Direct Interaction of Target SNAREs with the Kv2.1 Channel

Abstract

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)....

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.…”

Syntaxin 1A Regulates ENaC Channel Activity

“…Both effects may require or be influenced by interactions of S1A with other SNARE proteins. For example, Michaelevski et al (25) report that Kv 2.1 channel activity is differentially regulated depending on whether S1A is expressed alone or in combination with its partner t-SNARE, SNAP-25, and similar data have been obtained for S1A and SNAP-23 interactions with CFTR (31). At present, we do not know whether SNAP-23 modifies the action of S1A on ENaC trafficking or gating.…”

“…This interaction would provide a means to finely regulate vesicle membrane fusion and coordinate this process with Ca 2ϩ entry, which signals secretion (13). This so-called "excitosome" model has been extended to include other voltage-dependent channels of neuronal tissues such as Kv 2.1 (11,25). In addition, channels from non-excitable cells are regulated via interactions with SNARE proteins.…”

SNAP-25/Syntaxin 1A Complex Functionally Modulates Neurotransmitter γ-Aminobutyric Acid Reuptake

“…This study provides the possibility that the SNARE complex may functionally modulate those transporters. Indeed, the properties of two ion channels, Kv2.1 potassium channel and cystic fibrosis transmembrane regulator chloride channel, are reported to be directly affected by the target SNARE complex (23,24), as mediated by their direct interaction with the H3 domain of syntaxin 1A (45,46). Moreover, accumulating evidence also reveals that syntaxin 1A interacts with other membrane proteins and regulates their functions, and its H3 domain is the only reported binding site (45)(46)(47).…”

“…Increasing studies from the ion channels indicate that Kv2.1 potassium channel and cystic fibrosis transmembrane regulator chloride channel, which reside on the plasma membrane to control the intracellular or extracellular concentrations of respective ions, are functionally regulated by the t-SNARE complex (23,24). Because the t-SNARE complex acts on the ion channels, and syntaxin 1A physically interacts with and functionally regulates those neurotransmitter transporters, it is most likely that other SNARE proteins or the SNARE complex may modulate those transporters.…”