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...

Recently we suggested that direct interactions between voltage-gated K؉ channels and proteins of the exocytotic machinery, such as those observed between the Kv1.1/Kv␤ channel, syntaxin 1A, and SNAP-25 may be involved in neurotransmitter release. Furthermore, we demonstrated that the direct interaction with syntaxin 1A enhances the fast inactivation of Kv1.1/Kv␤1.1 in oocytes. Here we show that G-protein ␤␥ subunits play a crucial role in the enhancement of inactivation by syntaxin 1A. The effect caused by overexpression of syntaxin 1A is eliminated in the presence of chelators of endogenous ␤␥ subunits in the whole cell and at the plasma membrane. Conversely, enhancement of inactivation caused by overexpression of ␤ 1 ␥ 2 subunits is eliminated upon knock-down of endogenous syntaxin or its scavenging at the plasma membrane. We further show that the N terminus of Kv1.1 binds brain synaptosomal and recombinant syntaxin 1A and concomitantly binds ␤ 1 ␥ 2 ; the binding of ␤ 1 ␥ 2 enhances that of syntaxin 1A. Taken together, we suggest a mechanism whereby syntaxin and G protein ␤␥ subunits interact concomitantly with a Kv channel to regulate its inactivation.Voltage-gated K ϩ (Kv) 1 channels participate in a host of cellular processes, from setting the resting membrane potential and shaping action potential wave-form and frequency to controlling synaptic strength (1). Recently, we challenged the commonly accepted concept that presynaptic Kv channels participate in neurotransmitter release simply by virtue of their ability to shape action potentials that invade nerve terminals (2, 3), and suggested that the fine tuning of transmitter release might be attributable to direct interaction between Kv channels and proteins of the exocytotic machinery (4). We demonstrated that the Kv channel composed of the pore forming Kv1.1 and auxiliary Kv␤ subunits interact in fresh brain synaptosomes with syntaxin 1A, SNAP-25, and synaptotagmin, and this interaction is relieved following triggering of transmitter release. Furthermore, in insulinoma HIT-T15 ␤ cells the activity of Kv1.1 channel was inhibited by SNAP-25 (5). Also, we showed, in Xenopus oocytes, that the direct interaction of the Kv1.1/Kv␤1.1 (␣␤) channel with syntaxin 1A enhances the fast inactivation of the channel (4) that is conferred by the N-terminal part of ␤, in a mechanism termed "ball and chain" inactivation (6). The reciprocal effects of ␣, syntaxin 1A, and SNAP-25 are reminiscent of the finding that presynaptic Nand L-type voltage-gated Ca 2ϩ channels interact directly with proteins of the exocytotic apparatus in neurons, and that their interaction with syntaxin 1A and SNAP-25 causes feedback effects on the channel function in oocytes (reviewed in Ref . 7) and in synaptosomes (8). Recent studies have shown that disruption of the interaction with syntaxin 1A in neurons has functional implications for transmitter release, reducing the efficacy of both Ca 2ϩ -dependent (7, 9) and Ca 2ϩ -independent (10) release.Previous studies by our group have shown that the e...

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 ␤-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.The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 1 proteins syntaxin, SNAP-25, and VAMP are crucial factors in processes of transmitter and hormone release (1). They interact with a wide range of proteins, some of them (such as synaptotagmin) associated with vesicular membranes or with plasma membranes (for example, voltage-gated Ca 2ϩ channels) (1-5). We suggested previously (6) that SNARE proteins interact with a member of the Kv1 subfamily of the voltage-gated K ϩ (Kv) channels and that these interactions may play a role in synaptic efficacy and neuronal excitability. Our results showed that in brain synaptosomes the presynaptic Kv1.1 channel (7) interacts with some of the protein components of the exocytotic apparatus, including syntaxin 1A, SNAP-25, and synaptotagmin, in a manner that is sensitive to the exocytotic state of the synaptosomes. We also showed that Kv1.1 in complex with the auxiliary Kv␤ 1.1 subunits (8) interacts directly with syntaxin 1A, and the feedback effect of this interaction on the channel function enhances its fast inactivation in Xenopus oocytes. Involvement of G protein ␤␥ subunits was found to be a requirement for this interaction (9). These characteristics of the interaction of presynaptic Kv channels with syntaxin 1A are reminiscent of the interaction of the presynaptic N-type voltage-gated Ca 2ϩ channels (10 -12)....

Previously, we have demonstrated physical and functional interactions of the voltage-gated potassium channel Kv2.1 with the plasma membrane protein components of the exocytotic SNARE complex, syntaxin 1A, and the t-SNARE, syntaxin 1A/SNAP-25, complex. Importantly, the physical interaction of Kv2.1 with syntaxin was shown to be involved in the facilitation of secretion from PC12 cells, which was independent of potassium currents. Recently, we showed that also VAMP2, the vesicular SNARE, interacts physically and functionally with Kv2.1. Here, we first set out to test the interaction of the full SNARE, syntaxin/SNAP-25/VAMP2, complex with the channel. Using the interaction of VAMP2 with Kv2.1 in Xenopus oocytes as a probe, we showed that coexpression of the t-SNARE complex with VAMP2 abolished the VAMP2 effect on channel inactivation and reduced the amount of VAMP2 that coprecipitated with Kv2.1. Further, in vitro pull down assays showed that the full SNARE complex failed to interact with Kv2.1 N- and C-termini in tandem, in contrast to the individual SNARE components. This suggests that the interactions of the SNARE components with Kv2.1 are abolished upon their recruitment into a full SNARE complex, which does not interact with the channel. Other important findings arising from the in vitro study are that the t-SNARE complex, in addition to syntaxin, interacts with a specific C-terminal channel domain, C1a, shown to mediate the facilitation of release by Kv2.1 and that the presence of Kv2.1 N-terminus has crucial contribution to these interactions. These findings provide important insights into the understanding of the complex molecular events involved in the novel phenomenon of secretion facilitation in neuroendocrine cells by Kv2.1.