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Endocrinology, doi:10.1210/en.2006-1296
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Endocrinology Vol. 148, No. 5 2157-2167
Copyright © 2007 by The Endocrine Society

Targeting of Voltage-Gated K+ and Ca2+ Channels and Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Proteins to Cholesterol-Rich Lipid Rafts in Pancreatic {alpha}-Cells: Effects on Glucagon Stimulus-Secretion Coupling

Fuzhen Xia, Yuk M. Leung, Gregory Gaisano, Xiaodong Gao, Yi Chen, Jocelyn E. Manning Fox, Alpana Bhattacharjee, Michael B. Wheeler, Herbert Y. Gaisano and Robert G. Tsushima

Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Robert G. Tsushima, Department of Medicine, University of Toronto, 1 King’s College Circle, Room 7308, Toronto, Ontario, Canada M5S 1A8. E-mail: r.tsushima{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pancreatic {alpha}-cells secrete glucagon in response to low glucose to counter insulin actions, thereby maintaining glucose homeostasis. The molecular basis of {alpha}-cell stimulus-secretion coupling has not been fully elucidated. We investigated the expression of voltage-gated K+ (KV) and Ca2+ (CaV) channels, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in pancreatic {alpha}-cells and examined their targeting to specialized cholesterol-rich lipid rafts. In {alpha}-cells, we detected the expression of KV4.1/4.3 (A-type current), KV3.2/3.3 (delayed rectifier current), CaV1.2 (L-type current), CaV2.2 (N-type current), and the SNARE (synaptosomal-associated protein of 25 kDa, syntaxin 1A, and vesicle-associated membrane protein 2) and SNARE-associated proteins (Munc-13–1 and Munc-18a). We also detected caveolin-2, a structural protein of cholesterol-rich lipid rafts. Of these proteins, caveolin-2, KV4.1/4.3, CaV1.2, and SNARE proteins (syntaxin 1A, synaptosomal-associated protein of 25 kDa, and vesicle-associated membrane protein 2) target to lipid raft domains on {alpha}-cell plasma membranes. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-ß-cyclodextrin decreased the association of KV4.1/4.3, CaV1.2, and SNARE proteins with lipid rafts. This resulted in inhibition of A-type KV currents and enhancement of glucagon secretion from {alpha}-cells. Consistently, capacitance measurements of exocytosis of single {alpha}-cells showed enhanced exocytosis after membrane cholesterol depletion. Taken together, our results demonstrate the association of KV4, CaV1.2, and SNARE proteins with lipid rafts in pancreatic {alpha}-cells. Glucagon secretion from {alpha}-cells is regulated by lipid rafts, and the dissociation of SNARE proteins from cholesterol-rich lipid raft domains enhances glucagon secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCAGON-SECRETING {alpha}-CELLS AND insulin-secreting ß-cells are the most important cells in pancreatic islets for controlling glucose homeostasis. However, the precise molecular basis of stimulus-secretion coupling in {alpha}-cells has not been fully elucidated compared with our knowledge of the neighboring ß-cells. A model has been proposed by which low glucose stimulates glucagon release in mouse {alpha}-cells (1, 2, 3). However, recent data have demonstrated conflicting evidence on {alpha}-cell stimulus-secretion coupling. Single rat {alpha}-cell stimulus-secretion coupling was shown to mirror that of ß-cells, in which glucose stimulates glucagon secretion (4, 5). Similar findings have been shown in mouse islets and In-R1-G9 clonal {alpha}-cells (6). The action of glucose on {alpha}-cell glucagon secretion does not seem to be mediated by mitochondrial oxidative metabolism as in pancreatic ß-cells, because glucose does not provoke significant changes in {alpha}-cell metabolism (7) or in ATP/ADP ratio (8). There is additional evidence to support the theory that the cross-talk between islet {alpha}- and ß-cells plays an important role in regulating {alpha}-cell stimulus-secretion coupling. This suggests glucose itself as well as other contributing factors such as paracrine regulation by neighboring ß- and {delta}-cells are involved in controlling glucagon release (4, 9, 10).

Ion channel activity in {alpha}-cells has been characterized by their electrophysiological properties (1, 2, 3, 11, 12), but the molecular identity of these ion channels in {alpha}-cells has not been fully determined (13, 14, 15). Two types of voltage-gated K+ (KV) currents, A-type tetraethylammonium (TEA)-resistant transient KV currents and TEA-sensitive delayed rectifier KV currents, have been recorded in {alpha}-cells (1, 2, 3). In human {alpha}-cells, expression of mRNA transcripts of KV3.1 and silent KV6.1 have been reported, but corresponding A-type KV channel transcripts (KV1.4, KV3.4, and KV4) were not detected in these cells (15). KV4.3 have been found to be expressed in mouse {alpha}-cells by confocal microscopy (16), which would presumably carry the A-type KV currents in pancreatic {alpha}-cells.

High voltage-activated L-type and N-type voltage-gated Ca2+ (CaV) currents and low voltage-activated T-type CaV currents have been recorded in mouse and rat {alpha}-cells (2, 3, 17, 18, 19). Work by the Rorsman group (12) demonstrated blockade of N- but not L-type CaV channels impairs glucagon release and exocytotic activity in mouse {alpha}-cells. L-type CaV channels can regulate glucagon secretion but only when intracellular cAMP levels are elevated (18). However, a recent study by Vignali and colleagues (20) observed a lack of expression of N-type (CaV2.2) and T-type CaV channels in mouse {alpha}-cells by single-cell PCR and patch clamp experiments. The reason for these differences is not currently known but may be due to genetic differences in the mouse strains (NMRI vs. C57BL/6).

Lipid rafts are membrane microdomains composed mainly of cholesterol and sphingolipids (21, 22), which are insoluble to cold Triton X-100. These detergent-resistant properties of lipid rafts and their buoyancy on sucrose gradients have been used for purification of these plasma membrane microdomains. Lipid rafts have garnered much interest due to the targeting of numerous membrane proteins to these domains and the association of caveolin in normal cell biology and in the pathogenesis of a number of diseases including diabetes (22). We recently reported the expression of caveolin in pancreatic ß-cells and the targeting of KV2.1, CaV1.2, and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 1A, synaptosomal-associated protein of 25 kDa (SNAP-25), and vesicle-associated membrane protein 2 (VAMP-2) to lipid raft microdomains in ß-cells (23). That work indicated that membrane compartmentalization of ion channels and SNARE proteins in ß-cells play an essential role in regulating insulin release from ß-cells. In this present work, we explored whether cholesterol-rich lipid rafts play a fundamental role in regulating stimulus-secretion coupling in pancreatic {alpha}-cells to regulate glucagon secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Mouse {alpha}TC6 cells (kindly provided by Dr. Y. Moriyama, Okayama University, Japan) were grown and maintained in DMEM (Sigma Chemical Co., Oakville, Ontario, Canada) containing 25 mM glucose and supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine.

Pancreatic islet isolation and dispersion
Pancreatic islets from rats and mouse insulin promoter (MIP)-green fluorescent protein (GFP)-transgenic mice (kindly provided by Dr. M. Hara, University of Chicago) were isolated by collagenase digestion as described previously (23). Islets were dispersed into single cells with 0.25% trypsin in Ca2+- and Mg2+-free Hanks’ balanced salt solution (Invitrogen, Burlington, Ontario, Canada). Both intact islets and dispersed islet cells were cultured in RPMI 1640 medium containing 5 mM glucose supplemented with 10% fetal bovine serum, 0.25% HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cultured islet cells were used within 2 d.

RNA preparation and quantitative PCR
Total RNA was isolated from a monolayer of {alpha}TC6 cells using TRIzol (Invitrogen) following the manufacturer’s protocol. Subsequent DNase I treatment was performed to remove any residual DNA contamination (QIAGEN, Mississauga, Ontario, Canada). One microgram of the isolated RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase according to the manufacturer’s instructions (Invitrogen).

Quantitative PCR (qPCR) was performed as described previously (24). Primers were designed using Primer Express version 2.0 software (Applied Biosystems, Foster City, CA) (see Table 1Go), and sequence specificity confirmed before use. Master mix was aliquoted to a 384-well plate (Applied Biosystems) with each well containing 4 µl DNA, 2.85 µl H2O, 1 µl 10x PCR buffer, 0.2 µl Rox, 0.2 µl primer mix (or 0.1 µl forward, 0.1 µl reverse, 50 µM stock each), 1.2 µl 25 mM MgCl2, 0.2 µl dNTP mix (10 mM each), 0.025 µl Platinum Taq polymerase, 0.3 µl 1:1000 SYBR Green I (all components from Invitrogen). Ten nanograms of {alpha}TC6 cDNA per well were used as the template for amplification. The real-time PCR protocol employed was as follows: heat activation of polymerase at 95 C for 3 min, followed by 40 cycles of 95 C for 10 sec, 65 C for 15 sec, and 72 C for 20 sec. Readings were carried out on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) and compared against a standard curve created from mouse genomic DNA. Data were normalized to the expression of ß-actin in each sample.


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  		TABLE 1. Primer sequences of KV channels

 
Immunoblotting
Western blotting was performed to analyze the protein expression of ion channels and SNARE proteins. The cell lysates and sucrose gradient fractions were subjected to SDS-PAGE and transferred to polyvinylidene difluoride-plus membranes (Fisher Scientific Ltd., Nepean, Ontario, Canada). Membranes were probed with the indicated primary antibodies: anti-KV2.1, -KV3.2, -KV3.3, -KV3.4, -KV4.1, -KV4.2, -KV4.3, -CaV1.2, and -CaV2.2 from Alomone Laboratories (Jerusalem, Israel); anticaveolin and anti-Munc-18a from BD Biosciences (Mississauga, Ontario, Canada); anti-SNAP-25 and syntaxin 1A from Sigma; anti-VAMP-2 generated as described previously (25); and anti-Munc-13–1 kindly provided to us by Dr. N. Brose (Max-Planck-Institut für Experimentelle Medizin, Göttingen, Germany). The bound primary antibodies were detected with appropriate peroxidase-conjugated secondary antimouse or antirabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) and then visualized by chemiluminescence (ECL-Plus, GE Healthcare, Mississauga, Ontario, Canada) and exposure to x-ray films (Eastman Kodak Co., Rochester, NY).

Confocal immunofluorescence microscopy
Dispersed rat islet cells were fixed in 2% formaldehyde for 0.5 h at room temperature, blocked with 5% normal goat serum and 0.1% saponin for 0.5 h at room temperature, and then double immunolabeled with anticaveolin (1:200 dilution; BD Biosciences) and antiglucagon (1:400 dilution; Sigma) antibodies for 2 h at room temperature. The coverslips were rinsed with 0.1% saponin in PBS and then incubated with fluorescein isothiocyanate (FITC)-labeled antirabbit and Texas Red-labeled antimouse antibodies for 1 h at room temperature, and mounted on slides in a fading retarder (0.1% p-phenylenediamine in 90% glycerol). {alpha}TC6 cell lines and dispersed MIP-GFP mouse islet cells were used for the detection of membrane localization of SNAP-25 and syntaxin 1A. The same procedure was performed as above except different antibodies were used, anti-SNAP-25 and syntaxin 1A (Sigma) and FITC- (for {alpha}TC6) or tetramethylrhodamine isothiocyanate-labeled (for MIP-GFP islets) antimouse antibodies. Images were obtained using a Zeiss LSM 410 laser scanning confocal imaging system (Carl Zeiss, Oberkochen, Germany).

Lipid raft isolation
{alpha}TC6 cells were harvested and lysed by sonication with cold 1% Triton X-100 in 2-(N-morpholine)-ethane sulfonic acid (MES)-buffered saline [MBS, 25 mM MES, 150 mM NaCl (pH 6.5), supplemented with protease inhibitors]. Lysed cells were centrifuged at 2000 rpm for 15 min at 4 C. The supernatant was diluted with equal volume of an 80% sucrose solution in MBS (with 1% Triton X-100) and placed into the bottom of an ultracentrifuge tube. The 30 and 5% sucrose solutions in MBS were loaded on top of the 40% sucrose-Triton X-100 {alpha}TC6 lysate sequentially to form a discontinuous sucrose gradient, and the sample was centrifuged at 39,000 rpm in a Beckman SW41 rotor for 20 h at 4 C. Twenty gradient fractions (600 µl each) were collected from the top, and 10–30 µl of each fraction was loaded onto an SDS-PAGE gel for Western blot analysis. To deplete membrane cholesterol, {alpha}TC6 cells were incubated with 10 mM methyl-ß-cyclodextrin (MßCD) for 30 min at 37 C.

Glucagon secretion assay
Krebs-Ringer bicarbonate (KRB) buffer containing (in mM) 129 NaCl, 5 NaHCO3, 4.8 KCl, 1.2 KH2PO4, 2.5 CaCl2, 2.4 MgSO4, 10 HEPES, and 0.1% BSA was used for the glucagon secretion assay from mouse pancreatic islets. The isolated islets were cultured overnight in RPMI 1640 medium containing 11 mM glucose supplemented with 10% fetal bovine serum. The following day, 15–30 islets were washed once with KRB and preincubated for 30 min in 500 µl KRB supplemented with 16.7 mM glucose with or without 10 mM MßCD. After the preincubation period, islets were washed once and then stimulated with 1 mM glucose (in 500 µl KRB) for 1 h at 37 C, and the supernatants containing stimulated glucagon were then separated from the islets. Islets were washed with ice-cold PBS, harvested, lysed with 1% Triton X-100, and determined for protein concentration. Samples were kept at –20 C until assayed for glucagon using a RIA kit (Linco Research, Inc., St. Charles, MO), and the values of the released glucagon in the supernatants were normalized to the total protein in the cell lysate.

Electrophysiology
We recently reported the electrophysiological characterization of {alpha}- and ß-cells in MIP-GFP mice, where ß-cells express GFP (26). Primary {alpha}-cells in these MIP-GFP mice were selected from dispersed islet cells by being nongreen cells, with a size of 3 pF or less, and displayed characteristic voltage-gated Na+ currents at a holding potential of –80 mV (3). Single {alpha}-cells were voltage clamped in the whole-cell configuration using an EPC-10 amplifier and Pulse software (HEKA Electronik, Lambrecht, Germany). Patch electrodes were fabricated from 1.5-mm thin-walled borosilicate glass and polished to a tip resistance of 3–4 M{Omega} when filled with intracellular solution. The pipette solution for KV current measurements contained (in mM) 140 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, and 10 HEPES (pH 7.2, adjusted with KOH). For KATP current measurements, ATP was reduced to 0.1 mM. The bath solution for KV current measurements consisted of (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 5 D-glucose, and 10 HEPES (pH 7.4, adjusted with NaOH). TEA-chloride (20 mM) was included when measuring KATP currents. For the measurement of CaV currents, pipettes were filled with (in mM) 120 CsCl, 20 TEA-chloride, 5 EGTA, 5 MgATP, 1 MgCl2, and 10 HEPES (pH 7.2, adjusted with CsOH). The external solution for CaV currents comprised (in mM) 100 NaCl, 20 BaCl2, 20 TEA, 4 CsCl, 1 MgCl2, 5 D-glucose, and 10 HEPES (pH 7.4, adjusted with NaOH). TEA was used to block KV currents. Current recordings were performed at room temperature (~22 C) and normalized to cell capacitance. To elicit KV current, cells were held at –80 mV and depolarized from –80 to +60 mV in 10-mV increments using 500-msec step pulses. CaV currents were triggered by depolarizing voltage pulses (–70 to +70 mV, 500 msec) with membrane potential held at –80 mV.

Membrane capacitance measurement
Exocytosis of primary mouse {alpha}-cells from MIP-GFP mice was detected by the measurement of changes of cell capacitance. Recording electrodes were coated with orthodontic wax (Butler, Guelph, Ontario, Canada) close to the tips and fire polished. Pipette resistance ranged from 3–5 M{Omega} when pipettes were filled with the intracellular pipette solution, which contained (in mM) 125 K-glutamate, 10 KCl, 10 NaCl, 1 MgCl, 5 HEPES, 0.08 EGTA, 0.1 cAMP, and 4 MgATP (pH 7.1, adjusted with KCl). The extracellular solution consisted of (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 5 D-glucose (pH 7.3, adjusted with NaOH). Some {alpha}-cells were preincubated with the extracellular solution containing 10 mM MßCD for 30 min at 37 C before recordings. Membrane capacitance (Cm) was estimated by the Lindau-Neher technique (27), implementing the sine + DC feature of lock-in model (40 mV peak to peak and a frequency of 500 Hz) in the standard whole-cell configuration. Recordings were conducted using an EPC-10 patch clamp amplifier and Pulse software. Exocytotic events were elicited by a train of eight 500-msec depolarizing pulses (1-Hz stimulation frequency) from –70 to 0 mV. All recordings were performed at 30 C.

Statistical analysis
Data points represent mean ± SEM. An unpaired Student’s t test was used to compare control values from MßCD-treated-groups. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of caveolin in pancreatic {alpha}-cells
The presence of caveolin-2 in non-ß islet cells (23) motivated us to examine the function of lipid rafts in {alpha}-cells. We first demonstrate the expression of caveolin-2 in the mouse clonal {alpha}-cell line {alpha}TC6 by Western blot (Fig. 1AGo) and in primary rat pancreatic {alpha}-cells by confocal immunofluorescence microscopy (Fig. 1Go, B and C). In dispersed rat islet cells, caveolin-2 colocalized with glucagon in {alpha}-cells, and is also present in ß-cells (Fig. 1Go, B and 1C). We did not observe caveolin-1 or muscle-specific caveolin-3 in either {alpha}TC6 or primary rat {alpha}-cells (data not shown). Moreover, as we observed in ß-cells (23), caveolin-2 expression was primarily intracellular, possibly on glucagon granules. This, however, is not unexpected because both islet endocrine and exocrine secretory granular membranes are rich in cholesterol and caveolin (28, 29).


Figure 1
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  		FIG. 1. Expression of caveolin in glucagon-secreting {alpha}-cell lines and rat primary {alpha}-cells. A, Western blotting detection of caveolin-2 in {alpha}TC6 cells. Lysates from {alpha}TC6 cells (50 µg protein) and rat brain (25 µg protein) were loaded in each lane. B, Confocal microscopy of dispersed rat islet {alpha}-cells. Caveolin-2 (green) colocalized with glucagon (red), indicating the expression in {alpha}-cells. Note that ß-cells (larger cells) also express caveolin-2. C, Higher magnification of an {alpha}-cell showing the intracellular colocalization of caveolin-2 with glucagon. Scale bars, 20 µm (B) and 5 µm (C).

 
Depletion of membrane cholesterol enhanced low-glucose-stimulated glucagon secretion
To determine the roles of caveolin and lipid rafts in modulating glucagon secretion in {alpha}-cells, we depleted membrane cholesterol with MßCD, a cyclic oligosaccharide that is highly specific for cholesterol removal from membranes (30). To confirm the effect of cholesterol depletion with MßCD treatment, cholesterol content was measured from cultured {alpha}TC6 cells and normalized to the total cell protein. The content of cholesterol was reduced from 0.91 ± 0.05 pmol/µg protein to 0.38 ± 0.03 pmol/µg protein (n = 4; P < 0.01). MIP-GFP mouse islets were treated with 10 mM MßCD for 30 min before measuring glucagon secretion under low-glucose conditions. Remarkably, MßCD pretreatment enhanced glucagon secretion from 7.2 ± 1.3 pg/hr·µg protein to 15.0 ± 1.5 pg/hr·µg protein (n = 5; P < 0.05) after MßCD pretreatment. Given that MßCD enhances insulin secretion under high-glucose conditions, which itself will modulate glucagon release (10, 31), we did not perform the experiments in the presence of high glucose. Because ion channels and SNARE proteins are critical for the regulation of stimulus-secretion coupling in endocrine cells, we therefore proceeded to identify the specific ion channels and SNARE proteins in {alpha}-cells. Furthermore, we examine which of these {alpha}-cell ion channels and SNARE proteins are modulated by lipid rafts to influence glucagon secretion.

Expression of ion channels and SNARE proteins in {alpha}-cells
Because the characterization of {alpha}-cell ion channels has been based primarily on electrophysiological measurements, we first directed our effort to determine the molecular identities of {alpha}-cell KV and CaV channels in the {alpha}TC6 cell line. First, qPCR evaluation of {alpha}TC6 cells revealed high levels of KV2.1, KV3.3, KV3.4, and KV6.3 mRNA (normalized to ß-actin levels), with lower abundance of KV4.1 and KV4.3 (Fig. 2AGo). Barely detectable levels of the KV1 family of channels were observed. We confirmed the qPCR data showing the presence of KV3.2, KV3.3, KV4.1, and KV4.3 channel protein (Fig. 2BGo). Interestingly, KV2.1 and KV3.4 protein was not detected by Western blot analysis. One possibility is that either the protein expression level is not high enough to be detected by Western blotting or these two channel proteins in {alpha}TC6 cells were not recognized by the antibodies used. Another reason could be due to translational inhibition of these channels. Genomic and proteomic studies have recently indicated that there is a large discrepancy between mRNA and protein levels (32). Lastly, we show both CaV1.2 (L-type Ca2+ channels) and CaV2.2 (N-type Ca2+ channels) are expressed in these cells (Fig. 2CGo), as has been demonstrated in primary mouse {alpha}-cells (12, 33).


Figure 2
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  		FIG. 2. Expression of KV and CaV channels, and SNARE proteins in {alpha}TC6 cells. A, The qPCR results of KV channel mRNA levels in {alpha}TC6 cells normalized to message levels of ß-actin. Results are the mean ± SE from four to nine experiments from three independent RNA samples. B–D, Western blot analysis of KV (B), CaV (C), and SNARE (D) protein expression in {alpha}TC6 cells (50 µg cell lysate per lane). Rat brain (2.5–25 µg) was used as a positive control.

 
McGirr and colleagues (34) have shown the expression of syntaxin 1A, SNAP-25, and VAMP-2 in the {alpha}TC6 cells, which we now confirm, along with the presence of SNARE-associated proteins Munc-13–1 and Munc-18a (Fig. 2DGo). The expression of these exocytotic proteins implicates the important roles they may play in {alpha}-cell exocytosis, as has been well established in ß-cells (25).

KV4.1/4.3, CaV1.2, and SNARE proteins target to lipid rafts in {alpha}-cells
Cholesterol-rich lipid rafts are characterized by their resistance to cold Triton X-100 solubilization. Discontinuous sucrose gradient ultracentrifugation and Western blot were performed to isolate and verify the presence of targeted proteins to Triton X-100-resistant cholesterol-rich lipid rafts in {alpha}TC6 cells. Caveolin-2 migrated to the 5–30% sucrose interface (Fig. 3AGo), confirming its targeting and the identification of the cholesterol-rich lipid raft domains in {alpha}-cells. KV4.1/4.3, CaV1.2, and SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2 are also targeted to these cholesterol-rich lipid rafts (Figs. 3BGo and 4Go). In contrast, KV3.2, KV3.3, CaV2.2, and the SNARE-associated proteins Munc-13–1 and Munc-18a were not associated with lipid rafts (Figs. 3BGo and 4Go). To verify this membrane compartmentalization of ion channels and SNARE proteins with lipid rafts, cells were preincubated with 10 mM MßCD at 37 C for 30 min to reduce membrane cholesterol levels. This technique has been used to confirm the association of proteins with lipid rafts (35). Remarkably, MßCD treatment shifted KV4.1/4.3, CaV1.2, syntaxin 1A, SNAP-25, and VAMP-2 out of the Triton X-100-resistant fraction (Figs. 3BGo and 4Go, lower panels of each blot pair).


Figure 3
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  		FIG. 3. Targeting of ion channels to lipid rafts in {alpha}TC6 cells. {alpha}TC6 cells were lysed with 1% Triton X-100, and lipid raft fractions were isolated from a 5–40% discontinuous sucrose gradient ultracentrifugation followed by Western blot for the detection of the targeted caveolin (A) and ion channels (B). The interface between 5 and 30% sucrose denotes the Triton X-100-resistant lipid raft fractions. Western blots on cells treated with 10 mM MßCD for 30 min at 37 C are shown in the lower panels of each blot pair to confirm targeting to cholesterol-rich lipid rafts.

 

Figure 4
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  		FIG. 4. Targeting of SNARE proteins to lipid rafts in {alpha}TC6 cells. Cells were treated as described in Fig. 3Go, and cell lysate was subjected to ultracentrifugation on a discontinuous sucrose gradient. Membranes were probed with antibodies for the different SNARE and SNARE-associated proteins. Confirmation of SNARE protein targeting to lipid rafts was performed as described in Fig. 3Go.

 
Cholesterol depletion inhibits KV4 current amplitude but not CaV currents
We next examined the consequence of MßCD disruption of lipid rafts on MIP-GFP islet {alpha}-cell KV4 and CaV1.2 channel gating. We had previously characterized the {alpha}-cell ion channel properties in MIP-GFP mice (26). Whole-cell recordings revealed robust KV currents from control {alpha}-cells, displaying both inactivating TEA-insensitive (A-type) and noninactivating (delayed rectifying) TEA-sensitive KV currents (Fig. 5AGo). Depletion of membrane cholesterol from {alpha}-cells resulted in the disappearance of the A-type KV currents, whereas the delayed rectifying KV currents were not affected (Fig. 5BGo). The current-voltage relationship showed a significant reduction in peak outward KV currents after cholesterol depletion with MßCD at all voltages between –60 and +60 mV (P < 0.05, n = 6) compared with the control group (Fig. 5CGo). To verify whether the decreased A-type KV current amplitude was associated with the enhanced glucagon secretion, we tested the effects of heteropodotoxin, a highly specific toxin blocker of KV4 family channels (36). However, using this toxin (500 nM) on isolated mouse islets, we observed a reduction in static glucagon release from 2.96 ± 0.49 pg/hr·µg protein to 1.58 ± 0.53 pg/hr·µg protein (n = 5; P < 0.05). This is similar to the findings with the nonselective A-type K+ channel blocker, 4-aminopyridine, which decreased glucagon secretion from mouse islets (1). These results suggest the impaired A-type KV current function does not contribute to the increase in glucagon release after cholesterol depletion.


Figure 5
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  		FIG. 5. Effects of MßCD on KV currents in isolated mouse primary {alpha}-cells. Whole-cell currents were measured from dispersed islets cells from MIP-GFP mice. Cells were held at –80 mV, and currents were elicited by 10-mV depolarizing steps (500 msec) from –80 to +60 mV. A, Whole-cell recordings from a control {alpha}-cell shows robust KV currents displaying both inactivating (A-type) and noninactivating (delayed rectifying) KV currents. B, Incubation of islets cells with 10 mM MßCD for 30 min at 37 C before whole-cell recordings resulted in the disappearance of the A-type KV currents. The insets show KV currents measured from –80 to –20 mV. No A-type currents were observed at the more negative step potentials when pretreated with MßCD. C, Current-voltage relationship of control and MßCD-treated {alpha}-cells (n = 6). There is a significant reduction (*, P < 0.05 compared with the MßCD group) of KV currents from –60 to +60 mV, after cholesterol depletion.

 
Glucagon secretion is highly dependent on the activity of CaV and ATP-sensitive K+ (KATP) channels. To determine whether the enhanced glucagon release after membrane cholesterol depletion is associated with increased CaV channel activity and possible change of KATP channels, we recorded CaV and KATP currents from MIP-GFP mouse {alpha}-cells. However, we were unable to observe a significant change in CaV current amplitude or gating after treatment with MßCD (Fig. 6AGo), or changes of KATP currents (Fig. 6BGo). These results are similar to our observations with ß-cells, suggesting changes in the surrounding lipid environment do not influence CaV and KATP channel properties in either of these neuroendocrine cells.


Figure 6
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  		FIG. 6. Cholesterol depletion has no effect on CaV or KATP currents in mouse {alpha}-cells. Whole-cell currents were measured from MIP-GFP mouse {alpha}-cells. A, Whole-cell current-voltage relationship of CaV channels from control (n = 12) and MßCD-treated {alpha}-cells (n = 9); B, KATP channels (n = 3 in both control and MßCD-treated {alpha}-cells).

 
The integrity of SNAP-25 and syntaxin 1A clusters depends on cholesterol
Our findings demonstrating the targeting of the SNARE proteins to {alpha}-cell lipid rafts prompted us to further investigate the role lipid rafts may play on regulating these proteins. SNAP-25 and syntaxin 1A are important SNARE proteins in the secretory machinery. Confocal immunofluorescence microscopy was performed to visualize the SNAP-25 (Fig. 7AGo) and syntaxin 1A (Fig. 7BGo) on the plasma membranes. {alpha}TC6 cell lines and dispersed pancreatic islet cells from MIP-GFP mice were labeled with anti-SNAP-25 and anti-syntaxin 1A antibodies and fluorescent secondary antibodies. Primary {alpha}-cells were distinguished from the green ß-cells of the dispersed MIP-GFP mouse islet cells. Both SNAP-25 and syntaxin 1A demonstrated punctate labeling on the plasma membrane. Similar clustering patterns have been observed in ß-cells (37). To examine the roles of cholesterol on the integrity of SNAP-25 and syntaxin 1A membrane distribution, cells were incubated with 10 mM MßCD at 37 C for 30 min. MßCD treatment led to significant change in the labeling patterns of SNAP-25 and syntaxin 1A in both {alpha}TC6 cells and primary mouse {alpha}-cells. The punctate spots on the membranes were less apparent, leading to a more uniform labeling pattern on the membranes. This might be caused by the breakup of existing SNAP-25 and syntaxin 1A clusters, as has been observed in pancreatic ß-cells (37). These data, together with the Triton X-100 insolubility data, demonstrate that both SNAP-25 and syntaxin 1A were targeted to lipid rafts.


Figure 7
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  		FIG. 7. Integrity of SNAP-25 and syntaxin 1A clusters depends on cholesterol. Confocal immunofluorescence microscopy shows the clustering (punctate labeling) of SNAP-25 (A) and syntaxin 1A (B) on plasma membranes. {alpha}TC6 cell lines and dispersed pancreatic islet cells from MIP-GFP mice were labeled with anti-SNAP-25 or anti-syntaxin 1A antibodies, followed by the antibodies labeled with FITC (green, for {alpha}TC6 cells) or with tetramethylrhodamine isothiocyanate (red, for primary mouse {alpha}-cells, which can be distinguished from the GFP-transgenic green ß-cells). Treatment of the cells with 10 mM MßCD led to significant change in the labeling patterns of both SNAP-25 and syntaxin 1A in both {alpha}TC6 cells (top panels of A and B) and primary mouse {alpha}-cells (lower panels of A and B). Scale bar, 10 µm.

 
Depletion of membrane cholesterol-enhanced {alpha}-cell exocytosis
Because the depletion of cholesterol with MßCD disrupted SNAP-25 and syntaxin 1A on plasma membranes (Fig. 7Go), we examined how this would influence the kinetics of exocytosis. Cholesterol depletion has been shown to enhance exocytosis in other neuroendocrine cells in part by affecting the association of SNAP-25 with lipid rafts (38, 39). To examine exocytosis directly, we assessed the changes in Cm ({Delta}Cm) from dispersed single mouse {alpha}-cells evoked by a train of eight 500-msec depolarizing pulses (1-Hz stimulation frequency) from –70 to 0 mV, a protocol that reveals the initial size of the primed immediately releasable pool (first two pulses) and extent of refilling of this pool from the reserve pool (third to eighth pulses) (40). Figure 8Go shows that treatment of {alpha}-cells with 10 mM MßCD increased exocytosis of glucagon granules only from the readily releasable pool, but not the rate of granule mobilization from the reserve pool.


Figure 8
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  		FIG. 8. Single-cell exocytosis measured from {alpha}-cells. A and B, Representative changes in cell capacitance ({Delta}Cm) measured by a train of eight 500-msec depolarizing pulses (1-Hz stimulation frequency) from –70 to 0 mV from a control {alpha}-cell (A) and a separate {alpha}-cell pretreated with 10 mM MßCD for 30 min (B); C, summarized changes in Cm and plotted as a function of the pulse number. All values are mean ± SE of five to nine cells. *, P < 0.05 compared with the control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
KV and CaV channels in {alpha}-cells
The mechanism underlying glucose-dependent {alpha}-cell stimulus-secretion coupling has not been fully elucidated, and most of the data on {alpha}-cell ion channels have been based on electrophysiological characterization. We focused our attention on the expression of KV and CaV channels and how changes in membrane cholesterol regulate the function of these channels and impact on glucagon secretion. KV channels play an important role in regulating stimulus-secretion coupling in {alpha}-cells (1, 3). Whereas two types of KV currents, A-type TEA-resistant transient KV currents and TEA-sensitive delayed rectifier outward KV currents, have been measured (1, 2, 3), the precise identities of these KV channels in {alpha}-cells have not been elucidated. We detected KV4.1 and KV4.3 in {alpha}TC6 cells, suggesting possible heterotetramerization of these channel subunits and is consistent with the immunocytochemical identification of KV4.3 in mouse pancreatic {alpha}-cells (16). Therefore, KV4 subunits are the primary carrier of A-type KV currents in {alpha}-cells. We further show KV3.2 and KV3.3 to be the major KV isoforms contributing to the delayed rectifying KV currents in mouse {alpha}-cells. The {alpha}-cell delayed rectifying KV currents are highly sensitive to TEA blockade (IC50 = 0.25 ± 0.10 mM, n = 4; data not shown). Such high TEA sensitivity is a characteristic of KV3 family channels compared with KV1 and KV2 channels.

We confirmed {alpha}-cell expression of CaV1.2 (L-type Ca2+ channels) and CaV2.2 (N-type Ca2+ channels), which is consistent with the electrophysiological characterization of Ca2+ currents in these cells (2, 11, 12, 18). It has been proposed that glucagon secretion depends principally on Ca2+ influx through N-type CaV channels (12). L-type CaV channels can regulate glucagon secretion after activation of protein kinase A (18). Interestingly, we have determined the selective targeting of CaV1.2 but not CaV2.2 to cholesterol-rich lipid rafts. Moreover, removal of membrane cholesterol did not affect CaV currents. The difference in the membrane localization of these CaV channel isoforms is not known. CaV2.2 channels are modulated by both cholesterol and caveolin in neuroblastoma cells (41, 42). Furthermore, the closely related CaV2.1 channel is also associated with lipid rafts, but only in synaptosomal membranes and not cell soma membranes (43). Therefore, there appears to be selective association of the CaV channel isoforms to cholesterol-rich domains in {alpha}-cells. The importance of this selective membrane targeting remains to be determined.

Significance of ion channels and SNARE proteins targeting to lipid rafts
We demonstrate the related channel subunit, KV4.1/4.3, targets to lipid rafts in {alpha}-cells. Moreover, removal of membrane cholesterol has profound effects on KV4 channel function resulting in the absence of KV4-associated currents. Similarly, Wong and Schlichter (44) reported the targeting of KV4.2 channels to lipid rafts in hippocampal neurons. Changes in lipid composition or stiffness can have profound influences on channel gating or amplitude (41, 45). It is not known at this time whether the changes in KV4 currents are due to a decrease in surface protein levels or impaired channel activity. Additional studies are required to delineate the exact mechanisms by which cholesterol modulates KV4 channels. Interestingly, our data indicate the reduction in KV4 A-type K+ currents caused by lipid raft disruption is not responsible for the observed increase in glucagon release. More surprisingly, specific blockade of KV channels with heteropodatoxin reduced glucagon secretion. This contrasts the role of KV channels in pancreatic ß-cells, which enhance insulin secretion after genetic suppression or pharmacological blockade (46). These results suggest other factors than KV4 channel activity have a greater contribution in regulating stimulus-secretion coupling in {alpha}-cells.

Our observation of the increased glucagon secretion and the enhanced depolarization evoked exocytosis from single {alpha}-cells after cholesterol depletion are consistent with our previous finding on ß-cells (23). In that study, we observed augmented insulin secretion and single-cell exocytosis after lipid raft disruption. Furthermore, our work is consistent with the recent reports on the neuroendocrine PC12 cells, where membrane cholesterol depletion increased hormone secretion (38, 39). These studies demonstrated that less SNAP-25 was associated in lipid raft fractions (20% of total SNAP-25) compared with closely related protein SNAP-23 (54% of total SNAP-23). This difference was due to the presence of an additional cysteine residue in SNAP-23 in the palmitoylated membrane targeting region, which enhances its concentration in lipid rafts. Mutation of the corresponding phenylalanine residue in SNAP-25 to cysteine increased its association to lipid rafts and, more importantly, reduced exocytosis from PC12 cells (39).

The colocalization of lipid raft marker protein caveolin-2 with glucagon in {alpha}-cells suggest their expression on glucagon secretory granules. The enhanced glucagon secretion after cholesterol depletion with MßCD is not only a reflection of changes of plasma membrane proteins associated with lipid rafts, such as SNAP-25 and syntaxin 1A but also a reflection of the redistribution of the raft-associated proteins on vesicles, such as v-SNARE protein VAMP-2. We initially speculated that cholesterol-rich lipid rafts were the sites of exocytosis in {alpha}- and ß-cells. However, our data in pancreatic {alpha}-cells, as well as the recent findings in PC12 cells, suggest association of SNARE proteins with lipid rafts is a negative regulator of exocytosis, and movement out of the cholesterol-rich domains allows for improved spatial coordination of granule docking and fusion (38).

Detergent resistance of lipid rafts, cholesterol depletion, and glucagon secretion
Lipid rafts are characterized by their insolubility in cold Triton X-100 and low buoyancy on sucrose gradients, resulting in detergent-resistant membranes (DRMs) (47, 48, 49). Resistance to detergent has become a standard method of identifying proteins partitioning into DRM lipid rafts. Using this method, we demonstrated that KV4.1/4.3 and CaV1.2 as well as SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2 are associated to lipid rafts in pancreatic {alpha}-cells. However, the reliability of detergent resistance as a criterion for the existence of lipid rafts has been under debate. The detergent insolubility of the raft-associated proteins highly depends on the types of detergents and extraction conditions. In a complex cell membrane environment, detergent resistance at best represents indirect evidence for the existence of lipid rafts. Additional approaches are needed to provide more reliable information regarding the architecture of preexisting molecular organization on the plasma membrane and the functional relevance of localization of a protein in a particular microdomain.

Cholesterol is an inherent part of the lipid raft microdomain, and the depletion of cholesterol by agents such as MßCD perturbs the structure of lipid rafts, leading to the loss of raft association of a protein and probably functional changes related. Using confocal fluorescence microscopy, we demonstrated that both SNAP-25 and syntaxin 1A are clustered in plasma membrane. Depletion of cholesterol with MßCD causes the decrease of SNAP-25 and syntaxin 1A clustering and redistribution of these two SNARE proteins on the plasma membranes. This is in accordance with our detergent extraction data, in which depletion of cholesterol decreased the association of SNARE proteins with DRMs, further supporting the roles of lipid rafts in regulating SNARE protein functions. Consistent with our previous finding and others (23, 38, 39), depletion of cholesterol with MßCD leads the increase of glucagon secretion and the enhanced depolarization-evoked exocytosis from single {alpha}-cells. We speculate that the increased {alpha}-cell exocytosis is caused by the movement of SNAP-25 and syntaxin 1A out of lipid rafts after cholesterol depletion. However, additional approaches are required to rule out any possible direct effects of MßCD on {alpha}-cell secretion and to further understand the dynamic movement of the raft-associated proteins in pancreatic {alpha}-cells.

In summary, we identified the molecular isoforms of KV and CaV channels in pancreatic {alpha}-cells and showed for the first time that KV4.1/4.3 are targeted to lipid raft microdomains in addition to CaV1.2 and SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2. More importantly, we demonstrate that membrane compartmentalization of SNAP-25 and syntaxin 1A with lipid rafts is critical for regulating exocytosis of pancreatic {alpha}-cells.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research (MOP 77638 to R.G.T. and MOP 49521 to M.B.W.), Juvenile Diabetes Research Foundation (1-2005-1112 to H.Y.G.), a Premier’s Research Excellence Award to R.G.T., and equipment grants from James H. Cummings Foundation, J. P. Bickell Foundation, and the Banting and Best Diabetes Centre (BBDC) to R.G.T. F.X. was supported by a BBDC Studentship Award, Ontario Graduate Scholarship, and Canadian Diabetes Association Doctoral Student Research Award. Y.M.L. was supported by a Canadian Diabetes Association Fellowship. G.G. was supported by a BBDC Charles Hollenberg Summer Studentship.

Current address for Y.M.L.: Department of Physiology, China Medical University, Taichung 40402, Taiwan, Republic of China.

Current address for Y.C.: Central Laboratory, Guangzhou Children’s Hospital, Guangzhou 510120, Guangdong, China.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 15, 2007

Abbreviations: CaV, Voltage-gated Ca2+; Cm, membrane capacitance; DRM, detergent-resistant membrane; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; KATP, ATP-sensitive K+; KRB, Krebs-Ringer bicarbonate; KV, voltage-gated K+; MßCD, methyl-ß-cyclodextrin; MBS, MES-buffered saline; MES, 2-(N-morpholine)-ethane sulfonic acid; MIP, mouse insulin promoter; qPCR, quantitative PCR; SNAP-25, synaptosomal-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; VAMP, vesicle-associated membrane protein.

Received September 20, 2006.

Accepted for publication February 6, 2007.


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