Voltage-gated K؉ (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet ␤-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet ␤-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet ␤-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634 -853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca 2؉ channels. In contrast to Ca 2؉ channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.

Kv channels inhibit release indirectly by hyperpolarizing membrane potential, but the significance of Kv channel interaction with the secretory apparatus is not known. The Kv2.1 channel is commonly expressed in the soma and dendrites of neurons, where it could influence the release of neuropeptides and neurotrophins, and in neuroendocrine cells, where it could influence hormone release. Here we show that Kv2.1 channels increase dense-core vesicle (DCV)-mediated release after elevation of cytoplasmic Ca 2ϩ . This facilitation occurs even after disruption of pore function and cannot be explained by changes in membrane potential and cytoplasmic Ca 2ϩ . However, triggering release increases channel binding to syntaxin, a secretory apparatus protein. Disrupting this interaction with competing peptides or by deleting the syntaxin association domain of the channel at the C terminus blocks facilitation of release. Thus, direct association of Kv2.1 with syntaxin promotes exocytosis. The dual functioning of the Kv channel to influence release, through its pore to hyperpolarize the membrane potential and through its C-terminal association with syntaxin to directly facilitate release, reinforces the requirements for repetitive firing for exocytosis of DCVs in neuroendocrine cells and in dendrites.

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.

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Kv2.1, the prevalent delayed-rectifier K ϩ channel in neuroendocrine and endocrine cells, was suggested previously by our group to be modulated in islet ␤-cells by syntaxin 1A (Syx) and soluble N-ethylmaleimide-sensitive factor attachment protein-25 . We also demonstrated physical interactions in neuroendocrine cells between Kv2.1, Syx, and SNAP-25, characterized their effects on Kv2.1 activation and inactivation in Xenopus laevis oocytes, and suggested that they pertain to the assembly/disassembly of the Syx/SNAP-25 (t-SNARE) complex. In the present work, we established the existence of a causal relationship between the physical and the functional interactions of Syx with the Kv2.1 channel using three different peptides that compete with the channel for binding of Syx when injected into oocytes already coexpressing Syx with Kv2.1 in the plasma membrane: one peptide corresponding to the Syxbinding region on the N-type Ca 2ϩ channel, and two peptides corresponding to Syx-binding regions on the Kv2.1 C terminus. All peptides reversed the effects of Syx on Kv2.1, suggesting that the hyperpolarizing shifts of the steady-state inactivation and activation of Kv2.1 caused by Syx result from cell-surface protein-protein interactions and point to participation of the C terminus in such an interaction. In line with these findings, the effects of Syx were dissipated by partial deletions of the C terminus. Furthermore, the t-SNARE complex was shown to bind to the Kv2.1 C terminus, and its effects on the inactivation of Kv2.1 were dissipated by partial deletions of the C terminus. Taken together, these findings suggest that physical interactions of both Syx and the t-SNARE complex with the C terminus of Kv2.1 are involved in channel regulation.The soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNARE proteins) comprise three conserved families of membrane-associated proteins, syntaxin, synaptobrevin/vesicle-associated membrane protein, and SNAP-25, that participate in the fusion of internal membranes in eukaryotic cells (Bennett and Scheller, 1993;Rothman and Warren, 1994;Jahn and Sudhof, 1999;Gerst, 2003) and thus play a crucial role in transmitter and hormone release (Sudhof, 1995). They interact with a wide range of proteins, some of which (such as synaptotagmin) are associated with vesicular membranes or with plasma membranes (e.g., voltage-gated Ca 2ϩ channels) (Bajjalieh and Scheller, 1995;Bennett, 1995;Sudhof, 1995;Sheng et al., 1996;Linial, 1997). Recent studies by our group have suggested a physical and functional coupling of the SNARE proteins with the voltage-gated K ϩ (Kv) channel Kv1.1 (Fili et al., 2001;Michaelevski et al., 2002); for example, it is suggested that Kv1.1 in a complex with the auxiliary Kv␤1.1 subunit coprecipitates with syntaxin 1A (Syx), SNAP-25 (SNAP), and synaptotagmin from brain synaptosomes in a manner that de- CHAPS,dimethylammonio]-1-propanesulfonic acid; His 6-N 718-963 , the synprint peptide; C1, the proximal half of the Kv2.1 C terminus; C2, th...