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

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.

Apoptotic surface delivery of K+ channels

“…Previous work had shown that these t-SNAREs can bind to and regulate the gating of Kv2.1. [15][16][17][18] However, since the gating of the delayed rectifier does not change with apoptosis, 2 the neurotoxin results, in concert with our trafficking experiments, are most consistent with the requirement of t-SNARE proteins for the membrane fusion required for insertion of new channels into the cell surface. This conclusion also is in accordance with the observations that overexpression of syntaxin in cell lines inhibits exocytosis 19 and Kv2.1 surface expression.…”

Direct Interaction of Target SNAREs with the Kv2.1 Channel

“…Syx and SNAP Directly Bind Cytosolic Domains of Kv2.1-Previously, we have shown in an in vitro binding assay that Syx and SNAP bind to the Kv2.1 channel (14,28). In this study we carried out a more comprehensive in vitro binding assay to substantiate the physical interaction of the channel with these proteins.…”

“…Preparation of mRNAs was as described (23). DNAs of Kv2.1 fragments for production of GST fusion proteins were constructed as described before (14). Materials and enzymes for molecular biology were purchased from Roche Applied Science, Promega (Madison, WI), and MBI Fermentas (Vilnius, Lithuania).…”

“…pus Oocytes-Recently we showed that the Kv2.1 channel, which is the principal delayed rectifier K ϩ channel of islet ␤-cells and controls membrane repolarization and downstream insulin exocytosis, interacts functionally with ␤-cell SNAP and Syx (14). In the present study we report for the first time in neuroendocrine cells the existence of a physical interaction of the Kv2.1 protein with Syx, occurring, at least in part, within the context of a macromolecular complex containing also SNAP.…”

“…Recently, we focused on endocrine cells, and we showed that Kv1.1 interacts with SNAP-25 in islet ␤-cells (13) and that in these cells also the Kv2.1 channel, a member of the Kv2 subfamily of Kv channels, is modulated by SNAP-25 and syntaxin 1A (14). Interestingly, in ␤-cells the interaction of exocytotic proteins with the L-type voltage-gated Ca 2ϩ channels has also been described (15).…”