title: Direct interaction of target SNAREs with the Kv2.1 channel. Modal regulation of channel activation and inactivation gating.
abstract: Previously we suggested that interaction between voltage-gated K+ channels and protein components of the exocytotic machinery regulated transmitter release. This study concerns the interaction between the Kv2.1 channel, the prevalent delayed rectifier K+ channel in neuroendocrine and endocrine cells, and syntaxin 1A and SNAP-25. We recently showed in islet beta-cells that the Kv2.1 K+ current is modulated by syntaxin 1A and SNAP-25. Here we demonstrate, using co-immunoprecipitation and immunocytochemistry analyses, the existence of a physical interaction in neuroendocrine cells between Kv2.1 and syntaxin 1A. Furthermore, using concomitant co-immunoprecipitation from plasma membranes and two-electrode voltage clamp analyses in Xenopus oocytes combined with in vitro binding analysis, we characterized the effects of these interactions on the Kv2.1 channel gating pertaining to the assembly/disassembly of the syntaxin 1A/SNAP-25 (target (t)-SNARE) complex. Syntaxin 1A alone binds strongly to Kv2.1 and shifts both activation and inactivation to hyperpolarized potentials. SNAP-25 alone binds weakly to Kv2.1 and probably has no effect by itself. Expression of SNAP-25 together with syntaxin 1A results in the formation of t-SNARE complexes, with consequent elimination of the effects of syntaxin 1A alone on both activation and inactivation. Moreover, inactivation is shifted to the opposite direction, toward depolarized potentials, and its extent and rate are attenuated. Based on these results we suggest that exocytosis in neuroendocrine cells is tuned by the dynamic coupling of the Kv2.1 channel gating to the assembly status of the t-SNARE complex.
authors.affiliation: Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 69978 Ramat Aviv, Israel.
authors.forename: Izhak
authors.initials: I
authors.lastname: Michaelevski
authors.forename: Dodo
authors.initials: D
authors.lastname: Chikvashvili
authors.forename: Sharon
authors.initials: S
authors.lastname: Tsuk
authors.forename: Dafna
authors.initials: D
authors.lastname: Singer-Lahat
authors.forename: Youhou
authors.initials: Y
authors.lastname: Kang
authors.forename: Michal
authors.initials: M
authors.lastname: Linial
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
authors.forename: Oded
authors.initials: O
authors.lastname: Fili
authors.forename: Ilana
authors.initials: I
authors.lastname: Lotan
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D051668:Delayed Rectifier Potassium Channels; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000737:chemistry; D017830:Octoxynol / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D016716:PC12 Cells; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry*; D024642:Potassium Channels, Voltage-Gated; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D051669:Shab Potassium Channels; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D013997:Time Factors; D033921:Vesicular Transport Proteins; D014981:Xenopus
chemical_list: D000954:Antigens, Surface; D051668:Delayed Rectifier Potassium Channels; D003902:Detergents; 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; D050600:SNARE Proteins; C494212:STX1A protein, human; D051669:Shab Potassium Channels; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D017830:Octoxynol; D005982:Glutathione Transferase; D011188:Potassium