Formation of the Full SNARE Complex Eliminates Interactions of Its Individual Protein Components with the Kv2.1 Channel

Abstract

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.

VAMP721 Conformations Unmask an Extended Motif for K+ Channel Binding and Gating Control

“…These studies offered no further details of the binding domain(s) or whether binding might be associated with the SNARE domain, as has been reported in animals Tsuk et al, 2008). To resolve the extent of the binding site, we prepared truncated proteins of VAMP721 and VAMP723 and investigated their interaction with the KAT1 K + channel using the yeast mating-based split-ubiquitin system (mbSUS) assay, as described before (Grefen et al, 2007;Zhang et al, 2015).…”

“…1 and 2) showed that binding is retained with VAMP721 truncations that incorporated either the longin domain or the SNARE domain. Binding with the SNARE domain was unexpected, although interactions of the Kv2.1 K + channel in mammals, which depends on the SNARE motif, have been proposed to regulate channel activity during the fusion of dense core vesicles Tsuk et al, 2008). Such interactions with mammalian SNAREs have been questioned in part because, when isolated, the SNARE domains show a high propensity for promiscuous binding in vitro (Fletcher et al, 2003).…”

Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes

“…For example, Kv1.1 channels interact with syntaxin-1A in brain synaptosomes and with SNAP-25 in insulinoma HIT-T15 ␤ cells (124,430). When expressed in Xenopus oocytes, both syntaxin-1A and SNAP-25 led to increased fast inactivation of the Kv1.1/Kv␤1.1-mediated current.…”

“…In Kv2.1-expressing HEK293 cells, coexpression of syntaxin-1A inhibits channel trafficking to the surface and reduces the corresponding current (234). Kv2.1 also interacts with SNAP-25 in rat islet ␤-cells (234,249,274) and with VAMP-2 in secreting neuroendocrine cells (430). Fast inactivating channels such as Kv4.x, responsible in the myocardium for the initial repolarization phase of the cardiac AP, also interact directly with syntaxin-1.…”

Non-conducting function of the Kv2.1 channel enables it to recruit vesicles for release in neuroendocrine and nerve cells

“…Notably, it has recently been shown that, in the presence of VAMP2, the t-SNARE complexes are released from Kv2.1 and form ternary SNARE complexes that do not interact with the channel (Tsuk et al, 2008). This suggests a role for Kv2.1 protein in the regulation of vesicle docking and/or priming during LDCV release without any interference in the formation of the ternary SNARE complex.…”