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• W2077749295 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 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 SNAP (kindly donated by E. Isacoff, Berkeley, CA) cDNAs were cloned in pGEMHE. BoNT/A and BoNT/C light chain cDNAs cloned in pCDNA3 have been described previously (22Ji J. Tsuk S. Salapatek A.M. Huang X. Chikvashvili D. Pasyk E.A. Kang Y. Sheu L. Tsushima R. Diamant N.E. Trimble W.S. Lotan I. Gaisano H.Y. J. Biol. Chem. 2002; 29: 20195-20204Abstract Full Text Full Text PDF Scopus (43) Google Scholar). Preparation of mRNAs was as described (23Levin G. Chikvashvili D. Singer-Lahat D. Peretz T. Thornhill W.B. Lotan I. J. Biol. Chem. 1996; 271: 29321-29328Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). DNAs of Kv2.1 fragments for production of GST fusion proteins were constructed as described before (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). Materials and enzymes for molecular biology were purchased from Roche Applied Science, Promega (Madison, WI), and MBI Fermentas (Vilnius, Lithuania). The degenerate phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) (including 5′ and 3′ end capping of 2- and 4-phosphorothioates, respectively, and a phosphorothioate at every third internal position to enhance nuclease resistance) was targeted against the following degenerate nucleotide sequence: Asyx, 5′-GA(AG)(CU)U(N)CA(UC)GA(CU)AUGUU(CU)AUGGA(CU)AUG-3′ (encoding amino acids ELHDMFMDM) corresponds to amino acids 211–219 within the H3 helix of human syntaxin 1A. The ODN is expected to hybridize to syntaxins from human, rodent, bovine, chick, aplysia, leech, and sea urchin homologs as well as to rat syntaxins 3 and 4 (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 sequence ODN, 5′-ATCGTTTGTGAGCGCTTCGGCATCGGT-3′, was used as a non-sense oligomer. Oocytes and Electrophysiological Recording—Xenopus laevis oocytes were prepared as described (24Dascal N. Lotan I. Karni E. Gigi A. J. Physiol. (Lond.). 1992; 450: 469-490Crossref Scopus (32) Google Scholar). Oocytes were injected with 15 ng/oocyte or with 0.05–0.25 ng/oocyte Kv2.1 mRNA for biochemical or electrophysiological studies, respectively. Syx mRNA was injected at 0.5 ng/oocyte for electrophysiological experiments and at 1.25 ng/oocyte for the biochemical experiments. SNAP mRNA was injected at 15 ng/oocyte for biochemical experiments and at 15 or 5 ng/oocyte for electrophysiological experiments. BoNT/A or BoNT/C mRNAs at 5–15 ng/oocyte were injected for both biochemical and electrophysiological experiments. AS-ODN at 0.05 ng/oocyte was injected 2 days before the electrophysiological assay, which was done 3 days after the mRNA injection. Two-electrode voltage clamp recordings were performed as described (25Levin G. Keren T. Peretz T. Chikvashvili D. Thornhill W.B. Lotan I. J. Biol. Chem. 1995; 270: 14611-14618Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To avoid possible errors introduced by series resistance, only current amplitudes up to 4 μA were recorded. Net current was obtained by subtracting the scaled leak current elicited by a voltage step from –80 to –90 mV. Oocytes with a leak current of more than 3 nA/1 mV were discarded. Experimental protocols and data analyses are described in the figure and table legends. Immunoprecipitation in Oocytes—Oocytes were subjected to immunoprecipitation as described (25Levin G. Keren T. Peretz T. Chikvashvili D. Thornhill W.B. Lotan I. J. Biol. Chem. 1995; 270: 14611-14618Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, immunoprecipitates from 1% Triton X-100 homogenates of either plasma membranes (PM) or internal fractions (IF) (separated mechanically, as described in Ref. 26Ivanina T. Perets T. Thornhill W.B. Levin G. Dascal N. Lotan I. Biochemistry. 1994; 33: 8786-8792Crossref PubMed Scopus (60) Google Scholar) were analyzed by SDS-PAGE (8% polyacrylamide). Digitized scans were derived by PhosphorImager (Amersham Biosciences), and relative intensities were quantified by ImageQuant. Immunoprecipitation and Immunoblotting in PC12 Cells—Immunoprecipitation (IP) has been described in detail 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 following, only the changes relevant for PC12 cells are described. Cells (5–15 × 10–6) were suspended in 5 ml of lysis buffer (20 mm Tris, pH 7.5, 5 mm EDTA, 100 mm EGTA, 100 mm NaCl, freshly prepared 1% CHAPS (Roche Applied Science) supplemented with protease inhibitor mixture (Roche Applied Science)), incubated for 1 h at 4 °C, and centrifuged for 10 min at 4 °C at 14,000 rpm. After overnight incubation of the supernatant with antibodies at 4 °C, protein A-Sepharose beads (Zymed Laboratories Inc., South San Francisco, CA) were added, and the bound proteins were thoroughly washed (in phosphate-buffered saline with only 0.1% Triton X-100), separated by SDS-PAGE, and subjected to Western blot analysis using the ECL detection system (Amersham Biosciences). Confocal Microscopy in PC12 Cells—One day after the PC12 cells were seeded on a plastic coverslip (Thermanox, Nunc, Naperville, IL), the cells were washed twice with TBS solution (10 mm Tris and 135 mm NaCl) containing 2 mm CaCl2, fixated for 30 min in 4% paraformaldehyde, washed, permeabilized with 0.01% Triton for 10 min, and washed twice with TBS buffer containing 2 mm CaCl2. Nonspecific sites were blocked with donkey immunoglobulin G (IgG, whole molecule, Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. Each coverslip was incubated for 2 h with mouse antibodies (Abs) against syntaxin (1:2000; Sigma) and rabbit Abs against Kv1.1 (1:5; Alomone Labs). Residual Abs were removed by washing 3 times, each time for 5 min, with TBS containing 2 mm CaCl2 and 2% bovine serum albumin. This was followed by incubation for 30 min with the secondary Abs Cy3 donkey anti-rabbit IgG (1:400, The Jackson Laboratory) or Alexa Fluor 488 goat anti-mouse IgG (1:500, Molecular Probes, Eugene, OR). Free secondary Abs were then washed three times with TBS containing 2 mm CaCl2 and 2% bovine serum albumin, and the coverslips were mounted on a glass slide. The fluorescent labeling was examined by the use of a confocal laser scanning microscope (LSM 410 invert, Zeiss, Oberkochen, Germany) equipped with a 25-milliwatt krypton-argon laser (488 and 568 maximum lines). A 40 NA/1.2 C-apochromat water-immersion lens (Axiovert 135 M, Zeiss) was used for imaging. Cy3- or Alexa-conjugated Abs were excited at 488 or 568 nm, respectively, and the emitted light was collected using BP 515-540 or LP 590 filters, respectively. The fluorescent signals were analyzed using the microscope manufacturer's program (LSM, Zeiss). In Vitro Binding of GST Fusion Proteins with Syx and SNAP—The fusion proteins were synthesized and reacted with Syx 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, 27Jing J. Chikvashvili D. Singer-Lahat D. Thornhill W.B. Reuveny E. Lotan I. EMBO J. 1999; 18: 1245-1256Crossref PubMed Google Scholar). Briefly, purified GST fusion proteins (150 pmol) immobilized on glutathione-Sepharose beads were incubated either with 5 μl of the lysate containing 35S-labeled syntaxin or SNAP (translated on the template of in vitro synthesized mRNAs using a translation rabbit reticulocyte lysate kit (Promega)) or with 200 pmol of recombinant syntaxin peptide prepared from a GST fusion construct (amino acids 1–264) cleaved by thrombin, as described, in 1 ml of phosphate-buffered saline with 0.1% Triton X-100 or 0.5% CHAPS. The GST fusion proteins were eluted with 20 mm reduced glutathione and then subjected to SDS-PAGE (12% polyacrylamide). Statistical Analysis—Data are presented as means ± S.E. The statistical significance of differences between the two groups was calculated by the use of independent sample t test procedures assuming unequal variance (Mann-Whitney's rank-sum test). One-way analysis of variance was used to estimate the statistical differences in experiments comparing several groups. The Voltage-gated K + Channel Kv2.1 Interacts Physically with Syntaxin 1A and SNAP-25 in PC12 Cells—In view of the wide distribution of the Kv2.1 channel in neuroendocrine cells, we were interested in finding out whether it interacts in PC12 cells with syntaxin 1A (Syx) and SNAP-25 (SNAP), which are the target SNARE (t-SNARE) partners of the exocytotic machinery (Fig. 1A). By using an antibody against Kv2.1, we found that both Syx and SNAP co-precipitate with the Kv2.1 protein. The co-precipitation of both Syx and SNAP could be reduced substantially by preincubation of the antibodies with the peptide against which the antibodies were raised. To verify the specificity of the co-precipitation, we performed the reciprocal experiments, in which Kv2.1 was co-precipitated with Syx and SNAP, using antibodies against Syx or SNAP. Kv2.1 Co-localizes with Syx in PC12 Cells—The co-immunoprecipitation results indicated an interaction between the channel protein and Syx (the interaction with SNAP was suggested to be prevalently via Syx, see below). To evaluate the extent of the interaction, we carried out an immunocytochemical study in PC12 cells. A monoclonal antibody against syntaxin 1A and a polyclonal antibody against Kv2.1 were used for double staining of the corresponding proteins. The confocal fluorescence microscopic images are shown in Fig. 1B. Kv2.1 was found to be distributed both at perinuclear regions and at plasma membranes, whereas Syx was mostly at the plasma membranes. The overlay image (Fig. 1B, right panel) shows co-localization of the channel at the plasma membranes with Syx. Syx and SNAP Directly Bind Cytosolic Domains of Kv2.1— Previously, we have shown in an in vitro binding assay that Syx and SNAP bind 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, 28Leung Y.M. Kang Y. Gao X. Xia F. Xie H. Sheu L. Tsuk S. Lotan I. Tsushima R.G. Gaisano H.Y. J. Biol. Chem. 2003; 278: 17532-17538Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In this study we carried out a more comprehensive in vitro binding assay to substantiate the physical interaction of the channel with these proteins. First, immobilized glutathione S-transferase (GST) fusion proteins corresponding to the major cytoplasmic parts of the channel: the N terminus (amino acids 1–182; N), and the proximal and distal halves of the C terminus (amino acids 411–632 and 633–853; C1 and C2, respectively) (Fig. 2A). These fusion proteins and GST itself were incubated in the presence of 0.1% Triton X-100 with 35S-labeled full-length Syx or SNAP synthesized in reticulocyte lysate (Fig. 2, B and C). Both Syx and SNAP bound to the channel, Syx bound preferentially to the C1 domain, whereas SNAP bound to the N- and the C-terminal domains with some preference for C2. Notably, the binding of Syx was stronger by about 1 order of magnitude than that of SNAP which was rather weak. The binding of Syx to C1 was further evaluated by the use of the recombinant cytoplasmic part of syntaxin (corresponding to amino acids 4–264) cleaved by thrombin from its corresponding GST fusion protein in two different detergents (0.1% Triton X-100 and 0.5% CHAPS) (Fig. 2D). By using different concentrations of Syx, we estimated that under our binding conditions, the binding was half-maximal at ∼0.3–0.4 μm Syx and that ∼2.2 pmol of Syx were bound per 6 pmol of C1, at a saturating concentration of Syx (Fig. 2E). We concluded that the binding of Syx to the channel was devoid of detergent artifacts, strong, targeted to a defined region on the channel (C1), dose-dependent, and saturable. However, the binding of SNAP-25 was less conclusive, being weak and not defined to a distinct region on the channel. Syx and SNAP Affect the Voltage Dependence of Inactivation of Kv2.1 Expressed in Xenopus Oocytes—To study the modulation of the Kv2.1 currents by Syx and SNAP, we used Xenopus oocytes. Oocytes injected with Kv2.1 mRNA exhibited outward K+ currents, which inactivated slowly upon 25-s membrane depolarizations to different voltages (Fig. 3A, upper left panel). Co-injection of Syx or SNAP mRNAs affected the Kv2.1 current traces (Fig. 3A). The effects of Syx and SNAP on the voltage dependence of the Kv2.1 current inactivation were studied using depolarizing prepulses. We started with prepulses of 25-s duration, which induces full inactivation, but for practical reasons subsequent experiments were done with 5-s prepulses, which yielded qualitatively similar results. Co-injection of Syx mRNA with Kv2.1 resulted in a negative shift of the half-inactivation voltage (Vi ½) by ∼20 mV, with no effect on the residual current fraction (RCF), defined as the fraction of current remaining after a 5-s prepulse to +15 mV (Fig. 3B and Table I).Table IEffect of syntaxin 1A (Syx), SNAP-25 (SNAP), and the combination of Syx and SNAP on the voltage dependence of inactivation of Kv2.1Vi1/2aNormalized RCFmV%Effect of SyxKv2.1 (Control)-17.32 ± 0.9-5.08 ± 0.83100+ Syx-37.43 ± 6.15ap values denoting statistically significant differences from control were obtained by t test in two group experiments and by one-way analysis of variance in multigroup experiments. p < 0.001.-7.38 ± 1.499.28 ± 2.09Effect of SNAPKv2.1 (Control)-17.72 ± 0.79-6.57 ± 1.36100+ SNAP-8.95 ± 2.49bp < 0.05.-8.33 ± 1.45155.26 ± 8.06ap values denoting statistically significant differences from control were obtained by t test in two group experiments and by one-way analysis of variance in multigroup experiments. p < 0.001.Effect of the combination of Syx (0.5) and SNAPKv2.1 (Control)-17.86 ± 1.54-4.61 ± 0.28100+ Syx + SNAP-15.14 ± 1.96-6.94 ± 0.99155.09 ± 5.63ap values denoting statistically significant differences from control were obtained by t test in two group experiments and by one-way analysis of variance in multigroup experiments. p < 0.001.a p values denoting statistically significant differences from control were obtained by t test in two group experiments and by one-way analysis of variance in multigroup experiments. p < 0.001.b p < 0.05. Open table in a new tab Co-injection of SNAP with Kv2.1 mRNAs shifted Vi ½ to membrane potentials that were more depolarized by ∼9.5 mV and enhanced the RCF by ∼55% (Fig. 3C and Table I). Neither Syx nor SNAP had a significant effect on the slope factor (a) of the inactivation curve (Table I). Effect of a Combination of Syx and SNAP on Kv2.1 Voltage Dependence of Inactivation—The target membrane SNAREs syntaxin 1A and SNAP-25 have been shown to form a complex on presynaptic plasma membranes (29Zhang F. Chen Y. Kweon D.H. Kim C.S. Shin Y.K. J. Biol. Chem. 2002; 277: 24294-24298Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 30Xiao W. Poirier M.A. Bennett M.K. Shin Y.K. Nat. Struct. Biol. 2001; 8: 308-311Crossref PubMed Scopus (91) Google Scholar, 31Zhou Q. Xiao J. Liu Y. J. Neurosci. Res. 2000; 61: 321-328Crossref PubMed Scopus (41) Google Scholar, 32Poirier M.A. Xiao W. Macosko J.C. Chan C. Shin Y.K. Bennett M.K. Nat. Struct. Biol. 1998; 5: 765-769Crossref PubMed Scopus (419) Google Scholar). We therefore assessed the functional interaction of a combination of Syx and SNAP with Kv2.1. Concomitant expression of Syx and SNAP resulted in elimination of the leftward shift of Vi ½ by Syx. Rather, the combination of Syx and SNAP yielded effects similar to those observed with SNAP alone, namely a rightward shift of Vi ½ and an increase in RCF (Fig. 3D and Table I). The rightward shift in Vi ½ was smaller than in the case of SNAP alone. SNAP Affects the Kinetics of Kv2.1 Channel Inactivation— The kinetics of inactivation was characterized by the use of two experimental protocols. At potentials above the level where current activation became significant (above 10 mV), inactivation was measured as the rate of current decay during 25-s depolarizing pulses (Fig. 4A). At –10 mV, the onset of inactivation was determined by imposing prepulses of different duration to –10 mV, followed by a fixed test pulse to elicit the remaining current (Fig. 4B). In both protocols, the membrane was maintained at a negative potential between successive trials to allow complete recovery from inactivation before each measurement. The rate of inactivation was estimated by the inactivation time constant (τ) derived from a single exponential decay fit. At all voltages tested, increases of 85–90% in τ were observed in the presence of SNAP. Syx did not affect τ at any of the voltages tested. Recovery from inactivation was studied by a classic two-pulse procedure (Fig. 4C). Following a 25-s depolarizing test pulse to inactivate the current, recovery was assessed by application of a test pulse at a variable interval. The rate of recovery was estimated by the fast and slow time constants derived from a two-exponential rising fit. Neither Syx nor SNAP had any significant effect on the rate of recovery (Fig. 4C, legend). Syx Affects the Voltage Dependence of Activation of Kv2.1—As shown in Fig. 5, Syx affected the activation of a fraction of the channels. The activation curve of Kv2.1 co-expressed with Syx fitted well to a two-component Boltzmann function: one component resembled the conductance (G)-voltage curve of Kv2.1 expressed alone (with the same half-activation voltage (Va ½) and slope factor (a)) and the other component had a negative shift of ∼55 mV in Va ½ and a much smaller slope factor (1 compared with 16, indicating much steeper slope) (Fig. 5A and Table II). The two components probably represent two distinct fractions of channels, the one not affected and the other affected by Syx. The channel activation was not affected at all by SNAP (Fig. 5B and Table II). The combination of SNAP and Syx completely abolished the effect of Syx on channel activation (Fig. 5C and Table II). The maximal channel conductance (G max) was not affected significantly under any of the above conditions (not shown).Table IIEffect of Syx, SNAP, and the combination of Syx and SNAP on the activation of Kv2.1Va 1/2Ia IVa 1/2IIaIImVmVEffect of SyxKv2.19.36 ± 3.3716.87 ± 0.72+ Syx7.73 ± 0.9918.62 ± 0.91-46.81 ± 2.110.92 ± 1.098Effect of SNAPKv2.110.65 ± 0.8613.35 ± 0.69+ SNAP8.91 ± 0.6513.88 ± 0.65Effect of the combination of Syx and SNAPKv2.112.88 ± 1.9512.77 ± 0.77Syx + SNAP13.40 ± 0.6613.35 ± 0.69 Open table in a new tab Association of Syx and SNAP with the Kv2.1 in Oocytes Is Interdependent—Concomitantly with the functional experiments, we examined the physical interactions of Syx and SNAP with the Kv2.1 channel in the oocyte PM (manually dissected (26Ivanina T. Perets T. Thornhill W.B. Levin G. Dascal N. Lotan I. Biochemistry. 1994; 33: 8786-8792Crossref PubMed Scopus (60) Google Scholar)) and IF comprising the rest of the cell. Reciprocal co-immunoprecipitation analysis in the PM of oocytes from a single batch (Fig. 6A), using antibodies against Kv2.1, Syx, and SNAP, showed strong Syx binding and weak SNAP binding to the channel when Syx and SNAP were each expressed on their own with the channel. Their combined expression resulted in significant reduction in the binding of Syx to the channel (a decrease of ∼2-fold, as indicated by intensity ratios shown in the bar diagrams below the corresponding lanes in the middle panels of Fig. 6A). At the same time, the binding of SNAP to the channel was enhanced in the presence of Syx." @default.
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• W2077749295 date "2003-09-01" @default.
• W2077749295 modified "2023-09-27" @default.
• W2077749295 title "Direct Interaction of Target SNAREs with the Kv2.1 Channel" @default.
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• W2077749295 doi "https://doi.org/10.1074/jbc.m304943200" @default.
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