Direct Interaction of Target SNAREs with the Kv2.1 Channel

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 β-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. 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 β-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) 1The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SNAP, SNAP-25; Syx, syntaxin; t-SNARE, target SNARE; RCF, residual current fraction; PM, plasma membrane; IF, internal fraction; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TBS, Tris-buffered saline; Abs, antibodies; AS-ODNs, antisense oligodeoxynucleotides; BoNT/C, Botulinum neurotoxin C. proteins syntaxin, SNAP-25, and VAMP are crucial factors in processes of transmitter and hormone release (1Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar). 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 Ca2+ channels) (1Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar, 2Bajjalieh S.M. Scheller R.H. J. Biol. Chem. 1995; 270: 1971-1974Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 3Linial M. Parnas D. Biochim. Biophys. Acta. 1996; 1286: 117-152Crossref PubMed Scopus (29) Google Scholar, 4Bennett M.K. Curr. Opin. Cell Biol. 1995; 7: 581-586Crossref PubMed Scopus (108) Google Scholar, 5Sheng Z.H. Rettig J. Cook T. Catterall W.A. Nature. 1996; 379: 451-454Crossref PubMed Scopus (314) Google Scholar). We suggested previously (6Fili O. Michaelevski I. Bledi Y. Chikvashvili D. Singer-Lahat D. Boshwitz H. Linial M. Lotan I. J. Neurosci. 2001; 21: 1964-1974Crossref PubMed Google Scholar) that SNARE proteins interact with a member of the Kv1 subfamily of the voltage-gated K+ (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 (7Stuhmer W. Ruppersberg J.P. Schroter K.H. Sakmann B. Stocker M. Giese K.P. Perschke A. Baumann A. Pongs O. EMBO J. 1989; 8: 3235-3244Crossref PubMed Scopus (616) Google Scholar) 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 Kvβ 1.1 subunits (8Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Crossref PubMed Scopus (748) Google Scholar) 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 βγ subunits was found to be a requirement for this interaction (9Michaelevski I. Chikvashvili D. Tsuk S. Fili O. Lohse M.J. Singer-Lahat D. Lotan I. J. Biol. Chem. 2002; 277: 34909-34917Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). These characteristics of the interaction of presynaptic Kv channels with syntaxin 1A are reminiscent of the interaction of the presynaptic N-type voltage-gated Ca2+ channels (10Jarvis S.E. Magga J.M. Beedle A.M. Braun J.E. Zamponi G.W. J. Biol. Chem. 2000; 275: 6388-6394Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 11Jarvis S.E. Zamponi G.W. J. Neurosci. 2001; 21: 2939-2948Crossref PubMed Google Scholar, 12Bezprozvanny I. Zhong P.Y. Scheller R.H. Tsien R.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13943-13948Crossref PubMed Scopus (90) Google Scholar). Recently, we focused on endocrine cells, and we showed that Kv1.1 interacts with SNAP-25 in islet β-cells (13MacDonald P.E. Ha X.F. Wang J. Smukler S.R. Sun A.M. Gaisano H.Y. Salapatek A.M. Backx P.H. Wheeler M.B. Mol. Endocrinol. 2001; 15: 1423-1435Crossref PubMed Scopus (163) Google Scholar) and that in these cells also the Kv2.1 channel, a member of the Kv2 subfamily of Kv channels, is modulated by SNAP-25 and syntaxin 1A (14MacDonald P.E. Wang G. Tsuk S. Dodo C. Kang Y. Tang L. Wheeler M.B. Cattral M.S. Lakey J.R. Salapatek A.M. Lotan I. Gaisano H.Y. Mol. Endocrinol. 2002; 16: 2452-2461Crossref PubMed Scopus (72) Google Scholar). Interestingly, in β-cells the interaction of exocytotic proteins with the L-type voltage-gated Ca2+ channels has also been described (15Wiser O. Trus M. Hernandez A. Renstrom E. Barg S. Rorsman P. Atlas D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 248-253Crossref PubMed Scopus (246) Google Scholar). Kv2.1, a slow-inactivating delayed rectifier channel (16Frech G.C. VanDongen A.M. Schuster G. Brown A.M. Joho R.H. Nature. 1989; 340: 642-645Crossref PubMed Scopus (358) Google Scholar), although being widely distributed in the central nervous system, mainly on postsynaptic structures (17Trimmer J.S. FEBS Lett. 1993; 324: 205-210Crossref PubMed Scopus (49) Google Scholar, 18Lim S.T. Antonucci D.E. Scannevin R.H. Trimmer J.S. Neuron. 2000; 25: 385-397Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), is the prevalent Kv channel in neuroendocrine and endocrine cells (19Barry D.M. Trimmer J.S. Merlie J.P. Nerbonne J.M. Circ. Res. 1995; 77: 361-369Crossref PubMed Google Scholar, 20Sharma N. D'Arcangelo G. Kleinlaus A. Halegoua S. Trimmer J.S. J. Cell Biol. 1993; 123: 1835-1843Crossref PubMed Scopus (63) Google Scholar). It was shown that the Kv2.1 current repolarizes β-cell action potentials during a glucose stimulus to limit Ca2+ entry and insulin secretion (21MacDonald P.E. Sewing S. Wang J. Joseph J.W. Smukler S.R. Sakellaropoulos G. Saleh M.C. Chan C.B. Tsushima R.G. Salapatek A.M. Wheeler M.B. J. Biol. Chem. 2002; 277: 44938-44945Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In the present work we show that the Kv2.1 channel interacts directly with the two plasma membrane-associated SNARE proteins, syntaxin 1A and SNAP-25 in neuroendocrine cells. We further show that these interactions have functional implications observed in Xenopus oocytes. Both activation and inactivation of the channel are affected, depending on the assembly/disassembly of the binary complex SNAP-25/syntaxin 1A. On the basis of our results, we suggest a physiological relevance for these interactions in the release processes described in neuroendocrine cells. Constructs and Antibodies—The primary antibodies used were Kv2.1-C terminus (Alomone Labs, Jerusalem, Israel), polyclonal syntaxin 1A (Alomone Labs), monoclonal anti-HPC-1 (Sigma), and monoclonal SNAP-25 (Signal Transduction, Lexington, KY). Kv2.1 (kindly donated by O. Pongs, Zentrum fur Molecular Neurobiologie, University of Hamburg, Hamburg, Germany), Syx, and (kindly donated by E. were in and in have been described previously J. Tsuk S. Salapatek A.M. Chikvashvili D. Kang Y. L. Tsushima Lotan I. Gaisano H.Y. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar). of was as described G. Chikvashvili D. Singer-Lahat D. T. Lotan I. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of Kv2.1 for of proteins were as described (14MacDonald P.E. Wang G. Tsuk S. Dodo C. Kang Y. Tang L. Wheeler M.B. Cattral M.S. Lakey J.R. Salapatek A.M. Lotan I. Gaisano H.Y. Mol. Endocrinol. 2002; 16: 2452-2461Crossref PubMed Scopus (72) Google Scholar). and for were from and The antisense and of and and a internal to was the to the of syntaxin 1A. The is to to from and as as to and (6Fili O. Michaelevski I. Bledi Y. Chikvashvili D. Singer-Lahat D. Boshwitz H. Linial M. Lotan I. J. Neurosci. 2001; 21: 1964-1974Crossref PubMed Google Scholar). The was used as a and oocytes were as described N. Lotan I. E. A. J. Scopus Google Scholar). were with or with Kv2.1 for or was for and for the was for and or for or were for both and was the was the voltage clamp were as described G. T. T. Chikvashvili D. Lotan I. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). by current to were current was by the current by a voltage from to with a current of were and analyses are described in the and in were to as described G. T. T. Chikvashvili D. Lotan I. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). from of plasma membranes or internal as described in T. T. G. N. Lotan I. 1994; PubMed Scopus Google Scholar) were by were by and were by and in has been described in for synaptosomes (6Fili O. Michaelevski I. Bledi Y. Chikvashvili D. Singer-Lahat D. Boshwitz H. Linial M. Lotan I. J. Neurosci. 2001; 21: 1964-1974Crossref PubMed Google Scholar). In the the for cells are were in of with for and for of the with antibodies protein were and the proteins were with by and to using the in the cells were on a the cells were with and for in with for and with were with G for was for with antibodies syntaxin and Kv1.1 were by for with and This was by for with the The or Molecular were with and and the were on a The was by the of a with a and was used for or were or and the was using or The were using the In of with and proteins were and with as described (6Fili O. Michaelevski I. Bledi Y. Chikvashvili D. Singer-Lahat D. Boshwitz H. Linial M. Lotan I. J. Neurosci. 2001; 21: 1964-1974Crossref PubMed Google Scholar, J. Chikvashvili D. Singer-Lahat D. E. Lotan I. EMBO J. 1999; PubMed Google Scholar). proteins on were with of the syntaxin or on the of in vitro using a or with of syntaxin from a by as in of with or The proteins were with glutathione and to are as S.E. The of between the two was by the of of was used to the in The Kv2.1 with Syntaxin 1A and SNAP-25 in of the wide of the Kv2.1 channel in neuroendocrine cells, we were in interacts in cells with syntaxin 1A and SNAP-25 are the target SNARE of the exocytotic machinery using Kv2.1, we found that both and with the Kv2.1 The of both and be by of the antibodies with the the antibodies were the of the we the in Kv2.1 was with and SNAP, using antibodies or Kv2.1 with in co-immunoprecipitation results interaction between the channel protein and interaction with was suggested to be Syx, the extent of the we study in cells. monoclonal syntaxin 1A and a polyclonal Kv2.1 were used for of the The are shown in Kv2.1 was found to be distributed both and plasma was the plasma The of the channel the plasma membranes with and of we have shown in in vitro binding that and to the Kv2.1 channel (14MacDonald P.E. Wang G. Tsuk S. Dodo C. Kang Y. Tang L. Wheeler M.B. Cattral M.S. Lakey J.R. Salapatek A.M. Lotan I. Gaisano H.Y. Mol. Endocrinol. 2002; 16: 2452-2461Crossref PubMed Scopus (72) Google Scholar, Kang Y. F. H. L. Tsuk S. Lotan I. Tsushima R.G. Gaisano H.Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this study we a in vitro binding to the physical interaction of the channel with these glutathione proteins to the of the the terminus and the and of the terminus and and These proteins and were in the of with or in and Both and to the channel, to the to the and the with some for the binding of was by of that of was The binding of to was further by the of the of syntaxin to by from its protein in two and using of Syx, we that our binding the binding was and that of were of a of We that the binding of to the channel was of to a on the channel and the binding of SNAP-25 was being and to a on the and the of of Kv2.1 in Xenopus study the of the Kv2.1 by and SNAP, we used Xenopus oocytes. with Kv2.1 K+ to of or the Kv2.1 current The effects of and on the voltage of the Kv2.1 current inactivation were using We with of for were with of with Kv2.1 in a of the voltage by with no effect on the residual current as the of current a to and of syntaxin 1A SNAP-25 and the of and on the voltage of inactivation of of from were by in two and by of in of from were by in two and by of in of the of and from were by in two and by of in from were by in two and by of in in a of with Kv2.1 shifted to potentials that were depolarized by and the by and a effect on the factor of the inactivation of a of and on Kv2.1 of target syntaxin 1A and SNAP-25 have been shown to a complex on presynaptic plasma membranes F. Y. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, W. M.K. Biol. 2001; 8: PubMed Scopus Google Scholar, J. Y. J. Neurosci. Res. 2000; PubMed Scopus Google Scholar, W. Chan C. M.K. Biol. PubMed Scopus Google Scholar). We the functional interaction of a of and with of and in elimination of the of by the of and effects to observed with a of and in and The in was in the of the of Kv2.1 The of inactivation was characterized by the of two potentials the current activation inactivation was as the rate of current during the of inactivation was by of to by a to the current In both the was a between to from inactivation The rate of inactivation was by the inactivation from a of in were observed in the of of the from inactivation was by a a to the was by of a a The rate of was by the fast and from a effect on the rate of the of of shown in the activation of a of the The activation of Kv2.1 with to a the of Kv2.1 alone the voltage and factor and the a of in and a factor with and The two components probably two of channels, the and the by The channel activation was by and The of and the effect of on channel activation and The channel was of the of Syx, SNAP, and the of and on the activation of of of of the of and in a of and with the Kv2.1 in with the functional we the physical interactions of and with the Kv2.1 channel in the T. T. G. N. Lotan I. 1994; PubMed Scopus Google and the of the co-immunoprecipitation in the of oocytes from a using antibodies Kv2.1, Syx, and SNAP, showed binding and binding to the channel and were on with the combined in in the binding of to the channel of as by shown in the the in the of the the binding of to the channel was in the of In binding of to the channel was in oocytes the of Botulinum neurotoxin the of Sci. 2002; Full Text Full Text PDF PubMed Scopus Google It be that a of the was the of two It is from that the of channel protein in the in the of oocytes that the channel alone was in the of oocytes. This that of the channel or its in the was by or This was observed in two of a of results of the and in oocytes were with the binds strongly to the channel and shifts both and of the to the and binds weakly to the channel and a of and of The combined of and the binding of and enhances the binding of a the of of the inactivation that is by the effects of and is toward the and the of the of activation by is Moreover, the effect of on is The a that is on the that the binding of is of may be that of its binding to the channel and its effects on the are to the of and be of the This was as described of the of the that the effect of is by Syx, we the using two we used is of the the and of and was previously shown to be to (6Fili O. Michaelevski I. Bledi Y. Chikvashvili D. Singer-Lahat D. Boshwitz H. Linial M. Lotan I. J. Neurosci. 2001; 21: 1964-1974Crossref PubMed Google Scholar). of was used as of the effects of and and showed that of oocytes and Kv2.1 the effect of on and on The effect of the was no effect the channel was alone of of on the voltage of inactivation of by were by in and by of in were by in and by of in by were by in and by of in were by in and by of in were by in and by of in in a The we used to was to the of Botulinum neurotoxin Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of this the antisense is that the antisense the and the the the protein we the of on the effect and showed that its with the of both the and we the effect of in the of shown in and of with the effect of on and the of the channel was no effect on the or on the These results the that the of to and to the rate of of inactivation the of Syx, as The of to to the is by the of Syx, the of that the are to this or this effect of Kv2.1 with and in with in Xenopus we showed that the Kv2.1 channel, is the delayed rectifier K+ channel of islet β-cells and and insulin interacts with β-cell and (14MacDonald P.E. Wang G. Tsuk S. Dodo C. Kang Y. Tang L. Wheeler M.B. Cattral M.S. Lakey J.R. Salapatek A.M. Lotan I. Gaisano H.Y. Mol. Endocrinol. 2002; 16: 2452-2461Crossref PubMed Scopus (72) Google Scholar). In the present study we for the in neuroendocrine cells the existence of a physical interaction of the Kv2.1 protein with Syx, in the of a complex also Furthermore, we the physical and functional interactions of Kv2.1 with these two and in pertaining to the assembly/disassembly of the a t-SNARE F. Y. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, W. M.K. Biol. 2001; 8: PubMed Scopus Google Scholar, J. Y. J. Neurosci. Res. 2000; PubMed Scopus Google Scholar, W. Chan C. M.K. Biol. PubMed Scopus Google Scholar), using Xenopus oocytes and in vitro binding using co-immunoprecipitation and immunocytochemistry analyses in cells, we the of Kv2.1 with and and the of the channel with plasma Furthermore, we a and physical interaction of with the of the Kv2.1 the interaction of SNAP-25 with the channel was and was to a these results to suggest that in neuroendocrine cells the channel interacts directly with and that the interaction with is This was further in oocytes by concomitant and We showed that in plasma membranes the physical interaction of with the channel is the interaction of with the channel is in the of is for the functional effect of Furthermore, we characterized gating activation and of the channel in the of that are on the assembly status of the t-SNARE complex On the basis of these we suggest the of dynamic coupling of a voltage-gated K+ channel to the exocytotic of neuroendocrine cells that is on the assembly/disassembly of the t-SNARE complex the of the SNARE and that this coupling to the exocytosis of these cells. of of Kv2.1 with and the we a to for the results of the and the in plasma membranes of oocytes Syx, or SNAP, or the of with and and the we by and On the basis of the results, we a that the interactions of Kv2.1 with and In this the channel has two binding for and the for the complex to the of these the channel in of gating In in the of or SNAP, of the is In in the of and of SNAP, binds to is on the of the terminus of In in the of both and SNAP, to the complex that binds to and In the we present the basis for the Our that and are binding was on the functional effects by alone and by and in and that functional depending on is also to the and is for of in was from the that both the physical and the functional interactions of with the channel were on the binding of alone to the channel, both in vitro and in plasma membranes of oocytes was was in oocytes in the of and on the of formation of the effect of alone was of and the that in and to of Kv2.1 and channel gating The of in to of by was on the basis of two that the binding of was in the of was of the of the effects of both on inactivation and on activation of the channel, observed in the of and are with of from the The of for is probably the of for as the of that with Kv2.1 in the plasma of oocytes was in the of The characteristics of the channel in of the are by the of the channel from of the activation and inactivation the The of activation and inactivation that a of these channels are The characteristics of are from the in the of alone and and and The activation is that the of channels and a of the channels, as was the in binding of to the channel shifts from to and the of the channel is to hyperpolarized potentials as The characteristics of are from the in the combined of and and and and in the is shifted to depolarized potentials, to in the channel and the these potentials is These from the of inactivation to and the of the are by the binding of complex to the shifts of inactivation and activation that are in are to the concomitant of from of the of Kv2.1 with and the we to the that the Kv2.1 channels interact with and in neuroendocrine cells with the of the channels in that the interaction of the t-SNARE complex with the channels the of the exocytotic SNARE we to a that may to the of exocytosis in neuroendocrine cells for D. Res. Res. 2000; PubMed Scopus Google Scholar). On the basis of the of to the Kv2.1 channels and its is to that potentials the channels are associated with and in B. to the of this that hyperpolarized potentials to the are to of L-type Ca2+ channels and the consequent Ca2+ is associated with the assembly of SNARE of for release. these with the complexes, the Kv2.1 channels to C. The in this that is shifted to depolarized potentials of and the of Ca2+ the that have exocytotic In we suggest a of a Kv channel that is sensitive to the assembly status of the t-SNARE complex and to secretion in neuroendocrine cells. physiological of our is with the role of in syntaxin 1A, in of channel for the and channels to and of J. J.P. 2001; PubMed Scopus Google Scholar), and also suggested by for the Kv1.1 channel (6Fili O. Michaelevski I. Bledi Y. Chikvashvili D. Singer-Lahat D. Boshwitz H. Linial M. Lotan I. J. Neurosci. 2001; 21: 1964-1974Crossref PubMed Google Scholar). In the of Kv2.1 we that in the of or the of channel protein in the plasma is this effect of of Kv2.1 channel in the plasma by is opposite to the of the We for and for with the

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