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Synaptosome-Associated Protein of 25 Kilodaltons Modulates Kv2.1 Voltage-Dependent K⁺ Channels in Neuroendocrine Islet ß-Cells through an Interaction with the Channel N Terminus

Departments of Physiology (P.E.M., G.W., Y.K., L.T., M.B.W., A.M.F.S., H.Y.G.), Medicine (G.W., Y.K., L.T., M.B.W., A.M.F.S., H.Y.G.), and Surgery (M.S.C.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; Department of Physiology and Pharmacology (S.T., C.D., I.L.), Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel 69978; and Department of Surgery (J.R.T.L.), University of Alberta, Edmonton, Canada T6N 2N8

Address all correspondence and requests for reprints to: Herbert Y. Gaisano and Anne Marie F. Salapatek, University of Toronto, 1 Kings College Circle, Room 7226, Toronto, Ontario, Canada, M5S 1A8. E-mail: herbert.gaisano{at}utoronto.ca and annemarie.salapatek{at}utoronto.ca.

Insulin secretion is initiated by ionic events involving membrane depolarization and Ca²⁺ 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 ß-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 ß-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 ß-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 ß-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.

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