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

Author:

MacDonald Patrick E.1,Wang Guotang12,Tsuk Sharon3,Dodo Chikvashvili3,Kang Youhou12,Tang Lan12,Wheeler Michael B.12,Cattral Mark S.4,Lakey Jonathan R. T.5,Salapatek Anne Marie F.12,Lotan Ilana3,Gaisano Herbert Y.12

Affiliation:

1. Departments of Physiology (P.E.M., G.W., Y.K., L.T., M.B.W., A.M.F.S., H.Y.G.), Toronto, Ontario, Canada M5S 1A8

2. Medicine (G.W., Y.K., L.T., M.B.W., A.M.F.S., H.Y.G.), Toronto, Ontario, Canada M5S 1A8

3. Department of Physiology and Pharmacology (S.T., C.D., I.L.), Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel 69978

4. Surgery (M.S.C.), University of Toronto, Toronto, Ontario, Canada M5S 1A8

5. Department of Surgery (J.R.T.L.), University of Alberta, Edmonton, Canada T6N 2N8

Abstract

AbstractInsulin 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 β-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 β-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 β-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 β-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.

Publisher

The Endocrine Society

Subject

Endocrinology,Molecular Biology,General Medicine

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