Presynaptic voltage-gated K ϩ (Kv) channels play a physiological role in the regulation of transmitter release by virtue of their ability to shape presynaptic action potentials. However, the possibility of a direct interaction of these channels with the exocytotic apparatus has never been examined. We report the existence of a physical interaction in brain synaptosomes between Kv␣1.1 and Kv␤ subunits with syntaxin 1A, occurring, at least partially, within the context of a macromolecular complex containing syntaxin, synaptotagmin, and SNAP-25. The interaction was altered after stimulation of neurotransmitter release. The interaction with syntaxin was further characterized in Xenopus oocytes by both overexpression and antisense knockdown of syntaxin. Direct physical interaction of syntaxin with the channel protein resulted in an increase in the extent of fast inactivation of the Kv1.1/Kv␤1.1 channel. Syntaxin also affected the channel amplitude in a biphasic manner, depending on its concentration. At low syntaxin concentrations there was a significant increase in amplitudes, with no detectable change in cell-surface channel expression. At higher concentrations, however, the amplitudes decreased, probably because of a concomitant decrease in cell-surface channel expression, consistent with the role of syntaxin in regulation of vesicle trafficking. The observed physical and functional interactions between syntaxin 1A and a Kv channel may play a role in synaptic efficacy and neuronal excitability.

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)....

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.