows on complex neurological diseases. J. Neurosci. 28, 11768–11777 (2008).

  48. 48

    Schopperle, W.M. et al. Slob, a novel protein that interacts with the Slowpoke calcium-dependent potassium channel. Neuron 20, 565–573 (1998).

  49. 49

    Zheng, X., Yang, Z., Yue, Z., Alvarez, J.D. & Sehgal, A. FOXO and insulin signaling regulate sensitivity of the circadian clock to oxidative stress. Proc. Natl. Acad. Sci. USA 104, 15899–15904 (2007).

  50. 50

    Feng, Y., Ueda, A. & Wu, C.F. A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae. J. Neurogenet. 18, 377–402 (2004).

Download references


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.

Author information

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.

Correspondence to Kyunghee Koh.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 1230 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading