Kv2.1 Channel Activation and Inactivation Is Influenced by Physical Interactions of Both Syntaxin 1A and the Syntaxin 1A/Soluble N-Ethylmaleimide-Sensitive Factor-25 (t-SNARE) Complex with the C Terminus of the Channel (original) (raw)
Related papers
Biochemistry, 2008
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.
Journal of Biological Chemistry, 2003
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.
Syntaxin 1A Binds to the Cytoplasmic C Terminus of Kv2.1 to Regulate Channel Gating and Trafficking
Journal of Biological Chemistry, 2003
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.
Direct Interaction of Target SNAREs with the Kv2.1 Channel
Journal of Biological Chemistry, 2003
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.
Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca2+ channels
The EMBO Journal, 1996
We have used an electrophysiological assay to investigate the functional interaction of syntaxin 1A and SNAP-25 with the class C, L-type, and the class B, N-type, voltage-sensitive calcium channels. Co-expression of syntaxin IA with the pore-forming subunits of the Land N-type channels in Xenopus oocytes generates a dramatic inhibition of inward currents (>60%) and modifies the rate of inactivation (X) and steadystate voltage depentdence of inactivation. Syntaxin 1-267, which lacks the transmembrane region (TMR), and syntaxin 2 do not modify channel properties, suggesting that the syntaxin 1A interaction site resides predominantly in the TMR. Co-expression of SNAP-25 significantly modifies the gating properties of Land N-type channels and displays modest inhibition of current amplitude. Syntaxin 1A and SNAP-25 combined restore the syntaxin-inhibited N-type inward current but not the reduced rate of inactivation. Hence, a distinct interaction of a putative syntaxin lA-SNAP-25 complex with the channel is apparent, consistent with the formation of a synaptosomal SNAP receptors (SNAREs) complex. The in vivo functional reconstitution: (i) establishes the proximity of the SNAREs to calcium channels; (ii) provides new insight into a potential regulatory role for the two SNAREs in controlling calcium influx through Nand L-type channels; and (iii) may suggest a pivotal role for calcium channels in the secretion process.
Molecular Pharmacology, 2006
We have hypothesized that the plasma membrane protein components of the exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex, syntaxin 1A and SNAP-25, distinctly regulate different voltagegated K ϩ (Kv) channels that are differentially distributed. Neuroendocrine islet cells (␣, , ␦) uniformly contain both syntaxin 1A and SNAP-25. However, using immunohistochemistry, we show that the different pancreatic islet cells contain distinct dominant Kv channels, including Kv2.1 in  cells and Kv2.2 in ␣ and ␦ cells, whose interactions with the SNARE proteins would, respectively regulate insulin, glucagon and somatostatin secretion. We therefore examined the regulation by syntaxin 1A and SNAP-25 of these two channels. We have shown that Kv2.1 interacts with syntaxin 1A and SNAP-25 and, based on studies in oocytes, suggested a model of two distinct modes of interaction of syntaxin 1A and the complex syntaxin 1A/SNAP-25 with the C terminus of the channel. Here, we characterized the interactions of syntaxin 1A and SNAP-25 with Kv2.2 which is highly homologous to Kv2.1, except for the C-terminus. Comparative two-electrode voltage clamp analysis in oocytes between Kv2.2 and Kv2.1 shows that Kv2.2 interacts only with syntaxin 1A and, in contrast to Kv2.1, it does not interact with the syntaxin 1A/SNAP-25 complex and hence is not sensitive to the assembly/disassembly state of the complex. The distinct regulation of these closely related channels by SNAREs may be attributed to differences in their C termini. Together with the differential distribution of these channels among islet cells, their distinct regulation suggests that the documented profound down-regulation of islet SNARE levels in diabetes could distort islet cell ion channels and secretory responses in different ways, ultimately contributing to the abnormal glucose homeostasis.
Journal of Physiology-Paris, 1994
Voltage-gated K-channels of the Shaker related subfamily have two subunits, membrane integrated ix-and peripheral [3-subunits. tx-Subunits may assemble as tetramers and form in in vitro expression systems functional K-channels. 13-Subunits cannot form channels by themselves. Like for tz-subunits, the rat nervous system apparently expresses a family of 13-subunit proteins. We have demonstrated that one rat K-channel [3-subunit, Kv~l, contains an inactivating domain. Upon association of cz-and Kv[31-subunits, delayed-rectifier type K-channels are converted to rapidly inactivating A-type K-channels. The [3-subunit inactivation domain acts via a ball and chain type mechanism previously proposed for N-type inactivation of ct-subunits. The association of ct-and [3-subunits endows the nervous system with an unprecedented flexibility and diversity of K-channels which may play an important role in the regulation of nervous excitability.
The Journal of biological chemistry, 2018
The voltage-gated potassium channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) w...
A BK (Slo1) channel journey from molecule to physiology
Channels, 2013
Abbreviations: BK, big conductance voltage and Ca 2+ -dependent potassium channel; Charybdotoxin, ChTx; Iberotoxin, IbTx; regulator of the conductance of K + channels, RCK; voltage sensing domain, VSD; leucine-rich repeat proteins, LRRC; nitric oxide, NO; cyclic guanosin mono-phosphate, cGMP www.landesbioscience.com Channels 443
Proceedings of the National Academy of Sciences, 1996
Voltage-gated K ؉ channels are complexes of membrane-bound, ion-conducting ␣ and cytoplasmic ancillary () subunits. The primary physiologic effect of coexpression of ␣ and  subunits is to increase the intrinsic rate of inactivation of the ␣ subunit. For one  subunit, Kv1.1, inactivation is enhanced through an N-type mechanism. A second  subunit, Kv1.2, has been shown to increase inactivation, but through a distinct mechanism. Here we show that the degree of enhancement of Kv1.2 inactivation is dependent on the amino acid composition in the pore mouth of the ␣ subunit and the concentration of extracellular K ؉ . Experimental conditions that promote C-type inactivation also enhance the stimulation of inactivation by Kv1.2, showing that this  subunit directly stimulates C-type inactivation. Chimeric constructs containing just the nonconserved Nterminal region of Kv1.2 fused with an ␣ subunit behave in a similar fashion to coexpressed Kv1.2 and ␣ subunit. This shows that it is the N-terminal domain of Kv1.2 that mediates the increase in C-type inactivation from the cytoplasmic side of the pore. We propose a model whereby the N terminus of Kv1.2 acts as a weakly binding ''ball'' domain that associates with the intracellular vestibule of the ␣ subunit to effect a conformational change leading to enhancement of C-type inactivation.