title: Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus.
abstract: Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.
authors.affiliation: Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Patrick E
authors.initials: PE
authors.lastname: MacDonald
authors.forename: Guotang
authors.initials: G
authors.lastname: Wang
authors.forename: Sharon
authors.initials: S
authors.lastname: Tsuk
authors.forename: Chikvashvili
authors.initials: C
authors.lastname: Dodo
authors.forename: Youhou
authors.initials: Y
authors.lastname: Kang
authors.forename: Lan
authors.initials: L
authors.lastname: Tang
authors.forename: Michael B
authors.initials: MB
authors.lastname: Wheeler
authors.forename: Mark S
authors.initials: MS
authors.lastname: Cattral
authors.forename: Jonathan R T
authors.initials: JR
authors.lastname: Lakey
authors.forename: Anne Marie F
authors.initials: AM
authors.lastname: Salapatek
authors.forename: Ilana
authors.initials: I
authors.lastname: Lotan
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D051668:Delayed Rectifier Potassium Channels; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051669:Shab Potassium Channels; D050825:Synaptosomal-Associated Protein 25; D014162:Transfection
chemical_list: D051668:Delayed Rectifier Potassium Channels; C496988:KCNB1 protein, human; C496991:Kcnb1 protein, rat; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; C494208:SNAP25 protein, human; D051669:Shab Potassium Channels; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D005982:Glutathione Transferase