Uncoupling of Ca v 1.2 from Ca 2؉ -induced Ca 2؉ release and SK channel regulation in pancreatic...

Uncoupling of Cav1.2 from Ca2�-induced Ca2� releaseand SK channel regulation in pancreatic �-cells

Yuchen Wang*, Rachel E. Jarrard*, Evan P.S. Pratt, Marcy L. Guerra,Amy E. Salyer, Allison M. Lange, Ian M. Soderling, and Gregory H. Hockerman

Purdue University Life Sciences Graduate Program (R.E.J., E.P.S.P., A.M.L.) and Department of MedicinalChemistry and Molecular Pharmacology (Y.W., M.L.G., A.E.S., I.M.S., G.H.H.), Purdue University, WLafayette, IN 47907–2091

We investigated the role of Cav1.2 in pancreatic �-cell function by expressing a Cav1.2 II-III loop/GFP fusion in INS-1 cells (Cav1.2/II-III cells) to disrupt channel-protein interactions. Neither block ofKATP channels nor stimulation of membrane depolarization by tolbutamide were different inINS-1 cells compared to Cav1.2/II-III cells, but whole-cell Cav current density was significantlyincreased in Cav1.2/II-III cells. Tolbutamide (200 �M) stimulated insulin secretion and Ca2� tran-sients in INS-1 cells and Cav1.2/II-III cells were completely blocked by nicardipine (2 �M), butthapsigargin (1 �M) blocked tolbutamide-stimulated secretion and Ca2� transients only in INS-1cells. Tolbutamide-stimulated ER [Ca2�] decrease was reduced in Cav1.2/II-III cells compared toINS-1 cells. However, Ca2� transients in both INS-1 cells and Cav1.2/II-III cells were significantlypotentiated by 8-pCPT-2�-O-Me-cAMP (5 �M), FPL-64176 (0.5 �M), or replacement of extracellularCa2� with Sr2�. Glucose (10 mM) � GLP-1 (10 nM) stimulated discrete spikes in [Ca2�]i in thepresence of verapamil at a higher frequency in INS-1 cells than in Cav1.2/II-II cells. Glucose (18 mM)stimulated more frequent action potentials in Cav1.2/II-III cells and primary rat �-cells expressingthe Cav1.2/II-II loop than in control cells. Further, apamin (1 �M) increased glucose-stimulatedaction potential frequency in INS-1 cells, but not Cav1.2/II-III cells, suggesting that SK channelswere not activated under these conditions in Cav1.2/II-III loop-expressing cells. We propose the II-IIIloop of Cav1.2 as a key molecular determinant that couples the channel to CICR and activation ofSK channels in pancreatic �-cells.

Type 2 diabetes is typified by hyperglycemia resultingfrom insufficient insulin secretion from pancreatic

�-cells, diminished sensitivity of target tissues to the ef-fects of insulin, or both (1). Glucose-stimulated increasesin intracellular Ca2� concentration are the primary driv-ers of insulin secretion from �-cells (2), and derangementsin Ca2� signaling are observed in �-cells from diabeticrodents (3) (4). Uptake of glucose into pancreatic �-cellsand subsequent metabolism of glucose increases the ATP/ADP ratio within �-cells (5). ATP binds to and blocks�-cell KATP channels (6), which are composed of the sul-fonylurea receptor 1 (SUR1) and the inwardly rectifyingK� channel Kir6.2 (7), resulting in membrane depolariza-tion (8). If of sufficient magnitude, membrane depolariza-

tion activates voltage-gated Ca2� (Cav) channels (9).Ca2� influx via the L-type channels present in �-cells,Cav1.2 and 1.3 (10), is thought to be particularly impor-tant for driving insulin secretion in response to elevatedglucose or sulfonylurea stimulation (11). Sulfonylureadrugs have long been used to stimulate insulin secretionfrom pancreatic �-cells in type 2 persons with diabetes(12). Similar to ATP, binding of sulfonylureas to SUR1/Kir6.2 decreases K� efflux through these channels, andtriggers membrane depolarization (13). One role of Ca2�

influx via L-type Ca2� channels into pancreatic �-cells isto stimulate further release of Ca2� from the endoplasmicreticulum, via Ca2�-induced Ca2� release (CICR). Thisprocess is activated in �-cells depolarized with elevated

ISSN Print 0888-8809 ISSN Online 1944-9917Printed in U.S.A.Copyright © 2014 by the Endocrine SocietyReceived April 5, 2013. Accepted January 28, 2014.

Abbreviations:

O R I G I N A L R E S E A R C H

doi: 10.1210/me.2013-1094 Mol Endocrinol mend.endojournals.org 1

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concentrations of extracellular KCl (14), but apparentlydoes not contribute to [Ca2�]i oscillations observed in�-cells stimulated with high glucose concentrations in theabsence of other cosecretagogues (15) (16).

The SUR1/Kir6.2 channel is known to interact directlyor indirectly with several proteins that reside at theplasma membrane and play a role in insulin exocytosis.The scaffolding proteins RIM2 (17) and Piccolo (18) alsointeract with the intracellular II-III loop domain of Cav1.2(19). We have previously reported that endogenously ex-pressed RIM2 is present in lipid rafts, and is immunopre-cipitated by a Cav1.2/II-III-eGFP loop fusion protein, butnot by a Cav1.3/II-III loop-eGFP fusion protein, in INS-1cells (20). Further, we reported that overexpression of theCav1.2/II-III loop causes accumulation of Cav1.2, but notCav1.3, outside of lipid rafts in the rat insulinoma cellline, INS-1. Conversely, overexpression of the Cav1.3/II-III loop selectively excluded Cav1.3, but not Cav1.2, fromlipid rafts (20). The guanine nucleotide exchange factorexchange protein directly activated by cAMP 2 (EPAC2)interacts directly with both SUR1 and Piccolo (21) whilePiccolo forms a dimer with RIM2, in a Ca2�-dependentmanner (21). Thus, a network of protein-protein interac-tions connecting the main components of the pathwaythat leads to insulin secretion stimulated by sulfonylureasimplicates Cav1.2 in this process. This signaling complexof scaffolding proteins (RIM2, Piccolo), cAMP effector(EPAC2), and ion channel subunits (SUR1, Cav1.2) is ofspecial significance since membrane depolarization-de-pendent calcium influx via Cav1.2 channels has been im-plicated in triggering Ca2�-induced Ca2� release from theER in pancreatic �-cells (22), a process that is amplifiedby cAMP, at least in part, through EPAC2 (22, 23). How-ever, it is not clear whether interaction of Cav1.2 withother proteins via the II-III loop has functional signifi-cance in sulfonylurea stimulated insulin secretion or[Ca2�]i transients in pancreatic �-cells. We show herethat expression of the Cav1.2 intracellular II-III loop inthe pancreatic �-cell line INS-1 doesn’t disrupt SUR1 orEPAC2 localization to lipid rafts, but does disrupt CICRstimulated by the sulfonylurea tolbutamide, as assessedby several different experimental approaches.

In addition to modulation of insulin secretion, in-creases in intracellular Ca2� concentration in pancreatic�-cells may also modulate electrical activity stimulated byglucose. One potential mechanism for fine-tuning of glu-cose-stimulated action potentials is via activation of thesmall conductance Ca2�-activated K� channel, SK (24).SK subtypes 1–3 were detected in the rat insulinoma cellline INS-1 (25). Activation of SK channels occurs as in-tracellular Ca2� accumulates at the plasma membrane,and K� efflux via SK channels limits action potential fre-

quency (26). Indeed, block of SK channels with specificpharmacological agents such as apamin (27) or UCL1684 (28) can markedly increase glucose-stimulated ac-tion potential frequency in pancreatic �-cells, but the ma-jor source of the Ca2� that activates SK channels in pan-creatic �-cells is not clear. Here we report thatmodulation of glucose-stimulated action potential fre-quency by SK channels is disrupted in both INS-1 cellsstably expressing the Cav1.2/II-III loop and primary rat�-cells expressing the Cav1.2/II-III loop via adenoviraltransduction.

Materials and Methods

Chemicals. Antibodies were acquired from the following sourc-es: EPAC2 and eIF3e- Santa Cruz Biotechnology (Santa CruzCA), SUR1- Abcam (Cambridge, UK), Kir6.2- Millipore (Bil-lerica, MA), GFP and HRP-conjugated secondary antibodies-BioRad (Hercules, CA). ESCA-AM was from Biolog (Bremen,Germany). Apamin was from Tocris Cookson (Minneapolis,MN). All other chemicals were from Sigma-Aldrich (St. Louis,MO).

Cell Culture. INS-1 cells (29) and Cav1.2/II-III cells (30) werecultured in RPMI medium (Sigma-Aldrich, St. Louis, MO) sup-plemented with 10% fetal bovine serum (HyClone, Logan, UT),11 mg/ml sodium pyruvate, 10 mM HEPES, 100 u/ml penicillin,100 �g/ml streptomycin, and 50 �M �-mercaptoethanol at37°C, 5% CO2 (INS-1 RPMI). Cav1.2/II-III cells were culturedin 200 �g/ml G418 to maintain expression of the Cav1.2 chan-nel II-III loop. INS-1 cells used for current clamp recordingswere cultured in low glucose (2.5 mM) for 18–24 hours prior toexperiments.

Sucrose density gradient fractionation. INS-1 cells and Cav1.2/II-III cells were lysed in the presence protease inhibitors (800 nMaprotinin, 50 �M leupeptin, 1 �g/ml pepstatin, 1 mM benzimi-dine, 1 mM AEBSF, 10 �g/ml calpain I and II inhibitors; all fromSigma) and fractionated over a discontinuous 5%/30%/40%sucrose gradient as described previously (20). One ml fractionswere removed from the top of each gradient, for a total of 11fractions. Like fractions from multiple tubes were pooled andconcentrated using Amicon Ultra-4 concentrator tubes (Milli-pore, Billenca, MA). Protein concentrations were determinedusing the BCA assay (Pierce, Rockford, IL) before they wereanalyzed via western blot.

Immunoprecipitation Assays. Experiments in which the Cav1.2/II-III or Cav1.3/II-III loops fused to GFP were immunoprecipi-tated from INS-1 cell lysates with antibodies against GFP wereperformed as previously described (20). Immunoprecipitationof eIF3e was performed as previously described except that celllysates were incubated with antibodies against eIF3e over nightat 4°C, then with agarose-immobilized Protein A Plus (ThermoScientific, Rockford, IL) overnight at 4°C before washing. Pro-tein was eluted by incubating at 80°C in Laemmli buffer.

2 Cav1.2 function in pancreatic �-cells Mol Endocrinol


Western Blotting. 20 �g of protein from each fraction of sucrosegradients were separated by SDS-PAGE using 4% acrylamide(29:1 acrylamide;bisacrylamide) stacking gels, and 8% resolv-ing gels for all blots except for Kir6.2, for which 10% acrylamideresolving gels were used. 50 �l of fraction 1 were loaded on eachgel because it consistently lacked any protein as detected by theBCA assay. Proteins were resolved on polyacrylamide gels in 25mM Tris, 192 mM glycine, 0.1% SDS, then transferred to poly-vinylidene difluoride (PVDF) membranes using a BioRad Trans-blot Cell in 10 mM CAPS, 10% methanol, at 70 V for 35minutes to 2 hours, depending on the size of the protein to bedetected. PVDF membranes were blocked by soaking in 5%nonfat milk in PBS with 0.1% Tween-20 (PBST) for 2 hours atroom temperature. Blocked membranes were incubated withprimary antibodies overnight at 4°C at the following dilutions(EPAC2, 1:100; SUR1, 1:500; Kir6.2, 1:200; eIF3e 1:100, GFP1:1000), then washed three times with PBST, and incubatedwith secondary antibodies conjugated to horseradish peroxi-dase (1:1000) for 1 hour at room temperature. Excess secondaryantibodies were removed by washing membranes three times inPBST, and proteins were detected using enhanced chemilumi-nescence or chemifluorescence (Amshersham Biosciences, Pisca-taway, NJ).

Electrophysiological Assays. Electrophysiological measure-ments in INS-1 cells and Cav1.2/II-III cells were recorded atroom temperature using an Axopatch 200B amplifier (Molecu-lar Devices, Sunnyvale, CA) and filtered at 1 kHz (six-pole Bes-sel filter, –3 dB). Patch pipettes were pulled from borosilicateglass (VWR, West Chester, PA) and fire-polished to resistancesof 2 to 5 M�. INS-1 cells used for current clamp recordings werecultured in low glucose (2.5 mM) for 18–24 hours prior toexperiments. For current clamp experiments and voltage clampexperiments to measure KATP channel current, the intracellularsolution contained (in mM) 90 K2SO4, 10 NaCl, 1 MgCl2, 1.1EGTA, 0.1 CaCl2, 5 HEPES, 0.3 ATP, 0.2 GTP. The extracel-lular solution used for measuring KATP currents contained (inmM) 138 NaCl, 5.6 KCl, 11.1 glucose, 10 HEPES, 1.2 MgCl2,2.6 CaCl2, and the same solution with 2.5 mM glucose insteadof 11.1 mM glucose was used in current-clamp experiments.The pH of solutions was adjusted to 7.4 with NaOH, and theosmolality was adjusted to 290 - 300 mOsm. Whole-cell KATP

currents were elicited by 1.3 second steps of � 20 mV from aholding potential of –70 mV. Data were acquired at a samplingfrequency of 1 kHz. The membrane potential of INS-1 cells andCav1.2/II-III cells was measured using gap-free recording at asampling frequency of 1 kHz in I � 0 current clamp mode. TheKATP channel opener, diazoxide (300 �M), was transiently ap-plied to maximally open KATP channels, before application oftolbutamide. Tolbutamide solutions were prepared from stocksdissolved in 0.1 M NaOH, made fresh daily. Diazoxide solu-tions were prepared from stocks dissolved in DMSO. For re-cordings of voltage-gated Ca2� channel currents, the bath solu-tion contained (in mM) 150 Tris, 10 BaCl2, 4 MgCl2. Theintracellular solution contained (in mM) 130 N-methyl-D-glu-camine, 10 EGTA, 60 HEPES, 2 ATP, and 1 MgCl2. The pH ofboth solutions was adjusted to 7.3 with methanesulfonic acidand the osmolality was corrected to 290–300 mOsM. Current-voltage relationship data were collected by applying 100-milli-second test depolarizations from –50 to �60 mV in 10-mVincrements, from a holding potential of –70 mV. Data were

acquired at a sampling frequency of 10 kHz, and filtered at 1kHz. For current clamp recordings of membrane depolarizationwith glucose, the perforated patch technique was used to keepthe intracellular signaling environment intact. The bath solutioncontained (in mM) 138 NaCl, 5.6 KCl, 2.5 glucose, 10 HEPES,1.2 MgCl2,2.6 CaCl2. The intracellular solution contained (inmM) 10 KCl, 10 NaCl, 70 K2SO4, 2 MgCl2, 10 HEPES, 100–120 �g/ml amphotericin B. The pH of both solutions was ad-justed to 7.4 with NaOH and the osmolality was adjusted to290–300 mOsm. A 30 mg/ml amphotericin stock was preparedin DMSO from powder prior to all experiments. The stocksolution and the intracellular solution containing amphotericinwere sonicated for at least 15 minutes prior to use, were usedwithin one hour, and were protected from light. Recording pi-pettes were front-filled with intracellular solution without am-photericin and were back-filled with intracellular solution con-taining amphotericin. After formation of the cell-attachedpatch, the access resistance (Ra) was monitored. After 10–15minutes, when Ra was less than 50 M�, the patch was consid-ered to be perforated enough to begin current clamp recordings.Upon adequate access, the membrane potential was measuredand 300 �M diazoxide was briefly applied to fully hyperpolar-ize the membrane potential. Once a stable baseline membranepotential was achieved, 18 mM glucose was perfused into therecording chamber to stimulate membrane depolarization andaction potential spiking. Whole-cell SK channel current record-ing was performed using a holding potential of –70 mV withsteps to –50 mV. The intracellular and extracellular solutionsfor SK channel whole-cell recordings were made as describedpreviously (25). The intracellular solution consisted of (in mM):110 potassium-gluconate, 10 KCl, 10 NaCl, 1MgCl2, 3 Mg-ATP, 5 HEPES, 10 EGTA, and 9.6 CaCl2 to achieve 2 �M free[Ca2�] (MaxChelator: http://www.stanford.edu/�cpatton/maxc.html); pH and the osmolality were adjusted to 7.2 and290–310, respectively. Extracellular solution contained (inmM): 140 NaCl, 4 KCl, 2 NaHCO3, 1 NaH2PO4, 1 MgSO4, 5HEPES, 2.5 CaCl2, with pH and osmolality adjusted to 7.4 and290–310, respectively.

Insulin Secretion Assay. INS-1 cells or Cav1.2/II-III cells wereplated in 24-well tissue culture plates at 50%–70% confluencyand incubated overnight in RPMI medium with 2.5 mM glucoseat 37°C, 5% CO2. Immediately before the assay, cells werewashed twice in phosphate buffered saline (PBS: 137 mM NaCl,10 mM Phosphate, 2.7 mM KCl (pH 7.4)), and preincubatedwith a modified Krebs-Ringer buffer (KRBH: 134 mM NaCl,3.5 mM KCl, 1.2 mM KH2PO4, 0.5 mM MgSO4, 1.5 mMCaCl2, 5 mM NaHCO3, 10 mM HEPES (pH 7.4)) supple-mented with 0.05% fatty acid free BSA (KRBH buffer) alone, orcontaining indicated concentrations of inhibitors, for 30 min-utes at 37°C, 5% CO2. The buffer was decanted and replacedwith fresh KRBH buffer alone (basal condition) or KRBH con-taining the indicated concentrations of tolbutamide or glucosewith or without inhibitors for one hour at 37°C, 5% CO2.Secreted insulin was assayed using an ELISA for rat insulin(High-Range EIA kit. ALPCO Diagnostics, Salem, NH). Cellswere lysed in 20 mM Na2HPO4, 150 mM NaCl, 0.1% TritonX-100, 800 nM aprotinin, 50 �M leupeptin, 1 �g/ml pepstatin,1 mM benzamidine, 1 mM 4-(2-Aminoethyl) benzenesulfonyl-fluoride, 10 �g/ml calpain inhibitor I, 10 �g/ml calpain inhibitor

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II, (pH 7.4) and cellular protein in each well was determinedusing the BCA assay (Thermo Scientific, Rockford, IL).

Intracellular Ca2� Assays in 96 well plates. INS-1 cells orCav1.2/II-III cells were plated at 100% confluency in black-walled 96 well plates (Corning Life Sciences, Lowell, MA) inRPMI supplemented as described above, and incubated over-night at 37°C, 5% CO2. Cells were washed twice with PBS andincubated with 5 �M Fura-2AM (Molecular Probes, Eugene,OR) diluted in KRBH for one hour at 37°C, 5% CO2. TheKRBH containing Fura-2AM was then removed and the cellswere washed twice with KRBH, and equilibrated for 30 minutesat 37°C, 5% CO2 in the KRBH alone, or with indicated con-centrations of inhibitors or 8-pCPT-2�-O-Me-cAMP-AM. Cellswere stimulated by injection of the indicated concentration ofsulfonylurea or glucose (or buffer control) and changes in intra-cellular Ca2� concentrations were measured by recording theratio of fluorescence intensities at 508/20 nm resulting fromexcitation of Fura-2 at 340/11 nm or 380/20 nm (center/band-pass) using a Synergy 4 multimode microplate reader (BioTek,Winooski, VT). Ratios were acquired every 0.7 secs for 15 sec-onds before injection and two minutes after injection of tolbu-tamide. In experiments with 18 mM glucose as the stimulus,ratios were acquired every minute for 1 hour. Data were cor-rected for any injection artifact by subtracting the change influorescence ratio measured in cells injected with KRBH alone.

Single-cell intracellular Ca2� assays. Cells were seeded on apoly-D-lysine coated round glass coverslip and incubated over-night in INS-1 RPMI medium. Cells were washed with PBSbefore loading with 3 �M cell-permeant Ca2�-sensitive dyeFura-2 (Life Technologies, Carlsbad, CA) at room temperaturefor 1hr. The loading solution was removed and cells werewashed with KRBH followed by incubation in 0.5% BSAKRBH at room temperature for 30 minutes. The coverslip wasmounted on a perfusion chamber attached to the stage of anOlympus, IX50 inverted microscope (Olympus, Tokyo, Japan)equipped with a PlanApo 40X objective lens (0.95 na). Solutionwas perfused to the chamber through an in-line solution heater(Warner Instrument, Hamden, CT) at a constant flow rate (1ml/min) and constant temperature (37 � 1°C). Cells were firstperfused with KRBH for 30 seconds followed by 30 mM KCl todepolarize the cells for 5 minutes. 50 �M verapamil was addedduring the final 4 minutes with 30 mM KCl to bring the [Ca2�]ito basal. A combination of 10 mM glucose, 10 nM GLP-1 and50 �M verapamil was applied for the rest of the experiment toelicit CICR. Cells were alternatively excited at 340nm and380nm using a filter wheel (Sutter Instrument, Novato, CA),and the images (emission signal at 510nm) were recorded in timelapse (one ratio every 2 seconds) using a Clara CCD camera(Andor Technology, Belfast, UK) at the beginning of KRBHapplication. Single and isolated cells were selected as regions ofinterest (ROI) and the averaged intensities (340/380 nm ratio)for each ROI at different time points were measured using Meta-morph (Molecular Devices, Sunnyvale, CA). Basal [Ca2�]i levelof a cell was obtained by averaging the intensities of that cellwithin the first 30 seconds when only KRBH was present andthe basal was used to normalize the intensities at all time points.A single cell Ca2� transient trace was obtained by plotting all thenormalized intensities against lapsed time points. The slope of apeak was used to define a CICR peak. A spike in a trace is

considered as a CICR event if the slope of the peak is equal orgreater than that of the KCl-induced spike.

Measurement of ER Ca2� dynamics. INS-1 cells or Cav1.2/II-IIIcells were transiently transfected with the pCDNA3-D1ER plas-mid (Addgene, Cambridge, MA) encoding an ER-targeted Flu-orescence Resonance Energy Transfer (FRET)-based Ca2� sen-sor in which Ca2� binding enables FRET between cyanfluorescent protein (CFP) and citrine (31). Cells were split 24 to48 hours post-transfection into four chambered 35 mm glassbottom dishes (In Vitro Scientific, Sunnyvale, CA) and imaged48 to 72 hours post-transfection. Cell culture media was re-placed with Krebs-Ringer buffer 30 minutes prior to imaging.The basal FRET signal was collected for 1 minute prior to treat-ment and 10 minutes following the addition of the positivecontrol carbachol (500 �M) or tolbutamide (200 �M), whichwere diluted in Krebs-Ringer buffer. To determine the minimumFRET value, or Rmin, cells were treated with thapsigargin (1�M) for 30 minutes prior to imaging and subsequently stimu-lated with carbachol to exhaust ER calcium stores. The D1ERFRET signal was measured with a 20X objective lens using aNikon A1 Confocal System with a Perfect Focus Ti-E InvertedMicroscope. The CFP fluorophore of the D1ER FRET sensorwas excited at 457nm with an Argon laser. CFP and citrineemission were collected at 482nm and 525nm, respectively, us-ing 482/35nm and 525/50 filter cubes. The citrine signal inten-sity, or FRET signal, was normalized to the intensity of the CFPemission, which served as an expression of ER calcium levels.The time course of the FRET signal was analyzed using NISElements software. Only those cells which displayed a highFRET signal were chosen for analysis, and these values wereaveraged among the chosen cells for each experiment at all timepoints. Rmin was averaged among several separate experimentsand represents the point at which the D1ER sensor no longerdetects a decrease in ER calcium. This value was subtractedfrom each FRET time point in all experiments in order to nor-malize for the difference in Rmin between INS-1 and Cav1.2/II-III cells. To compare ER calcium changes among experimentsfor each cell type, the FRET/CFP values over the time coursewere then normalized to the average FRET/CFP ratio over thefirst minute of no treatment, yielding a baseline value over thefirst 60 seconds of 100% or Rmax. The peak change in theFRET/CFP ratio, or decrease in ER calcium levels, was calcu-lated based on the maximum difference from baseline over avariable time span following stimulation. Change in FRET wascalculated as a percentage of the dynamic ER calcium range, ordifference between the Rmax and Rmin, in either INS-1 orCav1.2/II-III cells.

Isolation and Adenoviral Transduction of Rat Pancreatic Islets.Pancreatic islets were isolated from 2-month-old, male Wistarrats (�225 g) via collagenase digestion by the Islet Core Facilityof the Indiana Diabetes Research Center, Indiana UniversitySchool of Medicine. Islets were maintained in islet RPMI me-dium (RPMI-1640 medium containing 8 mM glucose, 10 mMHEPES, 10% fetal bovine serum (Hyclone, Logan, UT), 11mg/mL sodium pyruvate, 100 U/mL penicillin, and 100 �g/mLstreptomycin) and kept at 37°C and 5% CO2 for 24 hours.Groups of 150 islets were handpicked into fresh islet RPMImedium for adenoviral transduction. Adenovirus encoding GFPalone or GFP fused to the C-terminal end of the Cav1.2/II-III



loop domain (30) was obtained from ViraQuest (North Liberty,IA). Islets were transduced at an MOI of 50 (assuming 1000cells/islet). After 24 hours with the virus, the islets were washedwith phosphate buffered saline (PBS) and transferred to freshislet RPMI medium. After another 24 hours, the islets weredissociated into single cells.

Islet Dissociation. Islets were digested in 1 mL of 0.25% Tryp-sin-EDTA for every 100 islets, and triturated repeatedly for 3minutes. Trypsin was neutralized with the addition of 3 mL ofRPMI medium, and cells were centrifuged at 800 rpm for 3minutes, resuspended in islet RPMI medium containing 2.5 mMglucose, and plated into poly-D-lysine coated 35 mm tissue cul-ture dishes. Dissociated cells were incubated at 37°C, 5% CO2

until they were used for electrophysiological experiments.

Data Analysis. Electrophysiological data were analyzed usingClampfit 8 (Molecular Devices). Area- underthe-curve (AUC)analysis and dose-response curve fits were performed using Sig-maPlot 11 (Systat Software, Chicago, IL). Single-cell calciumimaging data were analyzed with Metamorph software (Molec-ular Devices). Analyzed data are shown as mean values � stan-dard error. Statistical significance was determined using One-Way ANOVA and the Holm-Sidak post hoc test unlessotherwise indicated. Differences with P values � 0.05 were con-sidered significant.

Results

Sucrose-density gradient fractionation of proteins in-volved in depolarization-induced insulin secretion inINS-1 cells and Cav1.2/II-III cells. The KATP channel,composed of Kir6.2 and SUR1 subunits, plays a centralrole in the insulin secretion stimulated by sulfonylureasand glucose. We examined the localization of Kir6.2,SUR1 and EPAC2 in lipid rafts by fractionating the tritonX100-insoluble portion of INS-1 and Cav1.2/II-III celllysates on discontinuous sucrose gradients. EPAC2 is re-ported to interact directly with both Piccolo (21) andSUR1 (19), and we found that both EPAC2 and SUR1 arehighly concentrated in lipid raft fractions of sucrose gra-dients (the interface of 5% and 30% sucrose) in bothINS-1 cells and Cav1.2/II-III cells (Figure 1). We also as-sessed the localization of the KATP channel subunit Kir6.2and found that while it is present at the 5%/30% sucroseinterface, it was also distributed throughout the 40% su-crose fractions in both INS-1 cells and Cav1.2/II-III cells(Figure 1). The lipid raft-resident protein caveolin 1 wasdetected at the 5%/30% sucrose interface but also distrib-uted throughout the sucrose gradient in samples fromboth INS-1 and Cav1.2/II-III cells. This distribution ofcaveolin 1 is similar to that observed in a previous studyusing the pancreatic beta cell line HIT-T15 (32). Thus, theKATP channel subunits SUR1 and Kir6.2, along with theinteracting protein EPAC2 are present in lipid rafts in

INS-1 cells, and their distribution on discontinuous su-crose gradients is not perturbed by expression of theCav1.2 intracellular II-III loop.

Electrophysiological characterization of Cav1.2/II-IIIcells. Cav1.2 is reported to exist in a complex with pro-teins essential for stimulation of pancreatic �-cells by sul-fonylureas, so we compared the modulation of electricalactivity in INS-1 cells and Cav1.2/II-III cells by tolbut-amide. Figure 2A shows a whole-cell voltage-clamp ex-periment with a Cav1.2/II-III cell held at –70 mV, withalternating steps to –50 and –90 mV. Application of tol-butamide via external perfusion blocked both the inwardand outward K� current in a dose dependent manner.Plots of the percent current blocked by tolbutamide con-centrations between 100 nM and 500 �M are shown in

FIGURE 1. KATP channel subunits and the cAMP effector EPAC2 arepresent in lipid rafts in both INS-1 cells and Cav1.2/II-III cells. Westernblots detecting the indicated proteins are shown for each fraction ofthe sucrose-density gradients for cell lysates from INS-1 cells or Cav1.2/II-III cells. Fractions 2 and 3, at the interface of 5% and 30% sucrose,contained the Triton X-100 insoluble, low density material (lipid rafts).Kir6.2, the pore-forming subunit of the KATP channel, is detected in,but not restricted to, lipid raft fractions. This distribution is not affectedby expression of the Cav1.2/II-III loop. The dashed line across the Kir6.2blots indicate the boundary between two separate polyacrylamide gelscontaining fractions 1–9 (top) and fractions 10 and 11 (bottom) thatwere simultaneously transferred to a single PVDF membrane beforewestern blotting. EPAC2 and SUR1 are highly localized to the lipid raftfractions, and this localization was not affected by expression of theCav1.2/II-III loop. Caveolin 1 is present in lipid raft fractions, but alsodistributed into the 40% sucrose fractions in both INS-1 cells andCav1.2/II-III cells. Fractions 10 and 11 are not shown for the SUR1,EPAC2, and caveolin 1 blots because they contained little or none ofthe indicated proteins. Molecular weight standards are shown. Eachblot is representative of at least 3 independent experiments.



Figure 2A. Fits to these plots yielded EC50 values fortolbutamide of 2.6 � 0.7 �M and 3.8 � 0.2 �M for INS-1cells and Cav1.2/II-III cells, respectively. Since block ofKATP channels by tolbutamide leads to membrane depo-larization in pancreatic �-cells, we performed currentclamp experiments to compare the potency of tolbut-amide depolarizing the membrane potential in INS-1 cellsand in Cav1.2/II-III cells. As shown in Figure 2B, 200 �M

tolbutamide elicited strong membrane depolarizationleading to initiation of action potentials in both INS-1cells (left panel) and Cav1.2/II-III cells (right panel). Nei-ther the resting membrane potential, nor the membranedepolarization elicited by 10, 50, 200, or 500 �M tolbu-tamide were significantly different in INS-1 cells orCav1.2/II-III cells (Figure 2C). Finally, we compared thedensity of Ba2� current conducted by voltage-gated Ca2�

channels and the density of tolbut-amide-sensitive K� current in INS-1cells and Cav1.2/II-III cells. As wereported previously, the IBa densityis markedly increased in Cav1.2/II-III cells compared, to INS-1 cells(20); however, the KATP channelcurrent density is not significantlydifferent (Figure 2D). Thus, our datademonstrate that expression of theCav1.2 II-III loop increases voltage-gated Ca2� channel current density,but does not affect either KATP chan-nel current density, the sensitivity ofKATP channels to tolbutamide block,or the ability of tolbutamide to dose-dependently depolarize the mem-brane potential in INS-1 cells.

Insulin secretion and intracellularCa2� transients stimulated by tolb-utamide in Cav1.2/II-III cells don’trequire intact intracellular stores ofCa2�. Given the central role of theL-type channel Cav1.2 in the stimu-lation of insulin secretion, we exam-ined tolbutamide stimulated insulinsecretion in INS-1 cells and Cav1.2/II-III cells using the static incubationmethod (Figure 3). As expected fromthe electrophysiological character-ization of tolbutamide in each cellline, 200 �M tolbutamide stimu-lated insulin secretion that was sig-nificantly different from basal secre-tion (Figure 3A). The L-type-selective Ca2� channel blocker,nicardipine (2 �M) completely in-hibited secretion stimulated by tolb-utamide in each cell line (Figure 3A).To assess the role of internal storesof Ca2� in tolbutamide stimulatedinsulin secretion, the ability of 1 �Mthapsigargin, an inhibitor of the sar-

FIGURE 2. Tolbutamide induces similar KATP block and membrane depolarization in INS-1 andCav1.2/II-III cells. A, Left, representative trace of the dose-dependent tolbutamide block of KATP

current measured in Cav1.2/II-III cells using whole-cell voltage clamp. Whole-cell current waselicited by a � 20 mV step from a holding potential of –70 mV. Right, percentage of the whole-cell KATP current blocked by tolbutamide in INS-1 cells (open circles, n � 4–11) and Cav1.2/II-IIIcells (closed circles, n � 5–7). IC50 values were determined by fitting the data using the equation% Current Blocked � min � (max-min)/(1 � 10(logIC50 – [tolbutamide])). B, Representativetraces of the change in membrane potential elicited by 200 �M tolbutamide in INS-1 cells andCav1.2/II-III cells. C, dose-dependent effect of tolbutamide on membrane depolarization in INS-1cells (open bars, n � 8–18) and Cav1.2/II-III cells (closed bars, n � 7–13). EC50 values weredetermined by fitting the data using the equation membrane potential � 1/(1 � ([tolbutamide]/EC50)n). D, whole-cell current density comparison of voltage-gated calcium channels (Cav) andKATP channels in INS-1 cells (open bars) and Cav1.2/II-III cells (closed bars). IBa density (pA/pF) wasmeasured at 0 mV from a holding potential of –70 mV. The difference in total whole-cell IBa

density for Cav1.2/II-III cells compared with INS-1 cells is statistically significant (***, P � .001;n � 13–15). Current density of KATP channels was determined by measuring whole-cell currentelicited by a � 20 mV step from a holding potential of –70 mV in the presence of 300 �Mdiazoxide to maximally open KATP channels. There is no significant difference in KATP channelcurrent density between INS-1 cells and Cav1.2/II-III cells (n � 20).



coplasmic/endoplasmic reticulum Ca2�- ATPase(SERCA), to inhibit insulin secretion stimulated by tolb-utamide was determined. In INS-1 cells, we found thatthapsigargin significantly, but incompletely, blocked in-sulin secretion stimulated by 200 �M tolbutamide (Figure3A), suggesting that both influx of Ca2� via L-type Ca2�

channels and release of internal stores of Ca2� are re-

quired for maximal insulin secretion stimulated by tolb-utamide in INS-1 cells. In sharp contrast, thapsigargintreatment does not inhibit tolbutamide stimulated secre-tion in Cav1.2/II-III cells (Figure 3A). Therefore, our re-sults suggest that release of internal Ca2� stores is notrequired for maximal insulin secretion in response to tol-butamide in Cav1.2/II-III cells.

To compare changes in intracel-lular Ca2� concentration induced bytolbutamide, INS-1 cells andCav1.2/II-III cells in 96-well plateswere loaded with the Ca2� indicatorfura 2-AM and changes in the ratioof fluorescence intensity at 510 nmafter excitation at 340 nm and 380nm (340 nm/ 380 nm ratio) weremeasured upon injection of tolbut-amide (final concentration of 200�M). The changes in 340 nm/ 380nm ratio upon injection of tolbut-amide were corrected by subtractingthe changes in 340 nm/ 380 nm ratiomeasured in replicate wells of cellsupon injection of buffer only. Thenet change in fura 2 fluorescence ra-tio, reflecting the net change in intra-cellular Ca2� concentration stimu-lated by 200 �M tolbutamide isshown in Figure 3. Tolbutamide in-duced a biphasic rise in intracellularCa2� concentration in INS-1 cellsand Cav1.2/II-III cells. In INS-1cells, a rapid peak in intracellularCa2� concentration is followed bydecay to an elevated plateau levelthat persisted for the duration of thetwo-minute measurement (whitecircles: tolbutamide, Figure 3B).Tolbutamide induced Ca2� tran-sients in Cav1.2/II-III cells that roseto a similar peak as in INS-1 cells,but Cav1.2/II-III transients ap-peared to decay more slowly (whitecircles: tolbutamide, Figure 3B).Ca2� transients induced by tolbut-amide in INS-1 cells and Cav1.2/II-III cells are mediated by L-type Ca2�

channels since pretreatment with500 nM FPL-64176, an L-type-spe-cific Ca2� channel agonist, greatlyenhanced the level of intracellularCa2� in response to tolbutamide

FIGURE 3. Insulin secretion and Ca2� transients stimulated by tolbutamide are sensitive tothapsigargin in INS-1 cells but not Cav1.2/II-III cells. A, Insulin secretion stimulated by 200 �Mtolbutamide is completely blocked by 2 �M nicardipine (black bars) in both INS-1 cells andCav1.2/II-III cells. (***, P � .001 compared to basal; ###, P � .001 compared to nicardipine (n �9 in 3 separate experiments). Insulin secretion stimulated by 200 �M tolbutamide is partiallyinhibited by pretreatment with 1 �M thapsigargin (white bars) in INS-1 cells, but not in Cav1.2/II-III cells (***, P � .001, **, P � .01, *, P � .05 compared to basal; ##, P � .01 compared totolbutamide alone (n � 9 in 3 separate experiments)). B, Ca2� transients stimulated by 200 �Mtolbutamide are mediated by L-type Ca2� channels in both INS-1 cells and Cav1.2/II-III cells. Cellspretreated with 500 nM FPL 64176 (black circles) exhibit markedly greater increases inintracellular Ca2� concentration in response to tolbutamide stimulation than cells pretreated withKRBH buffer only (white circles). Pretreatment of INS-1 cells or Cav1.2/II-III cells with 2 �Mnicardipine (white diamonds) completely inhibits the Ca2� transients elicited by 200 �Mtolbutamide � 500 nM FPL 64176. Data shown are mean � SE from experiments representativeof 3 performed in quadruplicate. C, The rapid, initial phase of Ca2� transients stimulated by 200�M tolbutamide is inhibited by pretreatment with 1 �M thapsigargin in INS-1 cells but not inCav1.2/II-III cells. White circles represent the thapsigargin-sensitive portion of the tolbutamideresponse. Black circles represent the response to tolbutamide in the absence of thapsigargin. Incontrast, the rapid peak in [Ca2�]i stimulated by tolbutamide is greatly amplified in both cell linesby replacing extracellular Ca2� with 2 mM Sr2� (open diamonds). Data shown are mean � SEfrom experiments representative of 3 performed in quadruplicate.



stimulation (black circles: tolbutamide � FPL-64176,Figure 3B). When cells were pretreated with 2 �M nicar-dipine, the Ca2� transient stimulated by tolbutamide andFPL-64176 was greatly reduced (white diamonds: tolbu-tamide � FPL-64176 � nicardipine, Figure 3B). Thus, inboth INS-1 cells and Cav1.2/II-III cells, the increase inintracellular Ca2� concentration stimulated by tolbut-amide is controlled by influx of Ca2� via L-type Ca2�

channels.Since insulin secretion stimulated by tolbutamide was

differentially sensitive to thapsigargin in INS-1 andCav1.2/II-III cells, we examined the effect of thapsigarginon changes in intracellular Ca2� concentration stimu-lated by tolbutamide in these two cell lines (Figure 3C).Pretreatment of INS-1 cells with 1 �M thapsigargin be-fore stimulation with tolbutamide selectively inhibitedthe rapid peak in intracellular Ca2� concentration, buthad a minimal effect on the sustained plateau phase of thetransient. In the Cav1.2/II-III cells, pretreatment withthapsigargin was much less effective in inhibiting the peakin Ca2� concentration. The portion of the Ca2� responseto tolbutamide that was thapsigargin-sensitive [tolbut-amide response – (tolbutamide � thapsigargin response)]is shown as white circles in Figure 3C for INS-1 andCav1.2/II-III cells. The insensitivity of the Cav1.2/II-IIIcells to pretreatment with thapsigargin could indicate ei-ther an uncoupling of Ca2� influx via L-type Ca2� chan-nels from CICR, or a decreased sensitivity of the Ca2�

release channel RYR2 to gating by Ca2�. To test thesepossibilities, we measured the tolbutamide-stimulatedrise in intracellular Ca2� concentration with extracellularCa2� replaced with 2 mM Sr2�. Sr2� binds to fura-2 witha higher KD than Ca2� (33), is able to stimulate release ofCa2� from internal stores via activation of RYR2 in�-cells (14), and is not buffered as strongly in the cyto-plasm as Ca2� (34). We found that in the presence of 2mM extracellular Sr2�, 200 �M tolbutamide stimulatedmarked transient elevations of fura-2 340nm/380nm flu-orescence ratio in both INS-1 cells and Cav1.2/II-III cells(Figure 3C, open diamonds). Taken together, the resultspresented in Figure 3 suggest that influx of Ca2� via L-type channels in Cav1.2/II-III cells does not couple effi-ciently to release of Ca2� from internal stores, but thatSr2� influx strongly stimulates release of Ca2� from in-ternal stores in both INS-1 cells and Cav1.2/II-III cells.

Potentiation of tolbutamide-stimulated intracellularCa2� transients and insulin secretion by8-pCPT-2�-O-Me-cAMP-AM. Since tolbutamide-stimu-lated Ca2� transients were not sensitive to thapsigargin inCav1.2/II-III cells, we asked if Ca2� transients stimulatedby tolbutamide would be differentially potentiated by the

EPAC2-selective cAMP analog 8-pCPT-2�-O-Me-cAMP-AM in INS-1 and Cav1.2/II-III cells. We tested theability of 1 �M and 5 �M 8-pCPT-2�-O-Me-cAMP-AMto potentiate tolbutamide-stimulated Ca2� transients andinsulin secretion in INS-1 and Cav1.2/II-III cells. Cellswere loaded with fura-2-AM and were pretreated for 30minutes with 0,1, or 5 �M 8-pCPT-2�-O-Me-cAMP-AMprior to injection of either buffer only or 200 �M tolbu-tamide. Figure 4A shows the net change in fura-2 fluores-cence ratio (tolbutamide response – buffer only response)for representative experiments in INS-1 cells and Cav1.2/II-III cells. Pretreatment with 5 �M 8-pCPT-2�-O-Me-cAMP-AM increased the early peak of the Ca2� transientover pretreatment with buffer alone or 1 �M 8-pCPT-2�-O-Me-cAMP-AM in both INS-1 cells and Cav1.2/II-IIIcells. Area under the curve (AUC) analysis for the entirepostinjection time course of three independent experi-ments in INS-1 cells revealed that 5 �M 8-pCPT-2�-O-Me-cAMP-AM, but not 1 �M, significantly increased theCa2� transient induced by tolbutamide (Figure 4B). How-ever, AUC analysis in Cav1.2/II-III cells revealed that 1�M and 5 �M 8-pCPT-2�-O-Me-cAMP-AM significantlyincreased the Ca2� transient induced by tolbutamide (Fig-ure 4B). Because tolbutamide-stimulated Ca2� transientsin INS-1 cells and Cav1.2/II-III cells are potentiated by 5�M 8-pCPT-2�-O-Me-cAMP-AM and 1 and 5 �M8-pCPT-2�-O-Me-cAMP-AM, respectively, we nextasked if tolbutamide-stimulated insulin secretion wouldbe similarly potentiated by the same concentrations of8-pCPT-2�-O-Me-cAMP-AM. Insulin secretion was stim-ulated with 200 �M tolbutamide in the absence or pres-ence of 0, 1, or 5 �M 8-pCPT-2�-O-Me-cAMP-AM. Fig-ure 4C shows that 5 �M 8-pCPT-2�-O-Me-cAMP-AMsignificantly potentiated tolbutamide-stimulated insulinsecretion in INS-1 and Cav1.2/II-III cells.

Tolbutamide stimulationreducesERCa2� concentrationtoagreater extent in INS-1cells than inCav1.2/II-III cells.To further substantiate that CICR is disrupted in Cav1.2/II-III cells, we directly examined the change in ER [Ca2�]in response to tolbutamide using the ER-targeted calmo-dulin-based Ca2� sensor D1ER (31). D1ER is comprisedof the fluorescent proteins CFP and citrine connected bycalmodulin and a mutant calmodulin binding peptide insuch a way that Ca2� binding permits FRET between CFPand citrine. Thus, in the high [Ca2�] environment of theER, FRET is detected, and stimuli that reduce ER Ca2�

concentration will reduce D1ER FRET. INS-1 cells orCav1.2/II-III cells were transiently transfected with thepCDNA3-D1ER plasmid, and FRET between CFP andcitrine was clearly detected. The basal FRET value wastaken to be the FRET ratio measured under basal condi-



tions (Rmax) and the minimum FRET value was taken asthe FRET ratio measured in cells pretreated for 30 min-utes with thapsigargin and further stimulated with 500�M carbachol (Rmin). The FRET window for each cellline was taken as Rmax-Rmin. The FRET window forCav1.2/II-III cells was significantly lower than that mea-sured for INS-1 cells (Figure 5A). To normalize for thisdifference, changes in FRET ratios in subsequent experi-ments were expressed as a percentage of the FRET win-dow for that cell line. In both INS-1 cells and Cav1.2/II-IIIcells, application of 500 �M carbachol triggered a rapidand persistent reduction in FRET, indicating a decrease inER [Ca2�] (Figure 5B). In contrast, stimulation of bothINS-1 cells and Cav1.2/II-III cells with 200 �M tolbut-

amide resulted in a rapid but transient change in FRETratio that returned to basal levels approximately 1 minuteafter stimulation (Figure 5C). Comparison of the averagemaximum decrease in FRET ratio upon stimulation over7–8 experiments (246–271 cells for each condition) re-vealed that the decrease in ER [Ca2�] in response to car-bachol was not different between INS-1 cells and Cav1.2/II-III cells, but that the decrease in ER [Ca2�] in responseto tolbutamide was significantly reduced in Cav1.2/II-IIIcells compared to INS-1 cells (Figure 5D). Thus, the datain Figure 5C show a rapid, transient decrease in ER[Ca2�] corresponding with the rapid, transient thapsi-gargin-sensitive rise of [Ca2�]in stimulated by tolbut-amide shown in Figure 3C. Further, the tolbutamide-

stimulated transient drop in ER[Ca2�] and the corresponding rise ofcytosolic [Ca2�] detected withFura2AM in INS-1 cells are bothmarkedly reduced in Cav1.2/II-IIIcells.

Glucose and GLP-1in the presenceof verapamil stimulate Ca2�- in-duced Ca2� release events at a lowerfrequency in Cav1.2/II-III cells thanin INS-1 cells. To further examinethe ability of Ca2� influx across themembrane to activate Ca2� releasefrom internal stores in INS-1 cellsand Cav1.2/II-III cells, we measuredchanges in intracellular Ca2� con-centration in single cells stimulatedwith 10 mM glucose and 10 nMGLP-1 in the presence of 50 �M ve-rapamil (2). The rationale for thisprotocol is that glucose and GLP-1potentiate CICR, while verapamildecreases the frequency of L-typechannel openings on the plasmamembrane, allowing observation ofdiscrete CICR events (35). Figure 6Ashows five examples of INS-1 cells inwhich changes in intracellular Ca2�

concentration were measured usingfura 2. Cells were initially depolar-ized with application of 30 mM KClin KRBH for five minutes in the ab-sence of glucose, resulting in a large,transient rise in intracellular Ca2�

concentration. One minute into the30 mM KCl depolarization, perfu-sion of 50 �M verapamil was initi-

FIGURE 4. The EPAC-selective cAMP analog 8-pCPT-2�-O-Me-cAMP-AM potentiates insulinsecretion and Ca2� transients stimulated by tolbutamide in both INS-1 cells and Cav1.2/II-III cells.A, Ca2� transients stimulated by 200 �M tolbutamide in both INS-1 cells and Cav1.2/II-III cells arepotentiated by pretreatment of cells with 8-pCPT-2�-O-Me-cAMP-AM (ESCA). Data shown aremean � SE from representative experiments performed in quadruplicate. B, Area under thecurve (AUC: 2 minutes post injection) analysis of nine (INS-1 cells) or three (Cav1,2.II-III cells)independent experiments in which cells were pretreated with 0, 1, or 5 �M ESCA beforestimulation with 200 �M tolbutamide. ***, P � .001**, P � .01, *, P � .05 compared totolbutamide alone; ###, P � .001 compared to tolbutamide � 1 �M ESCA. C, Tolbutamide-stimulated insulin secretion is potentiated by 5 �M ESCA in INS-1 and Cav1.2/II-III cells (***, P �.001, **, P � .01 compared to basal; ###, P � .001 compared to 1 �M ESCA � tolbutamide).



ated, and maintained for the duration of the experiments.Four minutes after verapamil perfusion was initiated, 30mM KCl perfusion was terminated, and perfusion with10 mM glucose and 10 nM GLP-1 was initiated andmaintained for the duration of the experiments. Perfusionwith glucose and GLP-1 stimulated discrete, transientspikes in intracellular Ca2� concentration that arosefrom, and resolved to, baseline levels in INS-1 cells. Figure6B shows five examples of the same single-cell experimentperformed with Cav1.2/II-III cells. In both INS-1 cells and

Cav1.2/II-III cells, some cells displayed a gradual increasein base-line Ca2� concentration, while other cells dis-played little, if any, persistent increase in intracellularCa2� concentration. However, intermittent Ca2� spikeswere observed in both backgrounds. These intermittentCa2� spikes stimulated by glucose and GLP-1 in the pres-ence of verapamil were significantly more frequent inINS-1 cells than in Cav1.2/II-III cells (Figure 6C; P � .05).In addition, the average spike amplitude and slope wereboth significantly reduced in Cav1.2/II-III cells compared

to INS-1 cells (Figure 6D&E; P �.001 & P � .05, respectively). Takentogether, the data in Figure 6 suggestthat influx of Ca2� in response toglucose and GLP-1 stimulation inCav1.2/II-III cells is less efficientlycoupled to release of Ca2� from in-ternal stores than in INS-1 cells.

Glucose stimulatesactionpotentialsat a greater frequency in INS-1 cellsand primary rat �-cells expressingthe Cav1.2/II-III loop. Since glucoseuptake and metabolism stimulatemembrane depolarization and ac-tion potential firing in pancreatic�-cells, we measured changes inmembrane potential in intact INS-1cells and Cav1.2/II-II cells using theperforated patch configuration ofthe current clamp. Figure 7A showsrepresentative traces of membranedepolarization stimulated by appli-cation of 18 mM glucose (arrows in-dicate time at which glucose was ap-plied) to an INS-1 cell and a Cav1.2/II-III cell. The higher frequency ofaction potentials stimulated by glu-cose in the Cav1.2/II-III cells isclearly visible in these traces. Indeed,analysis of � 20 cells of each typerevealed that glucose-stimulated ac-tion potentials at a significantlyhigher frequency in Cav1.2/II-IIIcells than in INS-1 cells (P � .01)(Figure 7D). This increase in actionpotential frequency was not the re-sult of a stronger depolarization inresponse to glucose since neither theresting membrane potential (2.5mM glucose) nor the average base-line membrane potential after appli-

FIGURE 5. The drop in ER [Ca2�] in response to tolbutamide stimulation is diminished inCav1.2/II-III cells compared to INS-1 cells- A) Quantification of the D1ER FRET window (differencebetween the Rmax and Rmin) in INS-1 and Cav1.2/II-III cells. INS-1 cells have a significantly greaterdynamic range of ER calcium than Cav1.2/II-III cells. The Rmax was calculated prior to any type ofstimulation and represents the average of 26 experiments (total of 799 cells) in INS-1 cells and 21experiments (total of 727 cells) in Cav1.2/II-III in cells (INS-1 cells: 2.81 � 0.04; Cav1.2/II-III:2.33 � 0.04). The Rmin was calculated through depletion of ER calcium stores using 30 minutetreatment with thapsigargin followed by stimulation with carbachol. The minimal FRET ratiorepresents the average of 6 experiments (total of 259 cells) in INS-1 cells and 4 experiments (totalof 139 cells) in Cav1.2/II-III in cells (INS-1 cells: 2.11 � 0.07; Cav1.2/II-III: 1.99 � 0.03). The D1ERwindow was calculated by subtracting the minimal FRET value from the basal FRET value (***,P � .001; Student’s unpaired t test). B) Stimulation of INS-1 and Cav1.2/II-III cells with carbachol(500 �M; added at the 60 seconds time point) causes a rapid and prolonged decrease in theD1ER FRET signal. The INS-1 trace is a representative experiment which includes 49 cells. TheCav1.2/II-III trace is a representative experiment, which includes 83 cells. C) Stimulation of INS-1and Cav1.2/II-III cells with tolbutamide (200 �M; added at the 60 seconds time point) causes atransient decrease in the D1ER FRET signal followed by a rapid recovery to baseline. The peakreduction in ER calcium levels is greater in INS-1 cells than Cav1.2/II-III cells. Data shown arerepresentative experiments including 78 INS-1 cells, and 61 Cav1.2/II-III cells. D) Quantification ofthe decrease in D1ER FRET signal in INS-1 and Cav1.2/II-III cells following stimulation withcarbachol or tolbutamide. Stimulation of ER calcium release by tolbutamide but not carbachol issignificantly greater in INS-1 cells than in Cav1.2/II-III cells. Percent decrease in basal FRET ratiowas calculated by dividing the change in FRET ratio by (Rmax-Rmin), and this was averaged amongall experiments for both cell lines (Carbachol: INS-1 cells 22.29 � 1.79, Cav1.2/II-III cells 23.18 �2.39; Tolbutamide: INS-1 cells 8.43 � 1.06; Cav1.2/II-III cells 4.01 � 1.76)( *, P � .05, INS-1 cellscompared to Cav1.2/II-III cells, Student’s unpaired t test).



cation of 18 mM glucose (ie, the membrane potentialfrom which the action potentials arose) were differentbetween INS-1 cells and Cav1.2/II-III cells (Figure 7C).Activation of the small conductance, calcium-activatedpotassium channel (SK channel), is known to regulateaction potential frequency in pancreatic �-cells (27).Therefore, we examined whether or not dysregulation ofSK channel activity might play a role in the increasedaction potential frequency observed in Cav1.2/II-III cells.In the presence of the SK channel blocker apamin (1 �M),the frequency of 18 mM glucose-stimulated action poten-tials in INS-1 cells is significantly increased (Figure7B&D) but is not different from the frequency measuredin Cav1.2/II-III cells in the absence of apamin (Figure 7D),

suggesting that Ca2� activation of SK channels in Cav1.2/II-III cells is aberrant. Indeed, in Cav1.2/II-III cells, appli-cation of 1 �M apamin doesn’t significantly alter the fre-quency of action potentials stimulated by 18 mM glucose(Figure 7B&D). To further understand the mechanismleading to increased glucose-stimulated action potentialfrequency in Cav1.2/II-III cells, we compared the magni-tude of the action potential afterhyperpolarizations(AHPs) measured in INS-1 cells and Cav1.2/II-III cellsduring trains of action potentials stimulated with 18 mMglucose. Figure 7E, shows example traces of action poten-tials recorded from an INS-1 cell or a Cav1.2/II-III cell.Even though the initial action potentials both arise from amembrane potential of approximately –40 mV, the AHP

in the Cav1.2/II-III cell is of loweramplitude (ie, less negative) than theINS-1 cell. Consequently, within thetime frame of the traces, another ac-tion potential is generated in theCav1.2/II-III cells, but not in theINS-1 cell. The mean AHP ampli-tude was significantly reduced inCav1.2/II-III cells (-11.4 mV � 0.1mV; n � 16 cells, 1564 AHPs) com-pared to INS-1 cells (-13.5 mV � 0.1mV; n � 18 cells, 1087 AHPs; P �.001). Application of the SK channelblocker apamin (1 �M) significantlyreduced the AHP amplitude of glu-cose-stimulated action potentials inINS-1 cells (-10.8 � 0.1 mV; n � 8cells, 430 AHPs; P � .001 comparedto control INS-1 cells), but didn’tsignificantly affect the AHP ampli-tude in Cav1.2/II-III cells (-11.1 �0.1 mV; n � 7 cells, 476 AHPs). In-terestingly, the average AHP ampli-tude in Cav1.2/II-III cells in the ab-sence of apamin was notsignificantly different from that ob-served in INS-1 cells in the presenceof apamin. Taken together, the datain Figure 7 suggest that normal reg-ulation of action potential frequencyis disrupted in Cav1.2/II-III cells,and that loss of SK channel activa-tion could account for the signifi-cant increase in glucose-stimulatedaction potential frequency in thesecells.

Given the marked effects ofCav1.2/II-III loop expression on glu-

FIGURE 6. Discrete spikes in intracellular Ca2� concentration in response to glucose and GLP-1in the presence of verapamil are less frequent in Cav1.2/II-III cells than in INS-1 cells.Representative single cell Ca2� traces of INS-1 cells (A) or Cav1.2/II-III cells (B). Cells were loadedwith 3 �M Fura-2-AM, and perfused with KRBH for 30 seconds (basal) followed by 30 mM KClfor 1 minute. Coapplication of 50 �M verapamil for 4 minutes was used to bring the intracellularCa2� level ([Ca2�]i) to basal. 10 mM glucose (Glu) �10 nM GLP-1� 50 �M verapamil (verap)was perfused for the rest of the experiment to elicit CICR. C, The CICR peak numbers per cell forINS-1 and Cav1.2/II-III ( 2.261 � 0.509 (n � 46) and 0.925 � 0.290 (n � 40), respectively; *, P �.05, one-way ANOVA with the Tukey post hoc test). D, The average amplitude of CICR peaks(�340/380) is significantly greater in INS-1 cells than in Cav1.2/II-III cells (0.065 � 0.003 (n �108) and 0.03 � 0.003 (n � 40), respectively; ***, P � .001, one-way ANOVA with the Tukeypost hoc test). E, The initial slopes of CICR peaks in INS-1 cells are significantly greater thanthose of Cav1.2/II-III cells. The CICR peak slope is calculated as dividing the peak amplitude(�340/380) by the time (sec) from the lowest point to the highest point. The CICR peak slopes inINS-1 and Cav1.2/II-III cells are 0.02 � 0.002 (n � 108) and 0.012 � 0.001 (n � 40), respectively(*, P � .05, one-way ANOVA with the Tukey post hoc test).



cose-stimulated electrical activity observed in INS-1 cells,we asked if this could be recapitulated in primary rat�-cells. To this end, we constructed an adenovirus encod-ing the same Cav1.2/II-III loop-GFP fusion expressed inCav1.2/II-III cells, and used it to transduce primary rat�-cells. Figure 8A shows representative current-clamptraces recorded from primary rat �-cells transduced witheither a control adenovirus encoding GFP only (left panel)or with the adenovirus encoding the Cav1.2/II-IIII loop-

GFP fusion (right panel). The arrows indicate the time atwhich the perfusate was switched from 2.5 mM glucose to18 mM glucose. Neither the resting membrane potential(measured at 2.5 mM glucose) nor the baseline membranepotential measured at 18 mM glucose in cells transducedwith the Cav1.2/II-III-GFP encoding virus were differentfrom those measured in cells transduced with the controlvirus (Figure 8B). However, cells transduced with theCav1.2/II-III-GFP virus fired action potentials in response

to 18 mM glucose at a significantlygreater frequency than cells trans-duced with the control virus encod-ing GFP alone (Figure 8C)(P � .01).Analysis of AHP amplitude in GFPand Cav1.2/II-III-GFP transduced�-cells (Figure 8D) revealed that theAHP amplitude of glucose-stimu-lated action potentials was signifi-cantly reduced in cells expressingCav1.2/II-III-GFP (-8.6 � 0.1 mV;n � 9 cells, 432 AHPs) compared tocontrols expressing GFP only(-9.8 � 0.2 mV; n � 6 cells, 71AHPs) (P � .001, Student’s un-paired t test). Thus, consistent withwhat was observed in the INS-1 andCav1.2/II-III cell lines, acute expres-sion of the Cav1.2/II-III loop in pri-mary rat �-cells results in an increasein the frequency of glucose-stimu-lated action potentials.

SK-channel current is detected inboth INS-1 cells and Cav1.2/II-IIIcells. Given the apparent lack of SKchannel regulation of glucose-stimu-lated action potentials in �-cells ex-pressing the Cav1.2/II-III loop, weasked whether or not SK channel ac-tivity could be detected in Cav1.2/II-III cells. Whole cell K� currents weremeasured in INS-1 cells and Cav1.2/II-III cells using steps to –50 mVfrom a holding potential of –70 mV,with a calculated free Ca2� concen-tration of 2 �M in the recording pi-pette. Under these conditions, themajor K� channels available to con-duct current are the KATP channeland SK channels, since voltage-de-pendent channels are not activated.This protocol elicited an outward

FIGURE 7. SK channel modulation of glucose-stimulated action potentials is disrupted inCav1.2/II-III cells- A, Representative current-clamp traces from an INS-1 cell and a Cav1.2/II-III cellshowing the development of membrane depolarization and action potentials in response toperfusion with18 mM glucose (arrow). B, Representative current-clamp traces from an INS-1 celland a Cav1.2/II-III cell showing the effect of bath application of 1 �M apamin (arrow) on a trainof action potentials stimulated with 18 mM glucose. C, Membrane depolarization induced by 18mM glucose is similar in INS-1 cells (white bar; n � 40) and Cav1.2/II-III cells (black bar; n � 23).D, Action potentials induced by 18 mM glucose in Cav1.2/II-III cells (black bar) have a frequencysignificantly greater than the frequency measured in INS-1 cells (white bar) (**, P � .01; n � 20–22). The SK channel blocker apamin (1 �M) increases the frequency of action potentials in INS-1cells (*, P � .05; n � 8–22). The frequency of glucose-stimulated action potentials in Cav1.2/II-IIIcells is not significantly different in the presence or absence of apamin (n � 7–20). E,Afterhyperpolarizations of glucose-stimulated action potentials are reduced in Cav1.2/II-III cellscompared to INS-1 cells. Example action potentials stimulated by glucose in an INS-1 cell (bluetrace) or a Cav1.2/II-III cell (red trace) are shown. Analysis of 18 INS-1 cells (1087 AHPs) and 16Cav1.2/II-III cells (1564 AHPs) revealed a significant decrease in the amplitude of theafterhyperpolarization of glucose-stimulated action potentials in Cav1.2/II-III cells compared toINS-1 cells (***, P � .001). Apamin (1 �M) significantly reduces AHP amplitude in INS-1 cells (8cells, 430 AHPs; ***, P � .001), but not in Cav1.2/II-III cells (7 cells, 470 AHPs).



current at –50 mV in both INS-1 and Cav1.2/II-III cells(Figure 9A). Application of 100 nM apamin did not blocka significant fraction of this current in either cell line if thepipette solution did not contain free Ca2� (Figure 9 B). Incontrast, if 2 �M free Ca2� was included in the pipettesolution, 100 nM apamin blocked a significant portion ofthe K� currents measured using this protocol (Figure 9B).The density of apamin-sensitive outward K� current mea-sured under these conditions was not different betweenINS-1 and Cav1.2/II-III cells (Figure 9C). Thus, the lack ofmodulation of glucose-stimulated AP frequency byapamin in Cav1.2/II-III cells shown in Figures 7&8 is notthe result of the absence of functional SK channels. Wenext asked if block of SK channels with apamin couldmodulate glucose-stimulated insulin secretion in eitherINS-1 cells or Cav1.2/II-III cells reasoning it may also bedifferentially regulated by apamin in INS-1 cells andCav1.2/II-III cells. However, we found that glucose-stim-

ulated insulin secretion was significantly potentiated byapamin in both cell types (Figure 9D). To understand thisdifference in apamin modulation of glucose-dependentevents, we examined the time course of the rise in intra-cellular [Ca2�] stimulated by 18 mM glucose in INS-1cells and Cav1.2/II-III cells using Fura2. As shown in Fig-ure 9E, the rise in intracellular [Ca2�] was delayed overthe first several minutes of glucose stimulation in Cav1.2/II-III cells compared to INS-1 cells. Taken together, thedata in Figure 9 suggest that apamin does not modulateglucose-stimulated action potentials in Cav1.2/II-III cellsbecause the glucose-dependent rise in cytoplasmic [Ca2�]is not sufficient to activate SK channels within the timeframe of current-clamp experiments (ie, several minutesafter addition of glucose). However, within the longertime frame of the glucose-stimulated insulin secretion as-say (ie, 60 minutes of glucose stimulation) SK channels

are eventually activated in Cav1.2/II-III cells and exert the expected in-hibitory effect on secretion, which isrelieved by apamin blockade of thechannels.

IQGAP1 and eIF3e coimmunopre-cipitate with the Cav1.2/II-III loop.Given the marked effects of the ex-pression of the Cav1.2/II-III loop inINS-1 cells and primary �-cells onCa2� signaling, we examined theability of the Cav1.2/II-III loop to in-teract with proteins in INS-1 cellsusing coimmunoprecipitation ex-periments. The Cav1.2/II-III loopwas previously reported to bind di-rectly to the C2 domains of RIM2(36) and Piccolo (19), and to coim-munoprecipitate RIM2 from INS-1cell lysates (20). The GTP-bindingprotein CDC42 is reported to play akey role in regulating actin polymer-ization during glucose stimulated in-sulin secretion (37) (38). IQGAP1 isa scaffold protein that binds CDC42in a manner that’s regulated byCa2�-calmodulin (39). Further, theC-terminal regions of IQGAP1and 2were recently found to form apseudo C2 fold, capable of bindingphosphatidylinositol 3,4,5,-trispho-sphate (40). Therefore, we exam-ined if the Cav1.2/II-III loop couldinteract with IQGAP1 by immuno-

FIGURE 8. Glucose-induced action potential frequency is greater in rat �-cells expressingCav1.2/II-III compared to controls expressing GFP alone - A, Representative current clamp tracesfrom a rat primary �-cell transduced with adenovirus encoding GFP only (left panel) and a ratprimary �-cell transduced with adenovirus encoding Cav1.2/II-III-GFP (right panel) in response toperfusion with 18 mM glucose (arrow) which triggers membrane depolarization and actionpotentials. B, Resting membrane potential and membrane depolarization induced by 18 mMglucose are not different in �-cells transduced with the GFP adenovirus (white bar; n � 26) orthe Cav1.2/II-III-GFP adenovirus (black bar; n � 20). C, Glucose stimulates action potentials at agreater frequency in �-cells transduced with the Cav1.2/II-III-GFP adenovirus (black bar; n � 9)than in the control �-cells transduced with the GFP adenovirus (white bar; n � 6) (**, P � .01).D, The amplitude of afterhyperpolarizations measured during trains of glucose-stimulated actionpotentials were significantly reduced in primary rat �-cells transduced with the Cav1.2/II-III-GFPadenovirus compared to cells transduced with adenovirus encoding GFP alone (***, P � .001,Student’s t test).



FIGURE 9. SK channels are activated by intracellular Ca2� in both INS-1 and Cav1.2/II-III cells- A) Representative whole-cellvoltage clamp traces of INS-1 (right panel) and 1.2/II-III (left panel) cells demonstrating the Ca2�-dependent K� current blocked by apamin. Cellswere depolarized to –50 mV for 1 second from a holding potential of –70 mV. When the pipette solution contained 0 �M free Ca2�, no currentwas blocked by 100 nM apamin. Tolbutamide block of KATP channel current was detected both in the absence and presence of free Ca2� in thepipette, and was readily reversed upon washout. B) Comparison of fraction current remaining after 100 nM apamin between INS-1 (n � 8 at 2�M [Ca2�]in and n � 5 at 0 [Ca2�]in) and 1.2/II-III (n � 8 at 2 �M [Ca2�]in and n � 6 at 0 [Ca2�]in) cells. Data are shown as mean values � S.E.M.(0.65 � 0.08 (n � 8) and 1.05 � 0.08 (n � 5) at 2 �M [Ca2�]in and 0 �M [Ca2�]in, respectively) and in 1.2/II-III cells (0.69 � 0.05 (n � 8) and0.920 � 0.0651 (n � 6) at 2 �M [Ca2�]in and 0 �M [Ca2�]in, respectively). The fraction of current remaining in the presence of 100 nM apaminwas significantly lower with 2 �M free Ca2� in the pipette than in the absence of Ca2� (*, P � .05, one-way ANOVA with Tukey poc-hoc test). C)Comparison of SK current density between INS-1 (n � 8) and 1.2/II-III (n � 8) cells. Bar graphs represents the mean current density of apamin-sensitive current with 2 �M Ca2� in the pipette solution expressed as pA/pF � S.E.M (INS-1 cells: 1.280 � 0.314, n � 8; 1.2/II-III cells: 0.993 �0.381, n � 8)). The SK current density in Cav1.2/II-III cells is not different from that measured in INS-1 cells (P � .234, Mann-Whitney Rank SumTest). D) Glucose-stimulated insulin secretion is potentiated by 100 nM apamin in both INS-1 and Cav0.2/II-III cells. Cells were stimulated with 10mM glucose for one hour, and insulin secretion was assayed as described in Materials and Methods. The addition of apamin significantly increasedinsulin secretion compared to glucose alone (P� 0.05; Student’s unpaired T test). E) The rise in intracellular Ca2� in response to 18 mM glucose isdelayed in Cav1.2/II-III cells compared to INS-1 cells. Cells were loaded with fura2, and stimulated with by injection of 18 mM glucose. The 340nm/380nm ratio was measured every minute for one hour. Control experiments were performed with injection of KRBH containing no glucose. Thedata shown are corrected by subtraction of the slow, linear increase in 340/380 ratio observed in the absence of glucose. Data shown are themeans � S.E.M. of an experiment done in triplicate, and are representative of 3 independent experiments.



precipitating the loop with antibodies against GFP, andblotting for IQGAP1. As shown in Figure 10A, IQGAP1coimmunoprecipitated with neither GFP transiently ex-pressed in INS-1 cells, nor with the GFP-tagged Cav1.3/II-III loop stably expressed in INS-1 cells. In contrast,immunoprecipitation of the GFP-tagged Cav1.2/II-IIIloop from Cav1.2/II-III cell lysates coimmunoprecipitatedIQGAP1.

The translation initiation factor eIF3e was recently re-ported to mediate Ca2�- dependent internalization ofCav1.2 in INS-1 cells (41). eIF3e was previously reportedto bind directly to a three amino acid sequence towardsthe C-terminal end of the II-III loop of Cav1.1, 1.2, 1.3,2.2, and 2.3 (42). Disruption of a mechanism leading tointernalization of Cav channels could account for themarked increase in Cav current density we observed inCav1.2/II-III cells (Figure 2D). We therefore asked if theCav1.2/II-III loop was interacting with eIF3e using immu-noprecipitation of eIF3e and western blotting with anti-bodies to GFP to detect the Cav1.2/II-III loop fusion. Asshown in Figure 10B, immunoprecipitation of eIF3e didcoimmunoprecipitate the Cav1.2/II-III loop. We were un-able to detect coimmunoprecipitation of the Cav1.2/II-IIIloop and eIF3e using antibodies against GFP (data not

shown). It’s possible that eIF3e binding to the Cav1.2/II-III loop/GFP fusion hinders binding of antibodies to GFPsince the eIF3e binding motif and the GFP fusion site areboth on the C-terminal end of the loop. These data sug-gest that eIF3e and the Cav1.2/II-III loop interact inCav1.2/II-III cells in a manner that may result in accumu-lation of Cav channels in the plasma membrane of thesecells.

Discussion

Key proteins involved in triggering insulin secretion arepresent in lipid rafts in INS-1 cells. Lipid raft localizationof Cav1.2 has been reported in insulin secreting cells (20,32), but the functional significance of this localization islargely unclear. However, the scaffolding proteins RIM2and Piccolo were also detected in lipid rafts prepared us-ing sucrose gradient centrifugation (20), and are reportedto bind directly to the Cav1.2 II-III loop via their C2domains (19). Moreover, RIM2 is coimmunoprecipitatedfrom INS-1 cell lysates by the II-III loop of Cav1.2 but notthat of Cav1.3 (20). Further, the KATP channel subunitSUR1 binds to the cAMP effector EPAC2 (19). Activationof EPAC2 by cAMP in pancreatic �-cells is linked to am-plification of CICR via activation of PLC-� (43) and a2-APB-sensitive Ca2� influx (44). Here we show thatSUR1 and EPAC2 are highly enriched in lipid rafts pre-pared from INS-1 cell lysates (Figure 1), suggesting thatthese proteins, along with Cav1.2, may indeed colocalizeto lipid rafts on the membrane of INS-1 cells. In contrast,Kir6.2, while present in lipid rafts, is more widely distrib-uted throughout sucrose density gradients, consistentwith reports that it also resides within secretory granulesin �-cells (45) (46), and that Kir6.2 is present in secretorygranules in �-cells from Sur1-/- mice (47). Interestingly,expression of the Cav1.2/II-III loop did not appear toaffect the distribution of Piccolo and RIM2 (20), orSUR1, EPAC2, and Kir6.2 (this study) on sucrose densityfractions, in contrast to the marked shift of Cav1.2 tohigher density fractions in Cav1.2/II-III cells (20) reportedpreviously. Our finding that SUR1 and Kir6.2 are found inlipid rafts in INS-1 cells contrasts with a previous study,using HIT-T15 cells, that found them to be excluded fromlipid rafts, while Cav1.2 was found to be highly localizedto lipid rafts (32). The reason for this discrepancy is notclear; however, the colocalization of Cav1.2, SUR1,Kir6.2, Piccolo, RIM2, and EPAC2 in lipid rafts is consis-tent with reports that these proteins interact physicallyand functionally (19, 21).

FIGURE 10. The Cav1.2/II-III loop interacts with IQGAP1 andeIF3e in INS-1 cells- A) IQGAP1 coimmunoprecipitates with theCav1.2/II-III loop/GFP fusion peptide, but not the Cav1.3/II-III loopfusion peptide, from INS-1 cell lysates. Cell lysates were incubated withanti-GFP antibodies coupled to agarose, washed, and eluted with byincubation at 80°C in Laemmli buffer. Ly- 100 �g of cell lysate; Ub-unbound proteins removed after incubation with agarose-immobilizedantibodies; W- protein in the last wash fraction before elution; E-protein eluted by incubation at 80°C in Laemmli buffer. Samples wereblotted with antibodies against IQGAP1 (upper panel) or GFP (lowerpanel). Blots shown are representative of 3 separate experiments. B)The Cav1.2/II-III loop coimmunoprecipitates with eIF3e. Cav1.2/II-IIIlysates were incubated with antibodies to eIF3e and protein A-sepharose, washed, and eluted with Laemmli buffer. Lanes are labeledas described above Samples were blotted with antibodies against GFPto detect the Cav1.2/II-III loop/GFP fusion peptide (upper panel) or withantibodies against eIF3e (lower panel). Blots shown are representativeof 4 separate experiments.



Expression of the Cav1.2/II-III loop does not disrupt KATP

channel activity but increases Cav channel currentdensity. we compared the electrophysiological propertiesof the Cav1.2/II-III cells to those of INS-1 cells (Figure 2).We found no difference between Cav1.2/II-III cells andINS-1 cells in regard to either whole-cell KATP channelcurrent density and sensitivity to block by the sulfonyl-urea tolbutamide, or to the potency of membrane depo-larization and action potential frequency (data notshown) stimulated by tolbutamide in current clamp ex-periments conducted using the whole-cell configuration.However, as reported previously, (20) we did find thatwhole-cell Cav current density (measured using Ba2� asthe permeant ion) was significantly increased in Cav1.2/II-III cells compared to INS-1 cells. This increase in Cav

current density was not confined to L-type channels sincethe portion of current blocked by 10 �M nifedipine wasnot different in INS-1 cells and Cav1.2/II-III cells (20).The mechanism for this increase in Cav current density issuggested by the finding that the translation initiationfactor eIF3e binds to the II-III loop of multiple Cav chan-nels in a Ca2�-dependent manner to mediate internaliza-tion from the plasma membrane (42). Very recently, thismechanism has been reported to regulate the density ofCav1.2 channel on the plasma membrane of INS-1 cells(41). The three amino acid motif in the Cav1.2 II-III foundto be critical for interaction with eIF3e (33) is present inthe Cav1.2/II-III loop/GFP fusion used in this study. In-deed, we found that antibodies to eIF3e could coimmu-noprecipitate the Cav1.2/II-III loop. Thus, the marked in-crease in Cav current density is likely driven, at least inpart, by Cav1.2/II-III/GFP fusion interference with re-trieval of Cav channels from the plasma membrane via theeIF3e-dependent mechanism. However, even with the in-crease in Cav current density, CICR is diminished inCav1.2/II-III cells. It’s tempting to speculate that interrup-tion of the Cav1.2 channel/PCLO and or RIM2 interac-tions by expression of the Cav1.2/II-III loop excludesCav1.2 from a signaling complex critical for CICR, butdoes not otherwise interfere with insertion of Cav1.2 intothe plasma membrane. However, this study can’t excludethe possibility that loss of eIF3e modulation of Cav1.2plasma membrane expression, or interruption of aCav1.2/IQGAP1 interaction is contributing to the deficitsin CICR and SK channel activation observed in Cav1.2/II-III cells. Interestingly, IQGAP1 is an essential compo-nent of the Wnt-receptor-actin-myosin-polarity struc-ture, which recruits cortical ER to the plasma membraneduring directional cell movement (48). It will be of inter-est to determine the relative roles of Cav1.2 interactionswith scaffolding proteins and/or eIF3e in CICR and SKchannel activation in pancreatic �-cells.

Ca2�-induced Ca2� release is diminished in Cav1.2/II-IIIcells compared to INS-1 cells. Given that we detected nodeficits in KATP channel function or sensitivity to tolbut-amide in Cav1.2/II-III cells compared to INS-1 cells, weexamined both insulin secretion and Ca2� transients sim-ulated by tolbutamide in these cell lines (Figure 3). Secre-tion stimulated by tolbutamide in INS-1 cells is com-pletely inhibited by nicardipine, and partially inhibited bypretreatment with thapsigargin. This pharmacologicalprofile is consistent with contributions by both Ca2� fluxacross the membrane through L-type channels (thapsi-gargin-resistant portion) as well as from Ca2�-inducedrelease of Ca2� from internal stores (thapsigargin-sensi-tive portion) to tolbutamide-stimulated insulin secretion.In contrast, insulin secretion stimulated by tolbutamide inCav1.2/II-III cells was completely blocked by nicardipine,but completely resistant to pretreatment with thapsi-gargin, suggesting that release of Ca2� form internalstores does not contribute to tolbutamide-stimulated in-sulin secretion in these cells. Indeed, the strong inhibitionof the rapid tolbutamide-stimulated peak in intracellularCa2� concentration by thapsigargin in INS-1 cells wasmarkedly reduced in Cav1.2/II-III cells, supporting theconclusion that Ca2�-induced Ca2� release from internalstores is disrupted in these cells. This conclusion wasstrongly supported by measurements of ER [Ca2�]changes with the D1ER Ca2� indicator (Figure 5). Indeed,the kinetics of the decrease in ER [Ca2�] upon tolbut-amide stimulation closely reflected the corresponding in-crease in cytosolic Ca2� measured with Fura2 in INS-1cells, and both responses were similarly inhibited inCav1.2/II-III cells. Interestingly, the observation that theD1ER FRET window was significantly lower in Cav1.2/II-III cells compared to INS-1 cells suggests that localiza-tion of Cav1.2 via the II-III loop may contribute to main-tenance of ER Ca2� stores.

To determine that whether the above observationswere unique to sulfonylurea stimulation, we comparedCICR in INS-1 and Cav1.2/II-III cells stimulated withGLP-1 and glucose in the presence of 50 �M verapamil(Figure 6). The rationale behind these experiments is toenhance the sensitivity of RYR2-mediated release of Ca2�

from stores via the actions of GLP-l (49, 50), but minimizethe Ca2� influx via L-type Ca2� channels, which is alsoenhanced under these conditions (51), using the Ca2�

channel blocker verapamil to reduce the probability ofchannel opening (52). This protocol is reported to gener-ate discrete CICR events in pancreatic �-cells (2), andindeed, we observed, discrete, intermittent Ca2� spikesusing this protocol with INS-1 cells. When this protocolwas applied to Cav1.2/II-III cells, these discrete Ca2�

spikes were detected less frequently, and were of a signif-



icantly lower amplitude and slope compared to those de-tected in INS-1 cells. These data further support the con-clusion that Ca2� influx via Cav1.2 channels is notefficiently coupled to release of Ca2� from internal storesin Cav1.2/II-III cells during glucose � GLP-1 stimulation.

Given the increased Cav current density in Cav1.2/II-IIIcells, we propose that the observed disruption of CICR isthe result of a spatial uncoupling of Cav1.2 and RYR2 inCav1.2/II-III cells. An alternative explanation might be adeficit in the RYR2 response to increases in intracellularCa2� concentration in Cav1.2/II-III cells. However, ourfindings that replacement of extracellular Ca2� with Sr2�

restored the thapsigargin-sensitive, tolbutamide-stimu-lated early phase of the Ca2� transient in Cav1.2/II-IIIcells (Figure 3), and that the EPAC2-selective cAMP an-alog ESCA strongly potentiated both tolbutamide-stimu-lated insulin secretion and Ca2� transients in Cav1.2/II-IIIcells (Figure 4) argue against this interpretation.

Cav1.2/II-III cells exhibit an increased frequency of ac-tion potentials in response to glucose. If coupling of Ca2�

influx via Cav1.2 to intracellular signaling has, in fact,been disrupted in Cav1.2/II-III cells, then electrical excit-ability in response to glucose could be enhanced inCav1.2/II-III cells compared to INS-1 cells, if it contrib-utes to activation of small conductance Ca2� activatedK� (SK) channels in pancreatic �-cells. SKchannels areexpressed in mouse �-cells (53), INS-1 cells (25), andhuman �-cells (27). Pancreatic �-cells from SK-4 channelknockout mice exhibited an increase in glucose-stimu-lated action potential frequency (54), and block of SKchannels in human �-cells with the toxin apamin in-creases glucose-stimulated action potential frequency, re-flecting the role of SK channels in mediating the afterhy-perpolarization (25). Our finding that glucose-stimulatedaction potentials in both Cav1.2/II-III cells and in primaryrat �-cells transduced with an adenovirus encoding theCav1.2/II-III loop occurred at a frequency approximatelytwice that of their appropriate controls (Figures 7&8),supports the conclusion that interaction of Cav1.2 withother proteins via the II-III loop is required for normalregulation of SK channels. Loss of SK channel activationas the mechanism for this increase in glucose-stimulatedexcitability is strongly supported by the insensitivity ofglucose-stimulated action potentials in Cav1.2/II-III cellsto apamin, and the highly significant decrease in the mag-nitude of the AHP during trains of glucose-stimulatedaction potentials in Cav1.2/II-III cells and primary �-cellstransduced with the Cav1.2/II-III-GFP virus. Further, thedata in Figure 9 demonstrate that SK channels are acti-vated in Cav1.2/II-III cells if 2 �M Ca2� is provided in thepipette solution. Thus, the primary deficit leading to dys-

regulation of SK channels in Cav1.2/II-III cells is likely theprolonged delay in the rise of [Ca2�]in in response toglucose.

In conclusion, this study provides evidence that theCav1.2 II-III loop is critical for efficient CICR and regu-lation of SK channel activity in pancreatic �-cells. Specificinteractions between Cav1.2 and RIM2, Piccolo, IQ-GAP1, and eIF3e may contribute to maintenance of CICRand/or SK channel regulation, but the role that each ofthese individual proteins plays remains to be determined.In type 2 diabetes, defects in �-cell Ca2� responses toglucose are commonly observed (55). A recent study re-ported that transgenic mice expressing a phospho-mimic,gain of function mutant of RYR2 (S2814D) had dimin-ished Ca2� responses to glucose and KCl and developedglucose intolerance (56). Thus, it is conceivable that de-rangements in CICR could contribute to type 2 diabetes.Interestingly, exposure of INS-1 cells to 28 mM glucosefor 72 hours disrupted lipid rafts, and inhibited insulinsecretion in response to both KCl and glucose (57), aneffect that was attributed to glucose inhibition of choles-terol synthesis. In addition, exposure of pancreatic isletsto palmitate inhibited glucose-stimulated insulin secre-tion and dispersed microdomains of elevated intracellularCa2� concentration in response to brief episodes of mem-brane depolarization in pancreatic �-cells (58). It will beof interest to examine whether disruption of Cav1.2 lo-calization, and the loss of CICR efficiency and regulationof glucose-stimulated electrical excitability describedherein, plays a role in pancreatic �-cell dysfunction in-duced by glucotoxicity or lipotoxicity.

Acknowledgments

The authors acknowledge Dr. Li Li for assistance in analyzingthe FRET data, and Natalie Stull of the Indiana Diabetes Re-search Center Islet Core for isolation of rat pancreatic islets.

Received April 5, 2013. Accepted January 28, 2014.Address all correspondence and requests for reprints to:

Gregory H. Hockerman, 575 Stadium Mall Dr., West Lafayette,IN 47906, [email protected], 765–496-3874, fax765–494-1414.

*Y.W. and R.E.J. contributed equally to this work.The authors declare no conflicts of interest.This work was supported by a grant from the National In-

stitute of Diabetes and Digestive and Kidney Diseases (R01DK064736) to GHH.

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Uncoupling of Ca v 1.2 from Ca 2؉ -induced Ca 2؉ release and SK channel regulation in pancreatic...

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