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Departments of Physiology (P.E.M., G.W., Y.K., L.T., M.B.W., A.M.F.S., H.Y.G.), Medicine (G.W., Y.K., L.T., M.B.W., A.M.F.S., H.Y.G.), and Surgery (M.S.C.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; Department of Physiology and Pharmacology (S.T., C.D., I.L.), Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel 69978; and Department of Surgery (J.R.T.L.), University of Alberta, Edmonton, Canada T6N 2N8
Address all correspondence and requests for reprints to: Herbert Y. Gaisano and Anne Marie F. Salapatek, University of Toronto, 1 Kings College Circle, Room 7226, Toronto, Ontario, Canada, M5S 1A8. E-mail: herbert.gaisano{at}utoronto.ca and annemarie.salapatek{at}utoronto.ca.
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INTRODUCTION |
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SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are well-established mediators of membrane fusion, including exocytosis (13). SNARE proteins have also been found to bind and modulate membrane ion channels, particularly Ca2+ channels (14). In ß-cells, we and others (15, 16, 17, 18, 19, 20) have shown that SNARE proteins mediate insulin exocytosis. SNARE proteins are known to directly bind and modulate Ca2+ channels (14, 21, 22), are colocalized to sites of insulin granule exocytosis (23), and we have recently reported that multiple domains within SNAP-25 can modulate L-type Ca2+ channels in ß-cells (24). In brain synaptosomes, we recently reported that the SNARE proteins syntaxin 1A, SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptotagmin bind the Kv1.1/Kvß channel complex (25). In that study, syntaxin had effects on neuronal Kv1.1/Kvß1.1 channels expressed in Xenopus oocytes: a biphasic effect on current amplitudes, first increasing and then decreasing currents, and enhancement of fast inactivation. Taken together, these studies indicate that SNARE proteins mediate a complex regulation of neurotransmission and neuroendocrine secretion, including islet insulin exoctyosis, through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.
Here, we examined whether SNARE proteins could also regulate pancreatic islet Kv channels, specifically Kv2.1. We found that plasma membrane-bound SNAP-25 is capable of binding Kv2.1 protein and reducing this channel’s activity in rat pancreatic islet ß-cells and in a heterologous expression system. This effect is mediated at the Kv2.1 N terminus. Our further demonstration of the expression of Kv2.1 in human islets underscores the importance of this channel in regulating insulin secretion and implicates a role for this channel in human health and in diabetes.
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), which migrated to a size similar to Kv2.1 protein found in rat islets, rat brain, and MIN6 insulinoma cells. We also confirm the abundance of Kv2.1 protein in rat islets and insulin-secreting cell lines (10, 26). Although human islets appear to express Kv2.1 less abundantly than the other tissues (a result consistent within two separate human islet protein samples), it must be noted that the primary antibody was raised against a rodent Kv2.1 epitope (88.2% homologous to the human protein sequence) and differences in species selectivity cannot be ruled out. Previously, using specific oligonucleotide primers, we have identified Kv2.1 mRNA transcripts that were easily amplified from human islet total RNA (unpublished data). Additionally, and in agreement with previous results (20), the SNARE proteins SNAP-25 and syntaxin 1A were detected in human islet protein lysates (Fig. 1). Syntaxin 1A expression was demonstrated using antibodies raised against both human and rat syntaxin 1A epitopes.
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), indicating that the SNARE proteins SNAP-25 and syntaxin 1A physically interact with the Kv2.1 protein.
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) confirm direct binding between SNAP-25 and both the GST-Kv2.1N and GST-Kv2.1C fragments, but not GST itself.
SNAP-25 Blocks Kv2.1 Currents: An Effect Mediated at the Channel N Terminus
To determine whether SNAP-25 can modulate currents mediated by Kv2.1, we dialyzed a recombinant GST-SNAP-25 fusion protein (10-8 M) or GST alone (10-8 M) into HEK-293 cells expressing Kv2.1. Currents in control GST dialyzed cells were large and noninactivating over 250 msec (Fig. 3A), similar to Kv2.1 currents described previously (27) and at least 100-fold larger than endogenous currents in untransfected HEK-293 cells. Dialysis of GST-SNAP-25 (10-8 M) inhibited Kv2.1 mediated currents by 68.6 ± 11.7% (n= 10, P < 0.001; Fig. 3B) in a time-dependent manner compared with control (Fig. 2C). To confirm that the inhibitory effect of GST-SNAP-25 on Kv2.1 currents was not specific to solublized SNAP-25, we coexpressed SNAP-25 with Kv2.1 in HEK-293 cells to ensure the targeting of the expressed SNAP-25 to the plasma membrane (17). Importantly, the expressed membrane-bound SNAP-25 had a similar inhibitory effect (63.2 ± 6.6%, n= 9, P < 0.001) on Kv2.1 currents (Fig. 3D) as the dialyzed GST-SNAP-25 (Fig. 3B).
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), we examined which of these Kv2.1 domains would directly affect SNAP-25 actions on Kv2.1 channel activity. The inhibitory effect of GST-SNAP-25 on expressed Kv2.1 currents could be partially relieved (to 64.2 ± 4.8% of control, n= 7, P < 0.001) by codialysis of GST-Kv2.1N (10-8 M; Fig. 3B), which alone did not affect control currents (not shown). However, codialysis of GST-Kv2.1C (10-8 M) did not reverse the inhibitory effects of SNAP-25 on Kv2.1-mediated currents (n= 6; Fig. 3B).
To investigate whether SNAP-25 disrupts membrane targeting of Kv2.1, we performed experiments on HEK-293 cells coexpressing a C-terminal cyan fluorescent protein (CFP)-tagged Kv2.1 construct (Kv2.1-CFP) alone, with a dominant-negative Kv2.1 construct (DN-Kv2.1), or with SNAP-25. The C-terminal truncated DN-Kv2.1 construct lacks a pore-forming region, sixth transmembrane domain, and cytoplasmic C terminus and results in a functional knockout of Kv2 channel function (10, 28). Confocal microscopy revealed membrane localization of Kv2.1-CFP that was disrupted by coexpression of DN-Kv2.1 (Fig. 4, A and B), similar to what we have observed previously (unpublished data). Coexpression of SNAP-25, however, did not disrupt the membrane localization of the Kv2.1-CFP channel subunit (Fig. 4, A and B). Although the C-terminal CFP tag is not expected to affect the inhibitory effect of SNAP-25 mediated at the channel N terminus (see above), we nonetheless confirmed the effect of SNAP-25 on currents mediated by the Kv2.1-CFP construct (Fig. 4B). Currents mediated by Kv2.1-CFP were inhibited 60.0 ± 6.6% (P < 0.001, n= 6) by coexpression of SNAP-25 and 47.1 ± 4.3% (P < 0.001, n= 6) by coexpression of DN-Kv2.1 (at a 1:1 DNA ratio in transfection).
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, A and B). Similar to Kv2.1 expressing HEK-293 cells, codialysis of GST-Kv2.1N (10-8 M) acted to relieve the inhibitory effect of GST-SNAP-25 on the outward K+ currents (n= 6; Fig. 5B). Of note, in islet cells, this reversal of the inhibitory effect of SNAP-25 was complete, such that the difference between GST-dialyzed and GST-SNAP-25/GST-Kv2.1N dialyzed cells was not significant.
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). Secondly, we investigated whether the inhibitory effect of SNAP-25 can be modulated by [Ca2+]i. In the presence of 300 nM free [Ca2+]i, dialysis of GST-SNAP-25 (10-8 M) inhibited rat islet cell voltage- dependent outward K+ currents by the same degree (41.4 ± 6.6%, n= 6, P < 0.001) as in the absence of [Ca2+]i (Fig. 5C). The present data demonstrate that SNAP-25 inhibition of ß-cell voltage-dependent outward K+ currents does not strictly require that SNAP-25 be attached to the plasma membrane and is not sensitive to changes in [Ca2+]i within a physiological range. The latter is consistent with the binding data in Fig. 2B, which were performed in the absence of Ca2+ and presence of 2 mM EGTA.
SNAP-25 Specifically Blocks Kv2.1 in ß-Cells
To further determine whether the inhibitory effect of SNAP-25 on voltage-dependent outward K+ currents is specific to an interaction with Kv2.1, we examined the effect of intracellular dialysis of GST-SNAP-25 on outward currents from rat islet cells lacking functional Kv2.1 expression. In cells expressing the DN-Kv2.1 construct described above by adenovirus transfection, voltage-dependent outward K+ currents were reduced by more than 60% compared with control cells (not shown), similar to results reported previously (10). Intracellular dialysis of GST-SNAP-25 (10-8 M) in DN-Kv2.1 expressing cells was not able to reduce voltage-dependent outward K+ currents compared with DN-Kv2.1 expressing cells dialyzed with GST (10-8 M) alone (Fig. 6). These results, taken together with the ability of the Kv2.1N fragment to reverse the effect of SNAP-25, indicate that SNAP-25’s inhibitory effect is mediated by a specific interaction at the N terminus of Kv2.1, whereas other ß-cell Kv channels, which mediate the remaining outward currents (
40%), do not seem to be affected by SNAP-25.
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In islet ß-cells, we have reported Kv2.1 to be the major Kv channel mediating voltage-dependent outward K+ currents in rodent ß-cells (10). More specifically, we showed that functional knockout of Kv2.1 using a dominant-negative 
-subunit reduced voltage-dependent outward K+ currents by more than 60% and enhanced glucose-stimulated insulin secretion from rat islets (10). In the present study, we demonstrate for the first time, Kv2.1 protein expression in human islets (Fig. 1), providing further evidence for the importance of Kv2.1 in ß-cells. Blocking voltage- dependent outward K+ currents in insulin secreting cells results in prolongation of action potentials (4), enhanced depolarization (6, 7), enhanced [Ca2+]i fluxes (8), and increased insulin secretion (7, 9). We now show that SNAP-25 can associate with and regulate ß-cell Kv2.1 channels. Specifically, dialysis of a GST-SNAP-25 fusion protein or overexpression of SNAP-25 reduced Kv2.1 currents by 60–70% in HEK-293 cells overexpressing Kv2.1 (Fig. 2). In rat ß-cells, dialysis of GST-SNAP-25 or overexpression of SNAP-25 resulted in an approximately 40% reduction in Kv currents (Fig. 5), likely because Kv currents in these cells are mediated by a heterogeneous population of channels (60–70% Kv2.1) and SNAP-25 may not completely block the Kv2.1-mediated component. The inhibitory effect of SNAP-25 on Kv2.1 in ß-cells was not dependent on [Ca2+]i, suggesting that not only are their interactions per se not Ca2+ sensitive, but also that other Ca2+-dependent proteins may not be essential for the inhibitory action of SNAP-25 on Kv2.1. The inhibitory effect of SNAP-25 dialysis in both cell types was time dependent, with a maximum effect noted at approximately 4 min, which reflects the time necessary for the SNAP-25 protein to fully equilibrate in the cytosol.
Importantly, the inhibitory effect of SNAP-25 on Kv2.1 appears to be a direct effect on this channel, rather than by preventing channel surfacing to the plasma membrane or by channel endocytosis. Three lines of evidence in the present study support this. First, the inhibitory effect was the same regardless of whether SNAP-25 was cytosolic or membrane bound. Secondly, SNAP-25 did not alter the membrane localization of a CFP-tagged Kv2.1 construct even though it was able to antagonize currents mediated by this construct. Thirdly, the antagonist effect of SNAP-25 was specific to Kv2.1 in ß-cells compared with other Kv channels, ruling out a general inducement of endocytosis. Additionally, our previous studies demonstrate no effect of SNAP-25 expression on Kv1.1 biosynthesis and minimal effects on Kv1.1 membrane expression (30). These lines of data, together with the evidence for direct binding to Kv2.1, suggest that SNAP-25 directly regulates Kv2.1 channel function.
We have further determined that the inhibitory effect of SNAP-25 on Kv2.1 function is mediated at the channels N terminus, although both the Kv2.1N and C-terminal fragments bound SNAP-25 (Fig. 2B). Codialysis of GST-Kv2.1N partially alleviated the inhibitory effect of GST-SNAP-25 on Kv2.1 currents in HEK-293 cells (Fig. 3) but completely reversed the inhibitory effects on outward K+ currents in rat ß-cells (Fig. 5), whereas GST-Kv2.1C did not alter antagonism of Kv2.1 by GST-SNAP-25 (Fig. 3). The concentration of GST-Kv2.1N was equimolar to that of GST-SNAP-25 in both studies. Higher concentrations of GST-Kv2.1N were avoided as various N-terminal fragments of Kv2.1 and other Kv channels are known to have dominant-negative effects on channel function (10, 28). Although dialysis of Kv2.1N is not expected to act in a dominant-negative manner at the level of channel tetramerization (and we found no effect of the Kv2.1N fragment on control currents), potential effects of extremely high concentrations are nonetheless unknown. There are several explanations for the discrepancy between results obtained from the HEK-293 and rat ß-cells. In the HEK-293 cells, the Kv2.1N fragment may not be sufficient to totally compete with overexpressed Kv2.1 for binding with GST-SNAP-25, particularly if the SNAP-25 has a higher affinity for the intact membrane bound Kv2.1 protein. In the rat ß-cell, additional proteins associated with the Kv2.1 channel such as an unknown Kvß subunit or endogenous SNARE proteins (i.e. syntaxin 1A, synaptotagmin) may alter channel conformation that may allow the Kv2.1N fragment to compete more effectively for binding to SNAP-25. The significance of SNAP-25 binding to the Kv2.1 C-terminal is unclear but is not directly involved in channel modulation and may perhaps play a role in more complex interactions with other, undefined, proteins. Further studies are clearly needed to elucidate the specific action of the Kv2.1 C-terminal binding to SNAP-25.
Exogenous SNAP-25 suppressed voltage-dependent outward K+ currents in ß-cells by 40%, which may be entirely accounted for by interactions with Kv2.1 as demonstrated by the Kv2.1N-fragment experiment and our previous data (10). To conclusively prove that SNAP-25 is specifically interacting with and inhibiting Kv2.1 channels, as compared with other ß-cell Kv channels, we determined the effect of SNAP-25 on currents from cells lacking functional expression of Kv2.1. Because no change in voltage-dependant outward K+ currents was observed (Fig. 6), we conclude that SNAP-25 does not functionally interact, at least not independently, with other Kv channels expressed in rat ß-cells, such as Kv1.4, 1.6, and 4.2 (10). We have, however, just reported that SNAP-25 also binds and modulates the Kv1.1 channel protein (30), which is not a dominant Kv channel in the islet ß-cell. Further studies will be needed, however, to examine whether other SNARE proteins (i.e. syntaxins, synaptotagmins) can modulate these channels, and whether SNAP-25 could also indirectly modulate other ß-cell Kv channels.
The present study implicates SNAP-25 as a potential regulator of ß-cell membrane excitability through its interaction with Kv2.1, in addition to its roles in regulating L-type VDCCs (31, 32, 35) and insulin exocytosis (16, 17, 18, 19, 20). SNARE protein-ion channel complexes, therefore, may comprise functional units, perhaps with the SNARE exocytic complexes, to regulate insulin secretion, the modulation of which may have therapeutic implications for the treatment of type 2 diabetes mellitus.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Rat pancreatic islets were isolated by collagenase digestion as described previously (10). Islets were dispersed to single cells by treatment with 0.015% trypsin (Invitrogen Canada Inc.) in Ca2+- and Mg2+-free PBS (10). Islet cells were plated on glass coverslips in 35-mm dishes and cultured in low glucose Roswell Park Memorial Institute medium (2.5 mM glucose) supplemented with 7.5% fetal bovine serum, 0.25% HEPES (Sigma-Aldrich Canada Ltd., Oakville, Canada), and 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate (Invitrogen Canada Inc.). Islet cells were cultured for 3–5 d before transfection with pdDNA3.1-GFP-SNAP25 (see below). For intracellular dialysis experiments, islet cells were cultured for 1–2 d before electrophysiological recordings.
Human Islets
The Human Ethics Committee of the University of Alberta approved tissue procurement and experimental protocols. Pancreases were removed after in situ vascular perfusion using cold University of Wisconsin solution as part of a multi-organ procurement (36, 37). Pancreases were placed in cold University of Wisconsin solution on ice and immediately transported to the islet isolation laboratory for processing. Islets were isolated using the standard techniques of collagenase perfusion via the duct and gentle enzymatic dissociation using the Ricordi chamber (38, 39). Islets were purified from the exocrine tissue using continuous gradients of Ficoll using the Cobe 2991 blood cell processor and quantified using standard techniques (40).
DNA Constructs and Adenovirus Vectors
The vectors pcDNA3-Kv2.1 and pcDNA3-SNAP25 were generously provided by Dr. R. Joho (University of Texas, Southwestern Medical Center, Dallas, TX) and Dr. M. Wilson (Scripps Research Institute, La Jolla, CA), respectively. The Kv2.1-CFP construct (in pECFP-C1, CLONTECH Laboratories, Inc., Palo Alto, CA) was provided by Dr. O. T. Jones (University of Manchester, Manchester, UK). To facilitate transient expression of multiple membrane protein genes, we reconstructed the expression vector pcDNA3.1 (Invitrogen Canada Inc.) to pcDNA3.1-GFP by replacement of the neomycin resistance cassette with GFP from pEGFP-N3 (CLONTECH Laboratories, Inc.). We then subcloned Kv2.1 from pcDNA3-Kv2.1 and SNAP25 from pcDNA3-SNAP25 into this vector, respectively, to construct new vectors pcDNA3.1-GFP-Kv2.1 and pcDNA3.1-GFP-SNAP25, used to transiently express cytosol GFP and membrane proteins Kv2.1 and SNAP-25. The recombinant adenovirus vector expressing the dominant-negative Kv2.1 construct (DN-Kv2.1) was described previously (10). Islet cells were infected in 35-mm dishes 24 h after dispersion of islets to single cells.
Recombinant GST Fusion Proteins
The PCR primers for amplifying the cDNA fragments of N (amino acids 1–182) and C (amino acids 411–853) termini of Kv2.1 were (sense) 5'-atgacgaagcatggctcgc-3', (antisense) 5'-caccgacgagttgggct-3' and (sense) 5'-aacttctccgagttctacaag-3', (antisense) 5'-gatactctgatccctagtg-3', respectively. EcoRI and XhoI sites were added at the 5' ends of sense primers and antisense primers, respectively, for subcloning the PCR products into pGEX-5X-1 vector (Amersham Pharmacia Biotech, Piscataway, NJ). The pGEX-5X-1 vectors were generously provided by Dr. W. Trimble (Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada). GST-Kv2.1N, GST-Kv2.1C, GST-SNAP25 and GST alone were expressed in DH5a bacteria. The GST-fusion proteins were purified with glutathione beads and eluted with reduced glutathione (Sigma-Aldrich Canada Ltd.), according to the instructions of GST Gene Fusion System (Amersham Pharmacia Biotech). PAGE, Western blotting (for SNAP-25), and Coomassie blue staining (for Kv2.1 N- and C-terminal fragments) were used to confirm the identity and purity of these proteins.
Binding Studies
Xenopus laevis oocytes were prepared as described (19), injected (50 nl/oocyte) with the mRNAs, metabolically labeled for 3 d and subjected to immunoprecipitation, as described (19). Immunoprecipitates by polyclonal Kv2.1 antibody (Alomone Labs, Jerusalem, Israel) from 1% Triton X-100 homogenates of whole oocytes were analyzed by SDS-PAGE. Digitized scans were derived by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Kv2.1 cDNA in pBluescript was a gift from R. H. Joho. Preparations of mRNA have been previously described (41).
For binding to peptide fragments, purified Kv2.1N-GST fusion protein (200 pmol) or purified GST (200 pmol) was immobilized on gluthatione-Sepharose beads (Amersham Pharmacia Biotech). Forty microliters of beads were added to peptide fragments, incubated for 1 h at 4 C with gentle rocking, and washed four times in 1 ml of PBS with 0.1% Triton X-100. This was then incubated with 5 µl of the lysate containing 35S-labeled SNAP-25 [translated on the template of in vitro synthesized RNAs using a translation rabbit reticulocyte lysate kit (Promega Corp., Madison, WI)] in 1000 µl of PBS with 0.1% Triton X-100, with gentle rocking for 1 h at room temperature. Binding studies were performed in the absence of Ca2+ and the presence of 2 mM EGTA. After washing, the GST fusion proteins were eluted with 15 mM reduced gluthatione in 40 µl elution buffer (120 mM NaCl; 100 mM Tris-HCl, pH 8) and analyzed by 12% SDS-PAGE.
Western Blots
Immunoblotting was performed as previously described (17, 20). Islets were washed in ice-cold PBS, solublized in 2% SDS loading buffer, heated for 10 min at 80 C, then loaded and separated on a 8% (for Kv2.1) or 14% (for SNAP-25 and syntaxin) polyacrylamide gel. The protein was transferred to nitrocellulose membrane (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH) and incubated with primary antibody: Kv2.1 (Alomone; diluted 1:500), SNAP-25 (Sternberger Monoclonals, Lutherville, MD; 1:2000) or syntaxin (Calbiochem, San Diego, CA, 1:1000) for 1.5 h at room temperature. Detection was with appropriate secondary antibody (goat antirabbit or antimouse, peroxidase-conjugated IgG, Jackson ImmunoResearch, West Grove, PA) for 1 h. Visualization was by chemiluminescence (SuperSignal, Pierce Chemical Co., Rockford, IL) and exposure of the membranes to Kodak film (Eastman Kodak Co., Rochester, NY) for 5 sec to 10 min.
Electrophysiology
Cells were voltage-clamped in the whole-cell configuration using an EPC-9 amplifier and Pulse software (Heka Electronik, Lambrecht, Germany). Patch pipettes were prepared from 1.5 mm thin-walled borosilicate glass tubes using a two-stage Narishige (Tokyo, Japan) micropipette puller. Pipettes were heat polished and typically had tip resistances of 1–1.5 M
(for HEK-293 cells) or 3–5 M (for ß-cells) when filled with intracellular solution containing (in mM): KCl, 140; MgCl2·6H2O, 1; EGTA, 1; HEPES, 10; MgATP, 5; pH 7.25 with KOH. For experiments with a calculated free [Ca2+]i of 300 nM, the intracellular solution contained (in mM): KCl, 140; MgCl2·6H2O, 0.5; EGTA, 1; HEPES, 10; MgATP, 5; CaCl2, 3.87; pH 7.25 with KOH. The bath solution contained (in mM): NaCl, 140; CaCl2, 2; KCl, 4; MgCl2·6H2O, 1; HEPES, 10; pH 7.3 with NaOH. All electrophysiological measurements reported were made at room temperature (22–24 C) and normalized to cell capacitance. Outward currents were elicited with a 250-msec depolarization in steps of 20 mV to +70 mV from a holding potential of -70 mV. To minimize variation, maximum sustained current was determined as the average outward current over the final 95–99% of the 250-msec depolarizing pulse. For dialysis studies, the recombinant protein was added to the pipette solution and applied for at least 4 min before recording (the exception being the time-course experiments determining current inhibition as a function of time after break-in). Current-voltage curves were compared by one-way ANOVA followed by a Tukey posttest to compare currents at individual voltages. The time-course for current inhibition was analyzed by repeated-measures ANOVA and individual points were compared with time= 0 using a Bonferroni posttest. P < 0.05 was considered significant.
Confocal Microscopy
Confocal microscopy was performed on live HEK-293 cells bathed in the extracellular solution described above using an inverted Carl Zeiss (Carl Zeiss Inc., Thornwood, NY) laser scanning confocal microscope (LSM 410) with a 63x oil immersion lens (Carl Zeiss Inc.). Transfected cells were plated on glass coverslips 24 h before mounting in a bath chamber for microscopy. The CFP signal was visualized by excitation at a wavelength of 488 nm and measurement of emitted fluorescence through a 515- to 560-nm bandpass filter. The images presented are representative of 10 images from each group. Control cells expressing Kv2.1-CFP only were cotransfected with empty pcDNA-3.1 plasmid. The membrane to cytoplasmic fluorescence ratio (F[mem]/F[cyt]) was calculated using Scion Image (Scion Corp., Frederick, MD) and analyzed using the Student’s t test; P < 0.05 was considered significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 P.E.M., G.W., and S.T. contributed equally to this work. 
Abbreviations: CFP, Cyan fluorescent protein; GST, glutathione-S-transferase; Kv, voltage-dependent K+; SNAP-25, synaptosome-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; VDCCs, voltage-dependent Ca2+ channels.
Received for publication February 4, 2002. Accepted for publication June 24, 2002.
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