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Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv (T.W-G., I.M., D.C., I.L.); and Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada (L.S., H.Y.G.)
Received December 18, 2005; accepted May 31, 2006
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Abstract |
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, 
, 
) uniformly contain both syntaxin 1A and SNAP-25. However, using immunohistochemistry, we show that the different pancreatic islet cells contain distinct dominant Kv channels, including Kv2.1 in cells and Kv2.2 in and cells, whose interactions with the SNARE proteins would, respectively regulate insulin, glucagon and somatostatin secretion. We therefore examined the regulation by syntaxin 1A and SNAP-25 of these two channels. We have shown that Kv2.1 interacts with syntaxin 1A and SNAP-25 and, based on studies in oocytes, suggested a model of two distinct modes of interaction of syntaxin 1A and the complex syntaxin 1A/SNAP-25 with the C terminus of the channel. Here, we characterized the interactions of syntaxin 1A and SNAP-25 with Kv2.2 which is highly homologous to Kv2.1, except for the C-terminus. Comparative two-electrode voltage clamp analysis in oocytes between Kv2.2 and Kv2.1 shows that Kv2.2 interacts only with syntaxin 1A and, in contrast to Kv2.1, it does not interact with the syntaxin 1A/SNAP-25 complex and hence is not sensitive to the assembly/disassembly state of the complex. The distinct regulation of these closely related channels by SNAREs may be attributed to differences in their C termini. Together with the differential distribution of these channels among islet cells, their distinct regulation suggests that the documented profound down-regulation of islet SNARE levels in diabetes could distort islet cell ion channels and secretory responses in different ways, ultimately contributing to the abnormal glucose homeostasis.
; Spafford et al., 2003) and potassium channels, including Kv (Fili et al., 2001; Ji et al., 2002; Leung et al., 2003; Michaelevski et al., 2003) and KATP channels (Kang et al., 2004; Pasyk et al., 2004). Whereas these SNARE proteins bind highly conserved domains (II-III cytoplasmic loop) within the family of calcium channels (for review, see Spafford et al., 2003), we have noted that the SNARE proteins bind the different Kv channel members, particularly Kv1.1 and Kv2.1, at distinct domains of their cytoplasmic N and C termini (Fili et al., 2001; Michaelevski et al., 2002). These reports raise the hypothesis that these SNARE proteins are capable of distinct regulation of different Kv channel members that are differentially distributed within a single cell type (neuron) or within adjacent interacting cell types, which would thereby more versatilely influence their electrical plasticity and intercellular communication. The most notable examples of the latter are the brain and the pancreatic islet. The pancreatic islet consists of several cell types, including , , and cells, that assert intimate paracrine regulation over each other via their major secretory products to very finely regulate glucose homeostasis (Cejvan et al., 2003; Ishihara et al., 2003; Wendt et al., 2004). It is noteworthy that it has been suggested (based mainly on analysis at the mRNA level) that in the human pancreatic islet, the different islet cells contain distinct dominant Kv channels, including Kv3.1 in cells, Kv2.1 in cells, and Kv2.2 in cells, which would regulate secretion of glucagon, insulin, and somatostatin, respectively (Yan et al., 2004). All these islet cells contain syntaxin 1A and SNAP-25 (Wheeler et al., 1996); remarkably, in human and rodent type-2 diabetes, islet expression of these SNARE proteins are profoundly down-regulated, which we believe would distort the different islet cell ion channels and secretory responses, ultimately contributing to the abnormal glucose homeostasis (Chan et al., 1999; Gaisano et al., 2002; Ostenson et al., 2006). Here, we tested this hypothesis by examining the SNARE complex regulation of the closely related Kv2.1 and Kv2.2 channels. First, we demonstrated the localization of the Kv2.2 protein to human and rat and not cells and, vice versa, the localization of the Kv2.1 protein to and not cells. Islet cells are abundant, constituting 
70% of the islet cells, so that we could previously demonstrate the interaction of Kv2.1 channel with syntaxin 1A and SNAP-25 in these cells (MacDonald et al., 2002; Leung et al., 2003). However, cells are quite sparse, constituting <5% of islet cells, and are thus very difficult to examine (Kanno et al., 2002). We have therefore used the heterologous Xenopus laevis oocyte model to analyze the precise syntaxin 1A and SNAP-25 interactions with islet Kv channels Kv2.1 and Kv2.2.
We have previously characterized the interactions of syntaxin 1A and SNAP-25 with Kv2.1 (Michaelevski et al., 2003). We showed that both activation and inactivation of the channel were affected, depending on the assembly/disassembly of the syntaxin/SNAP-25 complex. On the basis of these results, we suggested a model that describes the modes of interaction of Kv2.1 with syntaxin 1A and with syntaxin 1A/SNAP-25. Furthermore, two sets of data indicated that the C terminus of Kv2.1 may play an important role in these interactions. First, in vitro binding assays have shown that syntaxin 1A and the syntaxin/SNAP-25 complex bind directly to the C terminus of the channel (Michaelevski et al., 2003; Tsuk et al., 2005). Second, the functional interactions were abolished by both C-terminal deletions and by the presence of dominant-negative C-terminal peptides (Tsuk et al., 2005). In this study, we characterized the functional and physical interactions of syntaxin 1A and SNAP-25 with Kv2.2, which is highly homologous to Kv2.1, except for its C terminus, which is the t-SNAREs interacting region. Comparison between Kv2.2 and Kv2.1 highlights different interactions of the t-SNAREs with these two channels. This study demonstrates that though these t-SNARE proteins are well conserved between interacting islet cells (and presumably adjacent distinct brain neurons), they nonetheless exert profoundly distinct actions on even closely related Kv channel isoforms.
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Materials and Methods |
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) were subcloned in pCDNA3. Preparation of mRNAs was as described previously (Levin et al., 1996). Materials and enzymes for molecular biology were purchased from Roche Diagnostics (Mannheim, Germany), 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) were targeted against the following degenerate (Xy denotes three parts of X and one part of y) nucleotide sequences corresponding to SNAP-25: ANSP-1, 5'GATGA(Ga)TC(Ca)(Ct)T(Gc)GA(GA)AG(CT)AC(Cg)CGNCGNATGCT3' (encoding amino acids DESLESTRRML); and ANSP-2, 5'A(CT)GA(Ag)(GA)A(TC)GA(AG)ATGGANGA(AG)AA(Ct)(Cta)T 3' (encoding amino acids REDEMEEN).
Characterization of Kv2.1- and Kv2.2-Specific Antibodies. We characterized a new affinity-purified antibody (N-terminal Ab) directed against an N-terminal peptide sequence of Kv2.1 (amino acids 4-26) that is highly homologous to a corresponding peptide in Kv2.2 (Alomone Labs, not available commercially), and compared it with the antibody directed against a unique C-terminal peptide sequence of Kv2.1 (C-terminal Ab; Alomone Labs). The C-terminal Ab, as expected (Michaelevski et al., 2003), recognized only Kv2.1 and not Kv2.2. In contrast, the N-terminal Ab, peculiarly, recognized only Kv2.2, not Kv2.1. Thus, in rat brain lysate (Fig. 1A, left) the N-terminal Ab did not recognize Kv2.1 that was recognized by the C-terminal Ab. However, it recognized higher molecular mass doublet of proteins [the calculated molecular mass of Kv2.2 (102.11 kDa) is higher than that of Kv2.1 (95.2 kDa)]; the lower band migrated as a Kv2.2 protein expressed in oocytes (Fig. 1B, left); the upper band may correspond to a post translationally processed (e.g., highly glycosylated) form of the protein. In addition, in PC-12 cells (Fig. 1A, right), the N-terminal Ab neither immunoprecipitated (IP) Kv2.1, which was precipitated by the C-terminal Ab nor immunoblotted (IB) Kv2.1 that was precipitated by the C-terminal Ab (at very long exposures, however, a very faint band corresponding to Kv2.1 could be resolved). However, the N-terminal Ab could both precipitate and blot what seems to be Kv2.2, as expected. In accord, in the heterologous expression system of Xenopus laevis oocytes, the N-terminal Ab blotted only expressed Kv2.2 but not Kv2.1 (Fig. 1B, right). However, it precipitated both Kv2.1 and Kv2.2 (Fig. 1B, left), in contrast to the results in PC-12 cells. A plausible explanation for the discrepancy may be that the large concentration of overexpressed channels in oocytes "overcomes" the low affinity of the N-terminal Ab to Kv2.1. We concluded that the N- and C-terminal Abs can differentially recognize Kv2.2 and Kv2.1 proteins in native tissues by immunoblotting, respectively.
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). In brief, cells were suspended in 5 ml of lysis buffer [20 mM Tris, pH 7.5, 5 mM EDTA, 100 mM EGTA, 100 mM NaCl, and 1% Triton X-100 supplemented with protease inhibitor cocktail (Boehringer-Mannheim)], 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, 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 Amersham ECL detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Immunoprecipitation in Oocytes. Oocytes were subjected to immunoprecipitation as described previously (Levin et al., 1995). In brief, immunoprecipitates from 1% Triton X-100 oocyte homogenates were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE; 8% polyacrylamide). Digitized scans were derived by PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and relative intensities were quantified by ImageQuant software.
Confocal Immunofluorescence Microscopy. This was done as previously described for HEK-293 cells transfected with Kv2.1 cDNA using lipofectamine 2000 (Invitrogen Canada Inc., Burlington, ON, Canada) (MacDonald et al., 2002) and for pancreatic tissue sections (Wheeler et al., 1996; Pasyk et al., 2004). Pancreatic tissue sections (10 µm thick) from male Sprague-Dawley rats (200 g) were plated on glass coverslips, fixed with 2% paraformaldehyde, and rinsed in phosphate-buffered saline for 5 min. These were then blocked with 5% normal goat serum with 0.1% saponin for 0.5 h at room temperature and then washed and incubated with the indicated paired primary antibodies (1:50 dilution) for 1 h, including rabbit antibodies to either Kv2.1-C terminus or Kv2.1/Kv2.2-N terminus, against guinea pig anti-insulin (gift from Ray Pederson, University of British Columbia, Vancouver, BC, Canada), mouse anti-glucagon (Sigma, St. Louis, MO), or mouse anti-somatostatin (GeneTex, San Antonio, TX) antibodies. This is then followed by appropriate paired fluorochrome (fluorescein isothiocyanate or Texas Red)-conjugated secondary antisera (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 1 h at 25°C. The coverslips were then mounted on slides in a fading retarder- 0.1% p-phenylenediamine in glycerol, and examined using a laser scanning confocal imaging system (LSM 410; Carl Zeiss, Oberkochen, Germany).
Oocytes and Electrophysiological Recording. X. laevis oocytes were prepared as described previously (Ivanina et al., 1994). Oocytes were injected with 0.05 to 0.25 or 10 ng/oocyte Kv2.1 and Kv2.2 mRNAs for electrophysiological and biochemical experiments, respectively. Syntaxin 1A mRNA was injected at 0.5 to 7.5 and at 5 ng/oocyte for electrophysiological and biochemical experiments, respectively. SNAP-25 mRNA was injected at 0.5 to 15 and at 15 ng/oocyte for electrophysiological and biochemical experiments, respectively. BoNT/A mRNA was injected for both biochemical and electrophysiological experiments at 5 to 15 ng/oocyte. 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 previously (Levin et al., 1995). 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 of more than 3 nA/mV were discarded. Experimental protocols are described in the figure legends.
Data Analysis. For inactivation, data from each oocyte were fitted to a Boltzmann equation: I/Imax = 1/1 + [exp(Vi1/2 - V)/a], and mean values for half-inactivation voltage (Vi1/2) and for the slope factor (a) were derived. For activation, data from each oocyte were fitted to a Boltzmann equation: G/Gmax = 1/1 + [exp(Va1/2 - V)/a]. Parameters describing the activation curves of groups injected with channel together with syntaxin 1A were obtained by fitting the data to a two-component Boltzmann equation: G/Gmax = b/(1 + [exp(Va1/2_1 - V)/a1]) + (1 - b)/(1 + [exp(Va1/2_2 - V)/a2]), where G/Gmax is normalized conductance, Va1/2_1 and Va1/2_2 are half-activation voltages, and a1 and a2 are slope factors.
Data are presented as means ± S.E.M. The statistical significance of differences between two groups was calculated by the use of independent sample t test procedures assuming unequal variance (Mann-Whitney's rank-sum test). One-way ANOVA was used to estimate the statistical differences in experiments comparing several groups.
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Results |
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cells, which are more abundant in the central core of the islet, costained with the C-terminal Ab (in f-h). Next, we examined the N-terminal Ab specificity in confocal microscopy analysis. Figure 2B shows that the N-terminal Ab specifically stained Kv2.2 channels overexpressed in HEK-293 cells, because the staining was completely blocked when the antibody was preabsorbed with the peptide against which the antibody was generated (in a-d). It is noteworthy that this antibody did not stain insulin-containing cells (in e-g), which we showed above to express Kv2.1, thereby verifying that Kv2.1, but not Kv2.2, is present in cells (MacDonald et al., 2001). We then examined which Kv isoform was present in the rat glucagon-containing cells and somatostatin-containing cells, both of which are predominantly present in the mantle or periphery of the pancreatic islet. Using the N-terminal Ab, we were surprised to find that Kv2.2 were present not only in the cells (in k-m), as would be expected, but also in the cells (in h-j). Kv2.2 mRNA has been consistently shown to be present in human islet cells, but the Kv2.2 protein was apparently not explored in the cells. Unfortunately, we could not get the N-terminal Ab to consistently stain the human pancreatic tissue well, probably because of degradation occurring during organ retrieval and preparation (data not shown). The and cells are rather sparse within the islets and are not readily distinguishable from the cells when the islets are dispersed into single cells. Whereas all islet cells contain t-SNARE proteins (Wheeler et al., 1996), each islet cell contains additional distinct Kv channels besides the dominant forms. Therefore, it would be extremely difficult to directly examine only the dominant Kv channels in these cells and their interactions with SNAREs. Hence, we have used the oocyte expression model to examine how the Kv2.1 and Kv2.2 channels in these different islet cells may be regulated by the t-SNARE proteins.
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). In this study, we examined the dependence of the effect on syntaxin 1A concentration (Fig. 3, A-D). We were surprised to find that the leftward shift of Vi1/2 caused by low syntaxin was replaced by a rightward shift in oocytes of the same batch expressing high concentration (2.5 ng/oocyte) of syntaxin 1A (high syntaxin). In addition, high syntaxin, in contrast to low syntaxin, increased the RCF. Neither low nor high syntaxin had a significant effect on the slope factor of the Kv2.1 inactivation curve.
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The effects of syntaxin 1A on the kinetics of onset of inactivation were compared between Kv2.1 and Kv2.2. Inactivation onset was measured as the rate of current decay during 25-s depolarizing pulses and estimated by the inactivation time constant (
i) derived from a single exponential decay fit. High syntaxin increased i (slowed down inactivation) of both Kv2.1 and Kv2.2 by approximately 40 to 50%, determined at +10 to +30 mV, whereas low syntaxin had no effect on i (data not shown).
Effects of Syntaxin 1A on the Voltage Dependence and Kinetics of Activation of Kv2.2 versus Kv2.1. In a previous work (Michaelevski et al., 2003), we have shown that low syntaxin affected the steady-state activation of the Kv2.1 channels. Thus, in oocytes coexpressing Kv2.1 with low syntaxin, the activation curve was fitted with two-component Boltzmann function. One component resembled that for the conductance (G)-voltage curve of Kv2.1 expressed alone [with the same half-activation voltage (Va1/2) and slope factor]. The other component had Va1/2, which is strongly shifted to hyperpolarized potentials with a strong decrease in the slope factor of the activation curve, indicating a steeper dependence on voltage (Fig. 4, A-C). In this study, we examined the effect of a high concentration of syntaxin 1A on the steady-state activation of Kv2.1 (Fig. 4, B and C). We were surprised to find that the hyperpolarizing shift by low syntaxin was eliminated in oocytes of the same batch expressing high syntaxin (Fig. 4, A-C).
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).
In contrast to Kv2.1, syntaxin 1A affected the activation rate of Kv2.2, (Fig. 4, compare E with A and H with D). Activation time constants were derived from exponential fits to current traces elicited at the denoted voltages. For oocytes expressing Kv2.2 alone, one activation time constant was derived, whereas for oocytes also expressing high syntaxin, two activation time constants were derived. One time constant, slow, was the same as that obtained in the absence of syntaxin 1A. The second time constant, fast, was significantly smaller and was not voltage-dependent in all voltages tested. The two time constants may indicate two populations of channels: affected and not affected by syntaxin. No significant changes in current amplitudes were observed in either Kv2.1 or Kv2.2 channels, in the presence of low or high concentrations of syntaxin.
Effects of SNAP-25, Alone and in Combination with Syntaxin, on the Inactivation of Kv2.2. We have shown previously that coexpression of SNAP-25 with Kv2.1 shifted the Vi1/2 to depolarized potentials, enhanced RCF, and increased 
(Michaelevski et al., 2003). Furthermore, we showed that combination of SNAP-25 with syntaxin 1A diminished the hyperpolarizing shift of Vi1/2 caused by low syntaxin and replaced it by a depolarizing shift, similar to the effect of SNAP-25 alone. Here, we examined the effects of SNAP-25, alone and in combination with high syntaxin (which is effective on Kv2.2), on Kv2.2 inactivation. In contrast to Kv2.1, Kv2.2 steady-state inactivation and i (data not shown) were not affected by SNAP-25 alone. In addition, SNAP-25, in combination with syntaxin, did not shift Vi1/2 to depolarized potentials (Fig. 5, A and B). Occasionally, however, the enhancement by syntaxin 1A of RCF was increased in the presence of SNAP-25.
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Effects of SNAP-25, Alone and Combined with Syntaxin 1A, on the Activation of Kv2.2. We have shown that SNAP-25 alone had no effect on Kv2.1 activation (Michaelevski et al., 2003); however, combined with syntaxin 1A, it diminished the hyperpolarizing shift caused by low syntaxin. Here, we examined the effects of SNAP-25, alone and in combination with high syntaxin, on Kv2.2 activation (Fig. 5, C and D). SNAP-25 alone had no effect on Kv2.2 activation, as with Kv2.1. However, in contrast to Kv2.1, in the presence of SNAP-25 and syntaxin 1A, the hyperpolarizing shift was apparent.
Physical Interactions of Syntaxin 1A and SNAP-25 with Kv2.2. Physical interactions were examined by coimmunoprecipitation analysis in oocytes, using antibody against Kv2.2. Figure 6 shows that in oocytes coinjected with Kv2.2 and syntaxin, syntaxin 1A coprecipitated with Kv2.2. However, in oocytes injected with Kv2.2 and SNAP-25, the coprecipitation of SNAP-25 was weak (if any).
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). Thus, because the effects by high syntaxin were similar to those exerted by the syntaxin/SNAP-25 complexes, we set out to examine whether they are dependent on endogenous SNAP-25 and thus may be attributable to complexes of syntaxin 1A with endogenous SNAP-25.
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To this end, we knocked down endogenous SNAP-25 using two approaches and assayed the effects of high syntaxin. First, we expressed the light chain of BoNT/A, which cleaves the last nine residues from the C terminus of SNAP-25 (Turton et al., 2002) and was shown by us to cleave SNAP-25 expressed in oocytes (Michaelevski et al., 2003). The functional efficiency of BoNT/A was verified by the large reduction of the effects of coexpressed SNAP-25: enhancement of the RCF was weakened by 70%, the depolarizing shift of Vi1/2 was abolished (Fig. 7A), and the increase in i was inhibited by 90% (not shown). Next, we examined the effect of high syntaxin in the presence of BoNT/A. As shown in Fig. 7B, coexpression of BoNT/A with high syntaxin abolished the depolarizing shift of Vi1/2, reduced by 60% the enhancement of RCF, and inhibited by 65% the increase in i (data not shown). When the channel was expressed alone, coexpression of BoNT/A had no significant effect on the RCF or on the Vi1/2 (data not shown). These results support the notion that the ability of high syntaxin to affect inactivation requires the presence of endogenous SNAP-25.
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3 mV) in oocytes coexpressing syntaxin 1A; data not shown). In oocytes expressing Kv2.1 alone, ASNP-2 had no effect on the RCF and had inconsistent effects on Vi1/2 (data not shown).
The results of these SNAP-25-knockout experiments were in accord with our notion (see above) that the effects of high syntaxin on Kv2.1 result from recruitment of endogenous SNAP-25 to the channel. Thus, the effects of high syntaxin, SNAP-25, or syntaxin+SNAP-25 reflect effects by the t-SNARE complex. This confirms our suggestion (Michaelevski et al., 2003) that the Kv2.1 channel is a target for either syntaxin 1A alone (low syntaxin) or the t-SNARE complex, because each exerts distinct effects on the activation and inactivation of the channel (Table 1).
As expected from the lack of any significant effect by SNAP-25 alone on Kv2.2 (Fig. 5 and Table 1, right), BoNT/A did not reduce the effects of high syntaxin on Kv2.2 (data not shown), in contrast to Kv2.1. Thus, it seems that Kv2.2 interacts with syntaxin 1A regardless of its association with SNAP-25. The ensuing effects are quite similar to those exerted on Kv2.1 by both syntaxin 1A alone and the syntaxin 1A/SNAP-25 complex (Table 1; in Kv2.2, and not in Kv2.1; in addition, the activation rate is enhanced).
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Discussion |
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The sites of interaction of syntaxin 1A and the syntaxin/SNAP-25 complex with Kv2.1 were previously mapped to the C terminus of the channel (Michaelevski et al., 2003). The site of syntaxin 1A was further mapped down to the proximal quarter of the C terminus (C1a domain) (Tsuk et al., 2005). Kv2.1 and Kv2.2 display high amino acid homology (84.2% amino acid identity) in their N-terminal cytoplasmic domains, core hydrophobic transmembrane regions, and their C1a domains. Thus, it is not surprising that Kv2.2, which harbors a C1a domain that displays 71.9% homology with that of Kv2.1, interacts functionally and physically with syntaxin 1A. However, the differences in the effects of syntaxin 1A between the two channels and the lack of interaction of the syntaxin/SNAP-25 complex with Kv2.2 versus Kv2.1 may be attributed to the involvement of amino acid sequences downstream to threonine 535 or 550 in Kv2.1 or Kv2.2, respectively, which display only 21% identity.
What would be the consequences of these differences on the physiology of cells expressing Kv2.1 versus Kv2.2? As discussed previously (Michaelevski et al., 2003), in cells expressing Kv2.1 (e.g., islet cells) there is a high probability that at resting membrane potential, the channel is associated with syntaxin 1A because of the the high affinity of syntaxin 1A to Kv2.1 and its high abundance. The association with syntaxin 1A shifts the window of voltages at which the channel conducts ("conductivity window"; derived from superposition of the activation and inactivation curves, see (Michaelevski et al., 2003) to hyperpolarized potentials close to the resting potential, stabilizing membrane potential and reducing cell excitability. However, assembly of syntaxin 1A with SNAP-25 (possibly within the context of the whole SNARE complex formation at spots of docked vesicles ready for release) and the interaction of the t-SNARE complex with Kv2.1 shifts the conductivity window to depolarized potentials, enhancing excitability and repolarization of action potentials, thereby facilitating bursting electrical activity. It is noteworthy that in cells expressing the Kv2.2 channel (e.g., islet - and cells), which is not sensitive to the assembly of syntaxin 1A with SNAP-25, the assembled t-SNARE complexes, at sites of docked vesicles, are not expected to relieve the membrane potential stabilizing effect conferred by syntaxin However, we expect that such cells will be less susceptible to these effects, either because of their relatively very high input resistance [e.g., islet cell; (Gopel et al., 2000a)] or because the voltage range of their resting potential and the threshold of action potential that is more negative than that of the conductivity window of Kv2.2 associated with the t-SNAREs [e.g., islet cells; (Gopel et al., 2000b)]. The hyperpolarizing shift of the conductivity window upon Kv2.2-t-SNAREs complex formation may affect the bursting pattern of action potentials by reducing the amplitude of individual action potentials and increasing intraburst intervals.
The distinct Kv2.1/Kv2.2 distribution among neighboring excitable cells in the brain, in various peripheral tissues and in neuroendocrine cells (Sharma et al., 1993; Barry et al., 1995), specifically among the different types of pancreatic islet cells (see Introduction and this study), suggests that these channels might affect interneuronal or interendocrine cell communication (paracrine control), which could be disrupted by impaired expression levels of SNARE proteins occurring in Diabetes (Ostenson et al., 2006) and a number of neurodegenerative diseases (for example, see Honer et al., 2002).
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Footnotes |
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T.W.-G., I.M., and L.S. contributed equally to this work.
ABBREVIATIONS: SNARE, SNAP receptor; t-SNARE, target SNARE; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; HEK, human embryonic kidney; BoNT/A, Botulinum neurotoxin A; AS-ODN, antisense oligodeoxynucleotide; Ab, antibody; IP, immunoprecipitated; IB, immunoblotted; PAGE, polyacrylamide gel electrophoresis; RCF, residual current fraction; NS, nonsense.
Address correspondence to: Prof. Ilana Lotan, Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 Ramat-Aviv. E-mail: ilotan{at}post.tau.ac.il
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