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J. Biol. Chem., Vol. 277, Issue 23, 20195-20204, June 7, 2002

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The 25-kDa Synaptosome-associated Protein (SNAP-25) Binds and Inhibits Delayed Rectifier Potassium Channels in Secretory Cells^*

Junzhi Ji^§, Sharon Tsuk^§¶, Anne Marie F. Salapatek^§, Xiaohang Huang^§**, Dodo Chikvashvili^¶, Ewa A. Pasyk, Youhou Kang, Laura Sheu, Robert Tsushima, Nicholas Diamant, William S. Trimble^§§, Ilana Lotan^¶, and Herbert Y. Gaisano^¶¶

From the Departments of  Medicine,  Physiology, and  Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the ^§§ Program in Cell Biology of the Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, ^** First Institute of Oceanography, State Ocean Administration, Qingdao 266061, China, and ^¶ Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel 69978

Received for publication, January 31, 2002, and in revised form, March 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Delayed-rectifier K⁺ channels (K_DR) are important regulators of membrane excitability in neurons and neuroendocrine cells. Opening of these voltage-dependent K⁺ channels results in membrane repolarization, leading to the closure of the Ca²⁺ channels and cessation of insulin secretion in neuroendocrine islet  cells. Using patch clamp techniques, we have demonstrated that the activity of the K_DR channel subtype, K_V1.1, identified by its specific blocker dendrodotoxin-K, is inhibited by SNAP-25 in insulinoma HIT-T15  cells. A co-precipitation study of rat brain confirmed that SNAP-25 interacts with the K_V1.1 protein. Cleavage of SNAP-25 by expression of botulinum neurotoxin A in HIT-T15 cells relieved this SNAP-25-mediated inhibition of K_DR. This inhibitory effect of SNAP-25 is mediated by the N terminus of K_V1.1, likely by direct interactions with K_V1.1 and/or K_V subunits, as revealed by co-immunoprecipitation performed in the Xenopus oocyte expression system and in vitro binding. Taken together we have concluded that SNAP-25 mediates secretion not only through its participation in the exocytotic SNARE complex but also by regulating membrane potential and calcium entry through its interaction with K_DR channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In islet  cells, glucose-mediated Ca²⁺-evoked insulin secretion is initiated by the closure of ATP-sensitive K⁺ channels, which in turn causes membrane depolarization and the resultant opening of L-type Ca²⁺ channels (1). The cessation of insulin secretion is brought about by the closure of these Ca²⁺ channels by membrane repolarization, which is primarily effected by the opening of a voltage-dependent K⁺ channels (K_V)¹ of the delayed rectifier subtype (K_DR) (2). Regulation of K_DR activity therefore directly affects the duration and extent of Ca²⁺ channel opening-ensuing Ca²⁺ influx. In neurons, the short duration of action potentials regulates rapidly activating and inactivating N-type Ca²⁺ channels, resulting in ultrashort (µs) Ca²⁺ fluxes acting on docked synaptic vesicles (3). In contrast to neurons that have a higher proportion of readily releasable docked vesicles (~10%), less than 5% of insulin-containing secretory granules in islet  cells are morphologically docked, with the vast majority of insulin granules located more distantly from the membrane within a reserve pool (4). A more sustained Ca²⁺ influx effected by a much longer train of action potentials would be necessary to reach and mobilize this reserve pool of insulin granules to the plasma membrane and then to effect their exocytosis (3, 4). This contributes to the more sustained phase of secretion, also observed in other neuroendocrine cells (5). In the islet  cell, since this glucose-sensitive sustained phase of insulin secretion is regulated by the K_DR channel, it would be an ideal drug target for the treatment of non-insulin-dependent or type 2 diabetes. In particular, drugs that interfere selectively with it would be superior to the current treatment with sulfonylureas that act on  cell K_ATP channels in a glucose-independent manner, often resulting in deleterious side effects such as hypoglycemia (6). However, very little is known about the K_DR channel or the identities of the molecules interacting with this channel (2).

The target-SNAREs, SNAP-25 and syntaxin, and the vesicleSNARE, vesicle-associated membrane protein, are thought to comprise the minimal machinery required for membrane fusion and exocytosis (7, 8). These proteins all possess -helical domains that interact to form a stable coiled-coil complex, the formation of which likely drives membrane fusion (7, 8). Clostridial neurotoxins, which specifically cleave these SNARE proteins, have been valuable tools in revealing SNARE protein functions (911). t-SNARE proteins syntaxin 1A and SNAP-25 can also directly interact with membrane ion channels involved in regulating the secretory process. Syntaxin 1A and SNAP-25 bind to (12, 13) and modulate neuronal (12-14) and neuroendocrine (pancreatic islet  cells) (15, 16) Ca²⁺ channels. More recently, syntaxin has also been shown to modulate epithelial Na⁺ (17, 18), Cl (19, 20), and voltage-gated K⁺ (21) channels.

In this study, we have begun to explore the molecular interactions between K_DR channels and the critical t-SNARE, SNAP-25. Here we show that SNAP-25 interacts with insulinoma HIT-T15 cell K_DR, specifically via the K_v1.1 subunit. Overexpression of SNAP-25 or exogenously applied recombinant SNAP-25 protein inhibits HIT cell K_DR activity. Cleavage of SNAP-25 by botulinum neurotoxin A (BoNT/A) light chain expression relieves the actions of endogenous and exogenous SNAP-25 on K_DR. Furthermore, the N-terminal domain but not C-terminal domain of K_V1.1 competitively reversed the effect of SNAP-25. This work demonstrates that SNAP-25 modulates secretion not only by its involvement in membrane fusion but also by its interaction with K_DR channels.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture, Transfection, and Plasma Membrane Fractionation

HIT-T15 cells (a gift from P. Robertson, Seattle, WA) were cultured at 37 °C in 5% CO₂, 95% air in RPMI 1640 medium supplemented with 20 mM glutamine, 10% fetal calf serum (Invitrogen), penicillin (100 units/ml), and streptomycin (100 mg/ml). These cells were then transfected with plasmids (pcDNA3) containing cDNAs of full-length SNAP-25 or BoNT/A light chain (a gift from H. Niemann, Hanover, Germany) exactly as we report (22). 48 h later, we noted the transfection efficiency to be ~30-40% as determined by visualization of the co-expressed green fluorescent protein (GFP) (CLONTECH, Palo Alto, CA), which was also used to identify the transfected cells for the electrophysiological studies. To determine the levels of Kv1.1 and SNARE proteins, these transfected HIT cells were further subjected to subcellular fractionation (i.e. whole-cell lysate and plasma membrane). The HIT-T15 cells were dislodged from the plates with trypsin and then washed twice with phosphate-buffered saline after alternate centrifugation (2000 rpm, 5 min). The resulting pellet was resuspended in a homogenization buffer (in mM: 150 NaCl, 20 Tris-HCl, 1 EDTA, 1 EGTA, and protein inhibitors) and homogenized using a sonicator. Large debris and nuclear fragments were removed by low centrifugation (2000 rpm for 5 min, IEC-8R centrifuge) at 4 °C, and the resulting pellet was resuspended and subjected to ultracentrifugation (50,000 rpm for 1 h, TL-100 ultracentrifuge). The pellet was then suspended in SDS-loading buffer and centrifuged at 2000 rpm at 4 °C for 5 min, and the supernatant containing the purified plasma membrane fraction was collected. Protein concentrations of the whole-cell lysate and plasma membrane fractions were determined by a modified Lowry method. About 15 µg of protein from cell lysate and 70 µg of protein from membrane preparation were separated on an 8 or 14% SDS-PAGE and transferred to a nitrocellulose membrane, and the blots were then identified with specific primary antibodies against SNAP-25 (1:1000) (22) or K_V1.1 (1:200, Alomone Labs, Jerusalem, Israel).

Immunoprecipitation

Rat Brain Homogenates-- Rat brain synaptosomal heavy membrane LP1 fraction was prepared exactly as described by Huttner et al. (23). LP1 protein (350-600 µg) was incubated in lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 0.1% Triton X-100 containing a mixture of protease inhibitors) to a concentration of >1 mg/ml in ice for 10 min, and then 1 µg of affinity-purified antibody (SNAP-25) was added. This mixture was then agitated overnight at 4 °C followed by the addition of 50-100 µl of 50% slurry of protein G-Sepharose and further agitated for 4 h. The Sepharose beads were then spun-down, washed 4 times with ice-cold lysis buffer, solubilized in sample buffer, and heated at 80 °C for 3 min. The proteins within the immunoprecipitated complexes were then separated on SDS-PAGE and identified by specific primary antibodies anti-SNAP-25 (1:1000) (22) and anti-K_V1.1 (1:1000, Alomone Labs).

Xenopus laevis Oocytes-- Oocytes were prepared as described (24), injected (50 nl/oocyte) with the mRNAs, metabolically labeled for 3 days, and subjected to immunoprecipitation, as described (25). Immunoprecipitates from 1% Triton X-100 homogenates of whole oocytes were analyzed by SDSPAGE. Digitized scans were derived by PhosphorImager (Molecular Dynamics/Sunnyvale, CA or Invitrogen). Here, the primary antibodies used were polyclonal anti-K_V1.1 and anti-SNAP-25 (Alomone Labs), K_v1.1, and K_v1.1 (a gift from O. Pong, Hamburg, Germany) cDNAs; their mRNA preparations have been previously described (26).

Binding of Glutathione S-Transferase (GST) Fusion Proteins

The fusion proteins were synthesized as previously described (27, 28). The protein concentration was estimated using the Bio-Rad protein assay kit. Purified GST fusion proteins (200 pmol) or purified GST (200 pmol) immobilized on glutathione-Sepharose beads (Amersham Biosciences; 40 µl of beads were added, incubated for 30 min at 4 °C, and washed 3 times in 1 ml of phosphate-buffered saline with 0.1% Triton X-100) were 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) according to the manufacturer's instructions) in 1000 µl of phosphate-buffered saline with 0.1% Triton X-100 for 1 h at room temperature with gentle rocking. After washing, the GST fusion proteins were eluted with 15 mM reduced glutathione in 40 µl of elution buffer (120 mM NaCl, 100 mM Tris-HCl, pH 8.0) and analyzed by 12% SDS-PAGE. DNAs of K_v1.1 and K_v1.1 fragments to create GST fusion proteins have been previously described (27).

Electrophysiology

Previously transfected GFP-positive HIT cells (passage 65-75) were studied with standard whole-cell and cell-attached patch clamp techniques (29) as we have previously described (30). The standard pipette solution contained the following (in mM) except where noted in the text: 140 KCl, 1 MgCl₂, 10 HEPES, 4 Na₂ATP, 0.3 EGTA, and 8 nM CaCl₂, pH 7.2. The external solution contained (in mM): 140 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, pH 7.4. Axopatch 1D and 200B patch clamp amplifiers were used together with pCLAMP software (v.6.0, Axon Instruments, Union City, CA) to record channel currents. Currents were typically elicited from a holding potential of 70 mV. Data were presented as mean ± S.E. and compared by Student's t test for single comparisons and by analysis of variance for multiple comparisons. p < 0.05 was considered to be statistically significant. Single-channel recording and analyses were performed in the cell-attached configuration in which HIT-T15 cells were bathed in the internal solution (high K⁺) and pipettes contained a normal external solution. Test pulses of 30s from 70 to 10 mV were applied every 3 min. Analog signals were filtered at 2 kHz using a Bessel filter. Single channel data were analyzed using Fetchan and pSTAT software (pCLAMP 6.0, Axon Instruments). Recombinant proteins including GST-SNAP-25, GST T1A, GST T1B, GST-K_V1.1_1-168, and GST-K_V1.1_411-495 were generated as previously described (27, 28) and dialyzed into a cell via the patch pipette. A single voltage step to +30 mV from a holding potential of 70 mV was used to assess the effects of these test proteins on K_DR.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Insulinoma HIT Cell K_DR Is Similar to That of Islet  Cells-- To determine whether the K_DR in HIT-T15 (HIT) cells are similar to those reported in islet  cells (2, 31, 32), we functionally and molecularly characterized this channel protein. Like islet  cell K_DR (2, 32), HIT cell K_DR possesses several similar functional properties (Fig. 1A). These properties include K⁺ ion selectivity, as demonstrated by an inhibitory effect of Cs⁺ ion, when intracellular KCl was replaced with CsCl (Fig. 1A, CsCl), a low threshold of activation (30 mV), and slow inactivation (>250 ms, shown in Fig. 1A, inset, and graphic summary). Characteristic of K_DR, the HIT cell K_DR was inhibited by high doses of the semi-selective K⁺ channel blocker tetraethylammonium (20 mM TEA in Fig. 1A, inset), which caused ~85% reduction (p < 0.05) in the outward current at +30 mV (31). Because large conductance Ca²⁺-sensitve K⁺ currents (K_Ca) also contributes to the outward currents in islet  cells (32), we determined the contribution of this channel to total outward currents. Like others (31), we also found that K_Ca contributed little to the HIT cell outward currents, as evidenced by (a) insensitivity to low intracellular Ca²⁺ concentrations (with 8 nM Ca²⁺, <8% reduction in outward currents, data not shown) or the Ca²⁺ channel blocker nifedipine (Fig. 1A), (b) insensitivity to low doses of TEA (<10% reduction at 1 mM TEA, not shown), or (c) inhibition by 100 nM iberiotoxin, a specific large conductance K_Ca blocker (Fig. 1A, iberiotoxin (IbTX)). Nonetheless, to negate even this small contribution of K_Ca to the outward currents, all pipette solutions contained low Ca²⁺ concentration (<8 nM).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   HIT-T15 cell KDR identification. Typical whole-cell KDR currents evoked with one-step voltage depolarization to +30 mV in a control cell before () and after it is inhibited by TEA (20 mM) application to the extracellular solution () (A, inset). A, effects of various K+ channel blockers: iberiotoxin (IbTX; 107 M, , n = 6), TEA (20 mM, , n = 6), CsCl (replaced KCl in the pipette solution, 140 mM, , n = 5), and a Ca2+ channel blocker, nifedipine (NIF; 105 M, , n = 6) on the mean steady-state current-voltage relationship recorded from control HIT cells (, n = 12). Whole-cell currents were evoked in response to a series of test pulses between 50 and +50 mV in 20-mV steps from a holding potential of -70 mV. KDR currents were remarkably reduced by TEA and CsCl. B, representative families of whole-cell KDR currents recorded from a control and a SNAP-25- and a BoNT/A-transfected cell. Currents were evoked in response to a series of test pulses from -50 to +50 mV in 10-mV steps from a holding potential of -70 mV. Steady-state current-voltage curves were obtained from control (, n = 16), SNAP-25- (, n = 18) or BoNT/A-transfected cells (, n = 14) (B, right panel). Data are presented as the mean ± S.E. The asterisk indicates p < 0.05 against control by analysis of variance. C, effects of DTX-K, a specific KV1.1 blocker, on KDR currents. Representative outward currents recorded from a control and a SNAP-25- and a BoNT/A-transfected cell before (, basal) and after 300 nM DTX-K addition (). Current traces were caused by one-step depolarization to +30 mV from a holding potential of -70 mV (C, left 3 panels). The bar graph shows the effects of DTX-K on the steady-state KDR currents (mean ± S.E.) in the control (n = 6) and SNAP-25- (n = 6) or BoNT/A-transfected cells (n = 6) (C, right panel). The asterisk indicates p < 0.05 versus basal. KDR current levels after the DTX-K addition in control or SNAP-25- or BoNT/A- overexpressing cells (see graph) were very close. The dotted lines are the zero current level. Currents are normalized to cell membrane capacitance.

SNAP-25 Inhibits Whole-cell K_DR Subtype, K_V1.1 Currents-- We and others previously showed that in pancreatic islets and insulinoma cell lines including HIT cells, SNAP-25 expression is required for insulin secretion (22, 33, 34). Furthermore, t-SNARE proteins syntaxin 1A and SNAP-25 can also directly interact with membrane ion channels involved in regulating the secretory process (15, 16). To determine whether SNAP-25 can modulate K_DR channels, we overexpressed SNAP-25 in HIT cells (Figs. 1B, 2B, and 3B). We already reported that overexpressed SNAP-25 proteins are appropriately targeted to the plasma membrane (22), which would allow for their interaction with membrane-spanning ion channel proteins such as K_V1.1. SNAP-25- or BoNT/A-transfected cells were identified by their expression of co-transfected GFP (22). All control cell studies were performed on cells transfected with GFP alone, and neither K_DR activity (data not shown) nor insulin secretion was altered by GFP transfection (22).

Fig. 1B shows representative whole-cell K_DR current traces after transfection and a graphical summary of the peak currents, which have been normalized by cell membrane capacitance for minimizing variations in cell size. Transfection of HIT cells with SNAP-25 resulted in a 3-fold increase in SNAP-25 expression in total HIT lysates (Fig. 2A, lane 3). Transfection efficiency as determined by the frequency of GFP-positive cells is consistently ~40%. With this transfection efficiency of ~40%, the transfected cells studied by patch clamp would be estimated to contain about a 5-6-fold increase in SNAP-25 compared with endogenous levels. In cells overexpressing SNAP-25, the peak K_DR current (86.9 ± 6.2 pA/picofarads, n = 18, p < 0.05) was inhibited at +30 mV by 22% compared with control cells (111.3 ± 7.9 pA/picofarads, n = 16) (Fig. 1B). We reasoned that if SNAP-25 genuinely acts to inhibit K_DR, then BoNT/A cleavage of SNAP-25 should enhance K_DR currents. We therefore overexpressed BoNT/A light chain in HIT cells. BoNT/A expression reduced endogenous SNAP-25 levels of HIT lysates to ~10% of control levels (Fig. 2A, lane 4). Therefore, in cells expressing BoNT/A, it can be assumed that all endogenous SNAP-25 would be cleaved (22). K_DR currents in these BoNT/A-expressing cells were augmented at +30 mV by 22% (135.4 ± 8.9 pA/picofarads, n = 14, p < 0.05) compared with control cells (Fig. 1B). Indeed, these effects of BoNT/A are opposite to the effects of SNAP-25 overexpression, which would therefore suggest that BoNT/A cleavage of SNAP-25 removes the inhibitory actions of full-length SNAP-25. BoNT/A cleavage of SNAP-25 also revealed a smaller band (Fig. 2A, lower unfilled arrowhead of the arrowhead doublets to the right of lanes 4 and 7), which is likely the SNAP-25 cleavage product representing amino acids 1-197 (22, 34).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effects of SNAP-25 and BoNT/A transfection on KDR channel expression and channel kinetics. A, expression of SNAP-25 (lower panel) and the voltage-activated K+ channel (KV) subtype, KV1.1 (upper panel) in HIT cells. HIT cells transfected with an empty vector (Control, lanes 2 and 5), SNAP-25 (lanes 3 and 6) or BoNT/A (lanes 4 and 7) were lysed by sonication or subjected to subcellular fractionation to obtain a purified plasma membrane fraction. Western analysis using specific antibodies to SNAP-25 (22) and KV1.1 (Alomone Labs) was performed on these HIT cell lysates (15 µg, lanes 2-4) and plasma membrane fractions (70 µg, lanes 5-7). Rat brain synaptosomal membrane fraction (LP1, 10 µg of protein) was used as the positive control. In BoNT/A-transfected HIT cells, we observed a slightly smaller (by ~1 kDa, indicated by lower open arrowhead on lanes 4 and 7) SNAP-25 immunoreactive band, which is likely the larger SNAP-25 cleavage product (amino acids 1-197). B (top left), representative whole-cell KDR activation current traces were evoked by depolarization to +10 mV for 300 ms from a holding potential of -70 mV and normalized to the same amplitude to compare their activation time course. Time constants of activation were obtained by fitting each current trace with a monoexponential equation over test voltages from +10 to +40 mV (B, bottom left). Each point was expressed as the mean ± S.E. The asterisk indicates p < 0.05 against control. SNAP-25 (, n = 8) decelerated KDR activation, whereas SNAP-25 cleavage by BoNT/A expression (, n = 8) accelerated KDR activation when compared with control cells (, n = 12). B (top right), representative whole-cell KDR inactivation current traces were evoked by a 10-s pulse to +10 mV from a holding potential of -70 mV, and these traces were normalized to the same amplitude to compare their decay. Time constants of inactivation were fitted by a biexponential equation to calculate fast (not shown) and slow time constants, represented by the histogram (B, bottom right). Data were the mean ± S.E. from control (n = 8), SNAP-25 (n = 6), and BoNT/A-transfected cells (n = 6), respectively. The asterisk indicates p < 0.05 against control. SNAP-25 accelerated KDR inactivation, whereas SNAP-25 cleavage by BoNT/A expression decelerated KDR inactivation when compared with control cell KDR inactivation.

To identify the K_DR subtype upon which SNAP-25 acted, we first applied dendrotoxin (DTX), which blocks K_v1.1 and K_v1.2 channels, and subsequently applied dendrotoxin-K (DTX-K), which more specifically blocks only K_v1.1 channels. We found that both toxins exhibited identical effects, and therefore, we show only the more specific DTX-K results in Fig. 1C. DTX-K (300 nM) application had no significant additional effects in SNAP-25-expressing cells compared with controls in which there was a 33% drop in K_DR current (p < 0.05). Thus, the K_DR subtype upon which SNAP-25 acted was K_v1.1. We confirmed the presence of the K_V1.1 channel protein in HIT cell lysates as shown in Fig. 2A in lane 2 (HIT Cell lysate, Control). This K_V1.1 antibody (Alomone Labs) has been depleted of antibodies that reacted with closely related K_V isoforms. K_V1.1 was previously identified in HIT cells (35) and also in human islets and human insulinoma  cells (2). Furthermore, HIT-T15 does not express the closely related K_V1.2 channel protein, as confirmed by us at the protein (by anti-K_V1.2 antibody, Alomone Labs) and mRNA (by reverse transcription-PCR) levels (36).

Because the K_DR current in HIT cells is likely to be contributed by K_V channels other than K_V1.1 (36), we next examined whether the SNAP-25-sensitive component of the K_DR current is predominantly K_V1.1. In Fig. 1C, we expressed BoNT/A to cleave endogenous SNAP-25, which predictably increased the K_DR currents (representative trace on the left, and summary on the right) over controls as was shown in Fig. 1B. More importantly, the application of DTX-K (300 nM) inhibited the K_DR current of the BoNT/A-transfected HIT cells to precisely the same extent as the control and SNAP-25-overexpressing cells (Fig. 1C), indicating that the SNAP-25-sensitive K_DR current in the HIT cells is indeed from K_V1.1 channels.

SNAP-25 Decelerates Activation and Accelerates Inactivation of Whole-cell K_DR Currents-- There are two possible explanations for the differences in whole-cell K_DR current magnitude observed in Fig. 1B. First, SNAP-25 or BoNT/A expression could affect K_DR synthesis and cell surface expression. In this way, changes in peak K_DR current would reflect a change in channel density rather than in K_DR activity. Fig. 2A (upper panel) shows that there is no obvious difference in the levels of K_V1.1 protein (indicated by rat brain positive control, 86 kDa) expression in whole-cell lysates of HIT cells overexpressing SNAP-25 (lane 3) or BoNT/A (lane 4) when compared with control HIT cell lysates (lane 2), indicating that there was no gross effects on K_V1.1 synthesis. To assess the effects on K_V1.1 expression at the cell surface, which could be due to changes in endocytotic or exocytotic events, we determined K_V1.1 levels in the plasma membrane fraction (Fig. 2A, upper panel, lanes 5-7). To our surprise, K_V1.1 expression in the plasma membrane preparation was reduced to 84% of control in the SNAP-25-overexpressing HIT cells (lane 6, Fig. 2A). This reduction of K_V1.1 expression at the cell surface would therefore at least contribute to the decreased current density observed in SNAP-25-transfected cells (Fig. 1B). However, if the primary action of SNAP-25 is to regulate K_V1.1 transport to the cell surface, we would expect an augmented expression of K_V1.1 in the plasma membrane after cleavage of endogenous SNAP-25 by overexpression of BoNT/A. This was not the case since BoNT/A overexpression, which reduced the membrane SNAP-25 levels to ~10% of controls, did not cause an increase in the plasma membrane levels of K_V1.1. In fact, the K_V1.1 levels in the plasma membrane (lane 7, Fig. 2A) were reduced to 49% of control, which we believe may in part be due to nonspecific proteolysis during the membrane preparation caused by the BoNT/A expression. Of note, the plasma membrane SNAP-25 levels of the SNAP-25-overexpressing HIT cells was greater than 5-fold normal levels, which is much greater in proportion to the reduction of membrane K_V1.1 levels, suggesting a pharmacological effect of the overexpressed SNAP-25. In the SNAP-25- and BoNT/A-transfected HIT cell lysates and plasma membrane, syntaxin 1A levels did not change (data not shown).

The most plausible explanation is that endogenous SNAP-25 physiologic actions are to inhibit K_DR channel activity. We tested this possibility by examining the effects of SNAP-25 on the activation and inactivation kinetics of the whole-cell K_DR current. Fig. 2B demonstrates that the overexpressed SNAP-25 acts to decelerate K_DR activation and accelerate K_DR inactivation, with the time constants for activation and inactivation at +10 mV of 21.5 ± 1.0 ms (n = 8) and 3.2 ± 0.3 s (n = 6), respectively. These values were significantly different (p < 0.05) than those for controls, which had activation and inactivation time constants of 16.3 ± 1.0 ms (n = 12) and 4.3 ± 0.3 s (n = 8), respectively. In contrast, cleavage of SNAP-25 by BoNT/A had the opposite effect, resulting in acceleration of activation and deceleration of inactivation (shown in Fig. 2B), with the time constants for activation and inactivation of 11.6 ± 0.7 ms (n = 8) and 5.4 ± 0.5 s (n = 6), respectively. These values were also significantly different (p < 0.05) than those for controls (as above). These differences in time-dependent kinetics of the K_DR channel effected by SNAP-25 and SNAP-25 cleavage by BoNT/A would contribute to the reduction or augmentation, respectively, of the peak K_DR current observed in cells overexpressing SNAP-25 or BoNT/A (shown in Fig. 1B).

SNAP-25 Inhibits Single K_DR Channels-- Single channel studies on cell-attached patches revealed single K⁺ channels in HIT cells that possess a unitary conductance of 10 picosiemens, as determined from the slope conductance (Fig. 3A), which is similar to that reported for K_DR in  cells (31). This conductance is like that reported for the K_V1.1 voltage-activated K⁺ channel isoform expressed in oocytes (37). We next attempted to determine whether SNAP-25 affected the probability of channel opening or the conductance of single K_DR channels in cell-attached patches (Fig. 3, B-D). Sample traces are shown in Fig. 3B. We examined these single K_DR channel currents evoked by a voltage pulse to 10 mV in all transfected cells. We found that neither SNAP-25 nor BoNT/A transfection had any effects on K_DR channel conductance, with all single channel slope conductances calculated to be 10 picosiemens. This is reflected by the observation that the mean unitary channel current amplitude at 10 mV for each of the transfections was not significantly different from control cells, ranging from 0.47 to 0.51 pA (Fig. 3C). However, SNAP-25 overexpression greatly reduced the number of K_DR channel openings (Fig. 3C), and furthermore, the mean K_DR channel open time (7.6 ± 3.7 ms, n = 5, p < 0.05) shown in Fig. 3D was greatly shortened by 59% compared with control cells (18.2 ± 2.3 ms, n = 5). In contrast, cleavage of endogenous SNAP-25 by BoNT/A transfection resulted in a profound increase in the number of channel openings (Fig. 3C) and a prolongation in K_DR channel open time by 150% (45.6 ± 3.2 ms, n = 5, p < 0.05 versus control in Fig. 3D). These altered single channel activities collectively contribute to the observed changes in the macroscopic, whole-cell currents arising from this population of K_DR channels shown in Figs. 1B and 2B.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   SNAP-25 regulation of single KDR channels. A (left), single channel KDR currents were recorded from membrane patches of control HIT cells in the cell-attached configuration. Currents were evoked in response to a random series of test pulses between -40 and +20 mV in 20-mV steps from a holding potential of -70 mV. Each test pulse lasted for 30s, with an interpulse interval greater than 3 min. A (right), current-voltage relationship obtained from single channel studies in which a mean single channel conductance of 10 picosiemens was calculated from the slope of line best-fitted to the data (n = 5). B, five representative single channel KDR currents recorded from cell-attached patches of control cells or cells overexpressing SNAP-25 or BoNT/A under the same experimental conditions described in Fig. 1B. Currents were evoked from transfected cells during a pulse from a holding potential of -70 to -10 mV recorded for 330 ms. C, amplitude histograms for each of these transfections were calculated. SNAP-25 overexpression greatly reduced the number of KDR channel openings over control cells. Conversely, cleavage of SNAP-25 by BoNT/A expression enhanced the number of KDR openings over both control and SNAP-25-transfected cells. KDR channel currents measured at 10 mV were the same magnitude for all transfections (ranging from 0.47 to 0.51 pA), suggesting that channel conductance was not changed. There was no change in KDR channel conductance with any transfection (data not shown). D, frequency versus lifetime histogram of channel openings. A total of greater than 1550 events were analyzed for each of the transfection and the open time distribution fitted to a single exponential with a time constant (). SNAP-25 (n = 5) shortened the duration of the KDR open time, whereas cleavage of SNAP-25 by expressed BoNT/A (n = 5) significantly increased the mean channel open time when compared with the mean KDR channel open time recorded from control cells (n = 5) (p < 0.05 against control cells for all test cells).

The Time-dependent Inhibitory Actions of Exogenous SNAP-25 on K_DR Are Relieved by BoNT/A Proteolysis-- To more clearly distinguish the effects of the overexpressed SNAP-25 on the cell surface expression of K_V1.1 (in Fig. 2A) and the direct effects of SNAP-25 on the K_V1.1 channel, we examined the effects of acutely applied exogenous recombinant SNAP-25 protein (introduced via the patch pipette after membrane rupture) in control (Fig. 4, A-C) and BoNT/A-expressing cells (Fig. 4C). This strategy also allowed us to examine the time course of action of SNAP-25 on K_DR. Furthermore, SNAP-25 overexpression in other cell types leads to chronic effects such as alteration in axonal growth (38) and vesicular trafficking processes (39). It has been previously shown (40) that for a 10-megaohm access-resistance pipette, a protein with a molecular mass of 50 kDa can diffuse into the cytosol with a time constant of 5 min. Because the diffusion time constant varies with the third root of the molecular mass of the diffusing substance, we expected that the concentration of the smaller SNAP-25 (25 kDa) in cytosol would be greater than 72% of the pipette concentration within 5 min. Therefore, we expected to see exogenously applied SNAP-25-mediated effects within 5 min, which was indeed the case.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Acute inhibition of KDR currents by intracellular dialysis of recombinant GST-SNAP-25. A, representative current traces show the reduction of KDR currents with recording time from a control HIT cell dialyzed with109 M GST-SNAP-25. KDR currents were caused by depolarization to +30 mV from a holding potential of -70 mV. B, graphical summary of dose-dependent and time-dependent inhibition of KDR currents by dialysis of GST-SNAP-25 into HIT cells. Steady-state KDR current amplitudes were normalized to corresponding maximal current levels (I/Imax). Data are expressed as mean ± S.E. (n = 5-6). The single asterisk indicates p < 0.05, and the double asterisk indicates p < 0.01 compared with 108 M GST alone. C, inhibitory effect of exogenous SNAP-25 on KDR is relieved by BoNT/A proteolysis. KDR currents were recorded over a 30-min time period with an initial interpulse interval of 12 s followed by a slower interpulse interval of 3 min after the first 3 min. To directly compare these effects, KDR currents obtained from GST-SNAP-25 dialysis in both control and BoNT/A-expressing cells were normalized as a percentage of the initial effect (immediately after cell membrane rupture) of inactive GST-alone dialyzed into control cells. Data are the mean ± S.E. Dialysis of 108 M GST into control cells (, n = 5) had no effect on KDR. GST-SNAP-25 dialyzed into control cells ( n = 4) resulted in prolonged inhibition of KDR currents. GST-SNAP-25 dialyzed into BoNT/A-expressing cells (, n = 5) resulted in an initial inhibition of KDR currents, which paralleled that seen in control cells. This inhibition reversed after the onset of BoNT/A cleavage of GST-SNAP-25, resulting in the complete removal of the inhibitory effect of GST-SNAP-25 on KDR currents after 30 min.

GST-SNAP-25 inhibited K_DR currents in a dose- and time-dependent manner. Fig. 4A shows a representative series of K_DR currents demonstrating a time-dependent reduction in K_DR currents in a control HIT cell dialyzed with 10⁹ M GST-SNAP-25. Fig. 4B is a summary (n = 5-6 cells each) of the dose-dependent inhibition of K_DR by SNAP-25. 10¹⁰ M GST-SNAP-25 had minimal effects on K_DR even 5 min after membrane rupture when compared with GST (10⁸ M) alone, which had no effect (even after up to 30 min, as shown in Fig. 4C). In contrast, 10⁹ M GST-SNAP-25 caused a significant 25% reduction of K_DR currents 4 min after membrane rupture (Fig. 4B). At a higher concentration of GST-SNAP-25 (10⁸ M), the inhibition reached a significant level just 2 min after membrane rupture, with a maximal level of 39% occurring at 5 min (Fig. 4B). In Fig. 4C, we recorded for up to 30 min and found that this maximal inhibition of K_DR by 10⁸ M GST-SNAP-25 was maintained. These acute changes on K_DR currents by exogenous application of SNAP-25 precisely mirrored those caused by SNAP-25 overexpression (Fig. 1B), which supports our thinking that a dominant action of the excess SNAP-25 is to inhibit K_DR currents.

We next wanted to examine the effects of the active cleavage of SNAP-25 by BoNT/A on K_DR activity. To demonstrate this, we dialyzed GST-SNAP-25 into BoNT/A-expressing HIT cells (Fig. 4C). In vivo expression of BoNT/A light chain ensured continued cleavage of SNAP-25 from both endogenous synthesis and exogenous application. The initial K_DR current recorded immediately after membrane rupture in BoNT/A-expressing cells was 23 ± 1% (n = 5, p < 0.05), higher than that recorded in control cells (Fig. 4C). The initial higher K_DR values in the BoNT/A cells is due to cleavage of endogenous SNAP-25, which is expected to be the case before dialysis of exogenous GST-SNAP-25 into the cytosol (Fig. 1B). As we dialyzed GST-SNAP-25 (10⁸ M) into these BoNT/A-transfected cells, we observed a decline in K_DR currents that paralleled the decline seen with GST-SNAP-25 dialyzed into control cells over a similar time period of 12 min after membrane rupture (Fig. 4C). This reached a maximum level of 84 ± 1% (n = 5, p < 0.05) of the initial control cell value, which when compared with the initial value of BoNT/A-transfected cells (123%), was a reduction of 39% (Fig. 4C). This percentage reduction is identical to that seen with GST-SNAP-25 dialysis into control cells. Of note, after 12 min, the K_DR currents in the BoNT/A-transfected cells began to recover as a result of BoNT/A action (Fig. 4C). The time course of this recovery of K_DR currents resulting from BoNT/A cleavage of the dialyzed SNAP-25 is consistent with the predicted enzymatic activity of BoNT/A, as reported by in vitro proteolysis of recombinant SNAP-25 by BoNT/A (41). By 30 min, K_DR activity completely recovered, reaching levels identical to those recorded immediately after initial membrane rupture (Fig. 4C). This indicates complete cleavage of SNAP-25, rendering it incompetent to modulate K_DR.

SNAP-25 Interacts with K_V1.1 and K_V1.1 Subunits-- These functional data therefore suggested the possibility that SNAP-25 could interact with K_v1.1 channel proteins. To test this possibility, we examined whether SNAP-25 is in a complex with the -subunit of the K_V1.1 channel (Fig. 5A). To demonstrate the general applicability of these findings to neurons and since these proteins are less abundant in HIT cells, we performed immunoprecipitation studies using rat brain LP1 synaptosomal fractions (s-LP1) shown in lane 1 to be enriched in SNAP-25 and K_V1.1 proteins (Fig. 5A). Of note, lane 2 shows that a rabbit anti-SNAP-25 antibody generated against the full-length SNAP-25 (22) immunoprecipitated SNAP-25 from the solubilized LP1 fraction and co-precipitated K_V1.1 proteins. In control studies, an ~50-kDa protein was detected that is likely an IgG heavy chain (lane 3) and which was also observed in lane 2. Neither SNAP-25 nor K_V1.1 proteins were precipitated by either the preimmune IgG (lane 3) or protein-G-Sepharose in the lysis buffer (lane 4).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   SNAP-25 interacts with KV1.1 proteins. A, co-precipitation of rat brain SNAP-25 and KV1.1. Lane 1, solubilized LP1 fraction (s-LP1) of rat brain synaptosomes shows clear bands for KV1.1 and SNAP-25 proteins. Lane 2 shows that the SNAP-25 antibody (SNAP-25) immunoprecipitated (IP) SNAP-25 from the solubilized LP1 fraction and co-precipitated KV1.1 channel proteins. Control studies were performed in lanes 3 and 4. Lane 3, control preimmune IgG precipitated an ~50-kDa protein (the IgG heavy chain). Neither protein-G-Sepharose in lysis buffer (lane 4) or preimmune IgG (lane 3) precipitated SNAP-25 or KV1.1 channel proteins. B and C, the KV1.1 () and KV1.1/KV1.1 () channels interact physically with SNAP-25 in oocytes. B, top, digitized PhosphorImager scan of SDSPAGE analysis of [35S]Met/Cys-labeled KV1.1 SNAP-25 proteins co-immunoprecipitated by KV1.1 antibody from homogenates of oocytes that were uninjected (control), injected with KV1.1 (10 ng/oocyte; ) mRNA only, co-injected with SNAP-25 (1.25 ng/oocyte; +SNAP-25), or injected with SNAP-25 alone. B, bottom, homogenates of each group of oocytes were subjected to immunoprecipitation by SNAP-25 antibody. The protein samples were analyzed on a 12% PAGE. The results shown are from one of three independent experiments. C, top, reciprocal co-immunoprecipitation using SNAP-25 antibody, carried out in oocytes that were either uninjected (control), injected with KV1.1 (10 ng/oocyte) together with KV1.1 (30 ng/oocyte) without or with SNAP-25 (10 ng/oocyte) mRNAs ( and +SNAP-25, correspondingly), or injected with SNAP-25 alone (SNAP-25). Bottom, homogenates of each group of oocytes were subjected to immunoprecipitation by KV1.1 antibody. Oocytes used in this experiment had a low level of endogenous proteins migrating as KV1.1 and precipitated by the KV1.1 antibody, as we report previously (42). The protein samples were analyzed on an 8% PAGE. Arrows indicate the relevant proteins. The electrophoretic mobilities of molecular mass standards (size in kDa) are shown along the right (B) or left (C) of each autoradiogram.

We showed earlier that K_v1.1 is in complex with K_v auxiliary subunits in brain synaptosomes and that this channel exists in multimolecular complex consisting also of the t-SNARE, syntaxin 1A (21). Also, we showed that the K_v1.1/K_v1.1 channel interacted physically with syntaxin 1A expressed in Xenopus oocytes. Thus, syntaxin 1A could mediate the interaction between K_V1.1 and SNAP-25 in synaptosomes. To examine whether K_v1.1 could interact directly with SNAP-25 without the mediation of other SNARE proteins or neuronal presynaptic proteins, we employed the heterologous expression system of Xenopus oocytes. We showed by SDS-PAGE analysis of metabolically labeled proteins that SNAP-25 expressed in oocytes co-immunoprecipitated using K_v1.1 antibody with K_v 1.1 () co-expressed alone (; Fig. 5B) or together with K_v1.1 (; not shown). The specificity of the channel interaction with SNAP-25 was verified by reciprocal co-immunoprecipitation of K_v1.1 and K_v1.1 with SNAP-25 using antibody against SNAP-25 (Fig. 5C). The faint immunoreactive bands corresponding to K_v1.1 and K_v1.1 in oocytes injected with without SNAP-25 are probably the result of coprecipitation with the endogenous SNAP-25 homolog(s)² (previously we also detected endogenous syntaxin homolog(s) (21)) of the channel proteins (which were better expressed in these oocytes than in oocytes coexpressing also SNAP-25; compare the expression of K_v1.1 in Fig. 5C, bottom panel, lanes +SNAP-25 and ). As previously shown (42), the K_v1.1 protein is expressed in oocytes mainly in the form of a doublet of ~57 and ~54 kDa polypeptides that are N-glycosylated and represent functional channels.

We then proceeded to determine the domain within the K_v1.1/K_v1.1 protein with which SNAP-25 would directly interact. This study is particularly important since we had detected a small amount of endogenous syntaxin homolog(s) in oocytes (21), which raised the possibility that SNAP-25 binding to the K_v1.1 could be mediated by the binding to the endogenous syntaxin. We have measured the in vitro binding of ³⁵S-labeled SNAP-25 synthesized in reticulocyte lysate to the following bacterially expressed recombinant proteins: GST C, corresponding to amino acids 411-495, i.e. the whole-length C terminus of K_v1.1; GSTT1A and GSTT1B, corresponding to amino acids 1-71 (containing 30 amino acids that comprise T1A domain) and 72-143 of the N terminus of K_v1.1, respectively (both are involved in tetramerization of K_v subunits (4345), and the latter is involved also in K_v subunit binding (46, 47); GST c, corresponding to amino acids 75-397, a region in K_v that is conserved among the different K_v isoforms and is involved in binding to K_v1.1; and full, corresponding to the whole length of K_v1.1 (amino acids 1-397). Fig. 6A shows that SNAP-25 bound to full-length K_v1.1 and to its C terminus, to the N terminus, and preferentially to the T1A domain but not to the C terminus of K_v1.1. Next we set out to test if there was a causative relationship between the direct interaction of the channel with SNAP-25 and the functional interaction that leads to inhibition of the current. To this end we tried to acutely rescue the channel in HIT cells overexpressing SNAP-25 from the functional effects of SNAP-25 by dialysis of a peptide corresponding to a domain of the channel that is involved in SNAP-25 binding. The N-terminal domain of K_v1.1 was chosen for the following considerations. The oocyte results demonstrated that K_v1.1 alone can bind SNAP-25, and the in vitro binding results pointed to its N terminus. Also, the in vitro binding results showed that the C-terminal domain of K_v, a region that is conserved among the different K_v isoforms, does bind SNAP-25. Because this domain binds the N-terminal domain of K_v1.1 (46, 47), we assumed that the impact of its possible physical interaction with SNAP-25 could be transferred to the N terminus of K_v1.1, conferring its functional effect. In any case, whether it interacts directly with SNAP-25 or indirectly via interaction with K_v, excess soluble N-terminal K_v1.1 is likely to interfere with the interaction of the endogenous membrane-bound K_v1.1 protein with SNAP-25. Indeed, dialysis of the recombinant T1A or N termini (amino acids 1-168) but not the T1B (data not shown), C- termini (amino acids 411-495), or GST resulted in the augmentation of the outward current (28-35% over control levels), presumably preventing the functional effect of SNAP-25 by disrupting its physical interaction with the channel proteins (Fig. 6B). We further showed that neither the N- nor C-terminal domains had any effect on K_DR currents of BoNT/A-transfected HIT cells (data not shown). Taken together, these binding and functional data point to a link between the functional effects of SNAP-25 on the K_v1.1 channel and its physical interaction with this channel protein.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Interactions of specific domains of KV1.1 and KV1.1 with SNAP-25. A, top, 200 pmol of GST fusion proteins corresponding to the indicated cytosolic parts of KV1.1 and KV1.1 (see precise sequences under "Results") immobilized on GSH-agarose beads were incubated with 5 µl of in vitro synthesized 35S-labeled SNAP-25 in a 1-ml reaction volume. After elution with glutathione, proteins were separated on 12% gel and analyzed by PhosphorImager to detect the co-precipitation of SNAP-25. A, bottom, Coomassie Blue staining of the same gel shows each of the eluted GST fusion proteins. Numbers refer to mobility of prestained molecular weight standards. Each panel is a representative experiment of three similar experiments. B, N-terminal domains of KV1.1 reverses the inhibitory effects of SNAP-25 on KDR currents in HIT cells. Dialysis of recombinant T1A (108 M, n = 5) or KV1.11-168 (108 M, n = 6) via the patch pipette enhanced the KDR current in SNAP-25-transfected cells. In contrast, dialysis of GST (108 M, n = 6) or the cytoplasmic C-terminus of KV1.1 (amino acids 411-495, KV1.1411-495, 108 M, n = 6) had no effect on the KDR current in SNAP-25-transfected cells. Whole-cell currents were evoked in response to +30 mV from a holding potential of -70 mV just after cell membrane rupture (t = 0) and 6 min later (t = 6 min). Data are the mean ± S.E. summarized in the bar graph (B, lower panel). The asterisk indicates p < 0.05 versus t = 0. pF, picofarads.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Here we present the first evidence that SNAP-25 interacts with and inhibits the K_DR (subtype K_v1.1) channel. Although the overexpressed SNAP-25 had a small effect in reducing the cell surface expression of K_v1.1 (Fig. 2A), dialysis of excess recombinant SNAP-25 protein acutely inhibited K_DR currents in both control and BoNT/A-transfected HIT cells (Fig. 4), which supports a primary inhibitory role of SNAP-25 on the K_v1.1 channel. More importantly, the experiments on BoNT/A cleavage of endogenous SNAP-25 (Figs. 1-3) demonstrated the specific actions of the endogenous SNAP-25 in inhibiting K_DR currents.

Our biophysical analysis showed that SNAP-25 inhibited K_DR activity in part by altering channel kinetics so that the channel assumes a reluctant state by slowing channel activation and enhancing channel slow (C-type) inactivation (Figs. 1-3). Biochemical analysis in Xenopus oocytes and in vitro binding studies revealed that the K_v1.1 channel interacts physically with SNAP-25 both via the membrane pore-forming K_v and the peripheral auxiliary K_v subunits (Figs. 5 and 6A). Both the functional and the physical interactions indicate that the cytosolic N-terminal fragment of K_v, particularly the T1A domain, acutely reverses the inhibitory effects of SNAP-25 on K_DR currents (Fig. 6B). The crystal structures of the K_v subunit and the T1 assembly domain of the N terminus of K_v subunit (48, 49) reveals that submembrane modulating complexes interact with the core domain of a channel responsible for the activation and C-type inactivation gating (50). Very recently it has been demonstrated that a cytosolic C-terminal complex conveys inhibition of HCN pacemaker channel gating (51). Cleavage of SNAP-25 by BoNT/A expression relieved inhibition of K_DR by SNAP-25 by reversing the SNAP-25 effects on K_DR kinetics. This indicates endogenous SNAP-25 has a clamping effect on K_DR.

Even though SNAP-25 can interact with the K_v1.1 channel directly, it is likely that the molecular complex also involves other proteins. In fact, we recently report direct interactions of syntaxin 1A with K_v1.1 channel enhancing its rapid inactivation (21). In that report, we showed that in brain synaptosomes, the K_v1.1 and K_v subunits exist in a macromolecular complex also comprising syntaxin 1A, SNAP-25, and synaptotagmin. Such an interaction between K_v1.1 and the exocytotic SNARE complex proteins is analogous to those described for the Ca²⁺ channels. For example, SNAP-25 inhibited P/Q-type Ca²⁺ channels, and this inhibition could be overcome by the formation of a SNARE complex of SNAP-25 with syntaxin 1A and synaptotagmin I (52). Similar modulation of N- and L-type Ca²⁺ channels by the molecular interactions of these SNARE proteins was also reported (13, 15). Further studies are required to examine whether these SNARE proteins and/or their associated proteins could modulate K_DR channels independently or in complex with SNAP-25. Notably, we also report that the G protein subunits directly interact with the N terminus of K_v1.1 and with K_v1.1 to affect fast (N-type) inactivation gating (27). It will be of interest to examine possible complex functional and physical interactions between SNAP-25, syntaxin 1A, and G protein subunits.

Delayed-rectifier K⁺ channels (K_DR) are important regulators of membrane excitability in neurons and neuroendocrine cells. Because the other major t-SNARE, syntaxin, has been shown to interact directly with and modulate other ion channels such as Na⁺ (17, 18), Ca²⁺ (12, 13, 15, 16), and Cl (19, 20) channels, our findings now extend the repertoire of known SNARE-channel interactions to a member of the important voltage-activated K⁺ channel (K_V) family. Our study shows that BoNT/A-mediated inhibition of secretion is likely due to both the inhibitory effects of SNAP-25 cleavage and also from the release of SNAP-25 inhibition of the K_DR channel protein, which ultimately leads to an inhibition of the sustained phase of Ca²⁺ influx. Wei et al. (53) show that the BoNT/A cleavage product SNAP-25C9 exhibited a novel distinct action on reducing the replenishment of the readily releasable pool in the neuroendocrine chromaffin cell, which also reduced the sustained phase of exocytosis as measured by membrane capacitance. Taken together, these studies suggest that SNAP-25 acts on multiple targets to orchestrate the sequence of events to effect optimal exocytosis, including modulation of the membrane potential as a mechanism to provide the appropriate feedback between these events.

	ACKNOWLEDGEMENTS

We thank Yong Song and Michael Wheeler for technical advice.

	FOOTNOTES

^* This work was supported by grants from the National Institutes of Health (DK55160), the Juvenile Diabetes Foundation, Canadian Diabetes Association, and the Canadian Institute for Health Research (to H. G.), and also by grants from the Israel Science Foundation (437/98) and USA-Israel Binational Foundation (1999396) (to I. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

^¶¶ To whom correspondence should be addressed: Rm. 7226, Medical Sciences Bldg., 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-1526; Fax: 416-978-8765; E-mail: herbert.gaisano@utoronto.ca.

Published, JBC Papers in Press, March 29, 2002, DOI 10.1074/jbc.M201034200

² S. Tsuk, D. Chikvashvili, and I. Lotan, unpublished results.

	ABBREVIATIONS

The abbreviations used are: K_V, voltage-dependent K⁺ channels; K_DR, delayed rectifier K⁺ channels; SNAP-25, synaptosome-associated protein of 25 kDa; t-SNARE, target-SNAP receptor; v-SNARE, vesicle-SNAP receptor; GST, glutathione S-transferase; BoNT/A, botulinum neurotoxin A; TEA, tetraethylammonium; DTX-K, dendrotoxin-K; GFP, green fluorescent protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES