J Physiol 582.3 (2007) pp 1205–1217 1205

Membrane cholesterol modulates Kv1.5 potassium channeldistribution and function in rat cardiomyocytes

Joelle Abi-Char1,2, Ange Maguy5, Alain Coulombe1,2, Elise Balse1,2, Philippe Ratajczak3,4,

Jane-Lise Samuel3,4, Stanley Nattel5 and Stephane N. Hatem1,2

1INSERM, Unite 621, Paris 75013, France2Universite Pierre et Marie Curie-Paris6, UMR-S621, Paris 75013, France3INSERM, U689, Paris 75018, France4Universite Denis Diderot-Paris7, Centre de recherche cardiovasculaire Lariboisiere, Paris 75018, France5Montreal Heart Institute Research Center and Universite de Montreal, 5000 Belanger St. E., Montreal, Quebec, Canada H1T 1C8

Membrane lipid composition is a major determinant of cell excitability. In this study, we

assessed the role of membrane cholesterol composition in the distribution and function of

Kv1.5-based channels in rat cardiac membranes. In isolated rat atrial myocytes, the application

of methyl-β-cyclodextrin (MCD), an agent that depletes membrane cholesterol, caused a

delayed increase in the Kv1.5-based sustained component, I kur, which reached steady state in

∼7 min. This effect was prevented by preloading the MCD with cholesterol. MCD-increased

current was inhibited by low 4-aminopyridine concentration. Neonatal rat cardiomyocytes

transfected with Green Fluorescent Protein (GFP)-tagged Kv1.5 channels showed a large

ultrarapid delayed-rectifier current (I Kur), which was also stimulated by MCD. In atrial

cryosections, Kv1.5 channels were mainly located at the intercalated disc, whereas caveolin-3

predominated at the cell periphery. A small portion of Kv1.5 floated in the low-density fractions

of step sucrose-gradient preparations. In live neonatal cardiomyocytes, GFP-tagged Kv1.5

channels were predominantly organized in clusters at the basal plasma membrane. MCD caused

reorganization of Kv1.5 subunits into larger clusters that redistributed throughout the plasma

membrane. The MCD effect on clusters was sizable 7 min after its application. We conclude

that Kv1.5 subunits are concentrated in cholesterol-enriched membrane microdomains distinct

from caveolae, and that redistribution of Kv1.5 subunits by depletion of membrane cholesterol

increases their current-carrying capacity.

(Resubmitted 18 April 2007; accepted after revision 18 May 2007; first published online 24 May 2007)

Corresponding author S. N. Hatem: INSERM UMR621, Faculte de Medecine Pierre-Marie Curie, 91 boulevard de

l’Hopital, 75013 Paris, France. Email: stephane.hatem@chups.jussieu.fr

Outward K+ currents are essential for shaping cardio-myocyte action potentials. These currents are carried bya range of voltage-gated potassium (Kv) channels thatare part of multiprotein complexes containing variousauxiliary subunits, scaffolding proteins and/or secondmessengers that regulate channel location and function(Rettig et al. 1994; Kim et al. 1995).

Lipids are other important determinants of channelfunction. Their composition greatly influence structuraland physical properties of the plasma membrane, suchas fluidity, curvature and stiffness, which are importantregulators of the gating of voltage-gated channels (Elinderet al. 1996; Lundbaek et al. 1996). Some lipids,

J. Abi Char and A. Maguy are to be regarded as joint first authors of this

paper.

like phospholipids, can also directly modulate channelfunction. Arachidonic acids and their amide anandamideintroduce rapid inactivation in otherwise non-inactivatingKv channels by binding to channels close to their selectivityfilter (Oliver et al. 2004).

Cholesterol and sphingolipids, two major lipids of theplasma membrane, can also pack tightly together to formmicrodomains called ‘lipid rafts’. Lipid rafts are dynamicplatforms important for the delivery of proteins to themembrane and for sequestering proteins in close physicalproximity to control their functional interactions (Pike,2004). An increasing number of channels has been foundto be targeted into these cholesterol- and sphingolipid-richmembrane microdomains, including Kv channels (Levitanet al. 2000; Martens et al. 2000; Martens et al. 2001;Yarbrough et al. 2002; Hajdu et al. 2003; Barbuti et al.2004; Pouvreau et al. 2004; Wong & Schlichter, 2004;

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society DOI: 10.1113/jphysiol.2007.134809

1206 J. Abi-Char and others J Physiol 582.3

Xia et al. 2004; Brainard et al. 2005; Maguy et al. 2006).However, most of the data available have been obtainedin heterologous expression systems and evidence of thelocalization of endogenous Kv channels in cholesterol-richmembrane microdomains of cardiac myocytes and itsfunctional importance are lacking.

Important clues regarding the regulation of channelfunction by cholesterol have been obtained owing tothe use of methyl-β-cyclodextrin (MCD). This moleculeremoves cholesterol from plasma membranes of live cells.MCD can be added to culture media or applied tosingle cells via the bath perfusate and is effective at bothphysiological and room temperatures (Christian et al.1997; Heino et al. 2000; Slimane et al. 2001; Barbutiet al. 2004). MCD application changes the properties ofseveral Kv channels in both native tissues and heterologousexpression systems (Martens et al. 2000, 2001; Hajduet al. 2003; Xia et al. 2004). In L-cells stably expressingKv1.5 channels, MCD shifts the activation and inactivationcurves of the current (Martens et al. 2001).

In atrial cardiomyocytes, the ultrarapid delayed-rectifiercurrent (IKur) is an important repolarizing current and isbelieved to be largely the functional expression of ShakerKv1.5 channels (Fedida et al. 1993, 2003; Wang et al.1993; Feng et al. 1997). The aim of this study was toexamine the effect of membrane cholesterol depletionon the distribution and function of Kv1.5 subunits inrat cardiomyocytes. We show here that MCD-inducedcholesterol depletion enhances IKur by modulating thefunction and clustering of Kv1.5 subunits.

Methods

Cardiac tissue samples and cardiomyocyte isolation

Animal handling was in accordance with the Guide forthe Care and Use of Laboratory Animals published bythe US National Institutes of Health. A 1/1 mixture ofxylazine (20 mg ml−1) and ketamine (100 mg ml−1) wasprepared and Wistar rats were anaesthetized using anintraperitoneal injection (0.1 ml (100 mg body weight)−1).Whole hearts were rapidly excised and thoroughlywashed in phosphate-buffered saline (PBS) to eliminateresidual blood. The left atria were then isolated,frozen in liquid nitrogen and stored at −80◦C forbiochemistry and immunohistochemistry. For electro-physiological studies, atrial myocytes were enzymaticallyisolated as previously described (Boixel et al. 2001).The left atrium was removed, cut up, and washed inCa2+-free Krebs–Ringer solution containing (mm): 35NaCl, 4.75 KCl, 1.19 KH2PO4, 16 Na2HPO4, 10 Hepes, 10glucose, 25 NaHCO3, 134 sucrose, and 30 2,3-butanedione2-monoxime (BDM) (pH was adjusted to 7.4 withNaOH), gassed with 95% O2–5% CO2, and maintainedat 37◦C. Pieces were re-incubated in this solution withoutBDM and containing bovine serum albumin (BSA)

(5 mg ml−1, Hoechst-Behring), 200 U ml−1 collagenase(type IV, Sigma Chemical Co.), and 6 U ml−1 protease(type XXIV, Sigma). After 30 min of digestion, theenzyme solution was replaced by the same solutioncontaining only collagenase (400 U ml−1). Isolatedmyocytes were resuspended in a bicarbonate-bufferedTyrode solution containing 2 mm Ca2+ and incubated at37◦C with continuous gassing with 95% O2–5% CO2 forat least 1 h before use.

One-day-old neonatal Wistar rats were killed bydecapitation with sharp scissors and hearts were rapidlyexcised and washed to remove blood and debris inpre-oxygenated Tyrode solution containing (mm): 135NaCl, 4 KCl, 2 MgCl2, 10 Hepes, 1 NaH2PO4, 20 glucose,2.5 pyruvate, adjusted to pH 7.4 with NaOH. The ventricleswere carefully minced and dissociated into single cellsby proteolytic enzymes in Tyrode solution containing0.1 mg ml−1 collagenase A (Roche Applied Science) and1% of bovine serum albumin, during repeated digestionswith gentle continuous stirring and aeration with 100%O2 at 37◦C for 10 min. Cell were centrifuged at 100 g for10 min and the pellet resuspended in growth mediumcontaining serum to inhibit proteolytic enzymes. Thisstep was repeated 6 times and pellets were pooled aftereach digestion. After 1 h of pre-plating to purify themyocyte population from fibroblasts, the cells werecounted, adjusted to the desired density (1 × 106 cellsper 9.6 cm2) for seeding on laminin-coated (Roche)LabTek borosilicate slides (Nunc) and grown in Dulbecco’smodified Eagle’s medium (Gibco) supplemented with10% horse serum (Biowest SAS), 5% fetal bovine serum(Biowest Ltd), 100 U ml−1 penicillin and 100 mg ml−1

streptomycin, in standard conditions (37◦C, 5% CO2).

Recombinant proteins and transfection

GFP-tagged human Kv1.5 (HKv1.5) cDNA was generatedby RT-PCR and inserted into the multicloning site ofthe expression vector pcDNA3 as previously described(Godreau et al. 2002, 2003). Twenty-four hoursafter cell isolation, neonatal ventricular cardiomyocytes(1 × 106 cells per 9.6 cm2) were transfected with 1 μgof GFP-tagged Kv1.5 expression vector using 4 μl ofFuGENE 6 Transfection Reagent (Roche Diagnostics)plus OptiMEM (Gibco) that yields ∼10% of transfectedcells.

Membrane microdomains separation and Westernblot analysis

Protein extraction. Frozen atrial appendages (n = 15)were powdered with a mortar in liquid nitrogen and homo-genized on ice with a glass Potter in 2 ml TNE solution(mm: 20 Tris; 150 NaCl; 1 EDTA, pH 7.4) to which weadded Complete EDTA-free Protease Inhibitor cocktail

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J Physiol 582.3 Membrane microdomains and cardiac channel function 1207

(Roche Applied Science). The homogenate was centrifugedat 1000 g for 5 min at 4◦C. The pellet was resuspendedin extraction buffer, re-homogenized with the Potter andcentrifuged again at 1000 g for 5 min at 4◦C. This latterstep was repeated three times to enhance protein extractionefficiency. All supernatants, corresponding to total proteinfraction, were collected and pooled in Eppendorf tubes.Triton X-100 was then added to the total protein fractionwith a final concentration of 1% in order to solubilizeproteins localized in the bulk plasma membrane. Notethat all steps of the protein extraction were performedat 4◦C; at this temperature lipid rafts are insoluble in 1%Triton X-100. After 30 min incubation on ice, the proteinconcentration was determined using a Bio-Rad proteinassay (Bio-Rad Laboratories). Then an Eppendorf tube wasfilled with 17.2 mg of the obtained protein fraction and thevolume was completed to 2 ml with cold extraction buffer(4◦C).

Sucrose gradient separation and SDS-PAGE. Two milli-litres of 80% sucrose solution was placed in a SW41centrifuge tube (Beckman); 2 ml of the total protein(17.2 mg) was placed on the sucrose solution and thepreparation was mixed with a vortex-agitator for 30 s.Four millilitres of 35% sucrose was gently poured onto themixture, followed by 4 ml of 5% sucrose. The gradient wasthen centrifuged in a Centrikon T-1170 ultracentrifuge(Kontron Instruments), for 18 h at 178 300 g and 4◦C,without braking. Fractions of 1 ml were collected fromthe top to the bottom of the centrifuge tube (12fractions kept at −80◦C). Each sample fraction wassonicated, its protein concentration measured, and thesame protein quantity loaded into each lane of 12.5%polyacrylamide-SDS gels. After separation, proteins weretransferred to PVDF membrane (NEN). The membranewas blocked with 5% non-fat milk and incubated withappropriate primary and secondary antibodies. Negativecontrols consisted in omitting the primary antibodyand in incubating membranes with only the secondaryantibody. Development was done using Western LightningChemoluminescence Reagent Plus (Perkin Elmer LifeScience Inc.) and BioFlex Scientific Imaging Films (ClonexCorporation).

Antibodies. Mouse anti-caveolin-3 was from BD Trans-duction Laboratories, rabbit anti-Kv1.5 was fromAlomone Laboratories and rabbit anti-connexin-43(Zymed Laboratories) was from Chemicon International.Sarcomeric α-actinin was from Sigma. Peroxidase-conjugated goat anti-mouse and FITC-conjugatedanti-rabbit IgG were from Jackson ImmunoResearchLaboratory. Texas Red-conjugated anti-mouse IgG andFITC-conjugated anti-rabbit IgG were obtained fromAmersham.

Immunohistochemistry–confocal microscopy

Cryostat sections (7 μm) of rat atrial tissue were fixed in4% formaldehyde for 10 min, permeabilized with 0.2%Triton X-100 for 5 min then pre-incubated for 30 minat room temperature (RT) in PBS containing 5% BSAto block non-specific binding sites. Tissues were rinsedin PBS (pH 7.4) between each step. Then slides wereincubated with rabbit anti-Kv1.5 polyclonal antibodies(pAbs) (1 : 20), with mouse anti-caveolin-3 monoclonalantibodies (mAbs) (1 : 50), with a mixture of mouseanti-caveolin-3 and rabbit anti-connexin-43 (1 : 50)antibodies, or with a mixture of mouse anti-α-actininsarcomeric and rabbit anti-Kv1.5 antibodies for 1 h at RT.After three washes in PBS, binding of primary antibodieswas detected with Texas Red-conjugated anti-mouse IgG(1 : 40) and FITC-conjugated anti-rabbit IgG (1 : 40). Allantibodies were prepared in PBS containing 2% BSA.To check for the specificity of the anti-Kv1.5 antibody,the immunostaining was repeated using a primary anti-body incubated overnight with the antigen at 4◦C. In thiscondition, no staining could be detected on cryosectionof atrial myocardium (data not shown). Sectionswere finally washed in PBS, mounted in Vectashieldmedium and observed with a Zeiss Axiovert-200 confocalmicroscope equipped with an argon laser. Images wereacquired and analysed with LSM510 version 3.2 software.

Neonatal cardiomyocytes were observed with theconfocal microscope using a ×63 oil immersion objectivewith pinhole set at 1 Airy. The 488 nm line of an argon lasersource (10% of full power) was used for GFP excitation. Allexperiments on neonatal cardiomyocytes were performedat 5% CO2 and 37◦C using a temperature-controlledstage.

The quantification of clusters in live myocytes wasperformed using the integrated morphometry analysisof the MetaMorph software to measure surface size ofclusters. This program uses a threshold tool to set a greylevel that discriminates clusters from background light andmeasures the area of signal above the threshold. Surfacearea of clusters were expressed in pixels and averaged foreach cell. The counting was performed on stack images,conducted in double blind and expressed as number ofclusters per myocyte.

Electrophysiology

Whole-cell patch-clamp currents were recorded aspreviously described (Godreau et al. 2002). Cells werebathed or perfused in solutions containing (mm): 135NaCl (or choline chloride), 4 KCl, 2 CaCl2, 2 MgCl2, 1Na2HPO4, 2.5 sodium pyruvate, 10 Hepes, 20 glucose;pH was adjusted to 7.4 with NaOH. Patch pipettes werefilled with a solution containing (mm): 115 potassiumaspartate, 10 KCl, 2 KH2PO4, 3 MgCl2, 5 EGTA, 5MgATP, 10 Hepes, 10 glucose and pH was adjusted to 7.2

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society

1208 J. Abi-Char and others J Physiol 582.3

with KOH. Activation plots were generated by dividingthe current by the difference between test and reversalpotential and were fitted by the Boltzmann distributionequation: G/Gmax = 1/[1 + exp(−(V − V 1/2

)/k)], whereG represents the conductance calculated at membranepotential V , V 1/2

the potential at which half the channelsare activated, and k the slope factor. All experimentswere carried out at RT (22–24◦C). Leak current was notcompensated.

Drugs

5-Methyl-β-cyclodextrin (MCD), 4-aminopyridine(4-AP) and tetraethylammonium (TEA) were obtainedfrom Sigma and dissolved in the extracellular solution.Cholesterol–MCD (8 : 2) complexes were obtained asfollows: 100 mg of cholesterol (Sigma) was dissolved in2 ml of a 1/1 chloroform–methanol solution and 12.8 μlof this solution was added to 10 ml of extracellularsolution and left overnight at 37◦C.

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Figure 1. Effects of cholesterol depletion on outward currents in native adult atrial myocytesA, direct and long period effects of MCD application on the outward current elicited as indicated by the commandwaveform at 0.2 Hz, shown in the inset. B, effects of MCD–cholesterol application. C, time course changes inIkur amplitude in control conditions, upon application of MCD and MCD–cholesterol complexes. D, bar graphssummarizing the effects of MCD and MCD–cholesterol complex, measured after 700 s application.

Statistical analysis

Data are presented as means ± s.e.m. When appropriate,Student’s paired or unpaired t tests were used to determinethe significance of differences. P values < 0.05 wereconsidered significant.

Results

Methyl-β-cyclodextrin increases IKur-relatedcomponent in atrial myocytes

We first examined the effect of the extracellular applicationof 2% MCD on the outward potassium currents of atrialmyocytes. In these cells, test pulses from −90 mV to+50 mV elicited an outward current characterized by a fastinactivating (I t) and a maintained component, the lattercorresponding to IKur. We observed a slow increase in IKur

(after ∼400 s of MCD application) to 15.6 ± 1.3 pA pF−1,from a baseline of 8.5 ± 1.2 pA pF−1, n = 20; P << 0.001)(Fig. 1A). It is worth noting that following 15–20 min

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J Physiol 582.3 Membrane microdomains and cardiac channel function 1209

applications of MCD a leak current was likely to occur.Thus, the application of MCD was limited to 20 minor when such a current was noticeable, the data of thisrecording were rejected. In around 50% of cells, a rapidinhibition of the fast outward current amplitude wasobserved just after MCD application (from 2.1 ± 0.7 nAto 1.8 ± 0.6 nA, n = 10, P < 0.05). We first checked thatprolonged current recording did not affect the kineticsand amplitude of the outward current by repeating theexperiments using a control external solution (n = 5;Fig. 1C and D). In order to determine the specificity ofMCD effects on current, the molecule was saturated withcholesterol at a ratio of 8 : 2 (ratio previously shown toprevent its capacity to buffer lipids: Christian et al. 1997;Heino et al. 2000; Slimane et al. 2001). As illustrated inFig. 1B, prolonged cell exposure to the cholesterol–MCDcomplex caused no increase in the outward K+ current(n = 10) while the fast inhibition was still observed. TheMCD-activated current was sensitive to 4-AP as indicatedby its inhibition with 50 μm 4-AP (46.3 ± 3.7%, n = 8)and was further suppressed at 2 mm 4-AP (74.6 ± 8.1%,n = 6; Fig. 2A and B). The outward current was poorlysensitive to TEA (4.7 ± 1.2%, n = 5) (Fig. 2C). FollowingMCD exposure, there was a slight but statisticallynon-significant (n.s.) change in steady-state activation ofthe maintained current, as indicated by the values of theslope factor (11.8 ± 5.5 mV in control versus 4.1 ± 1.5 mVin MCD, n = 7, n.s.) and the V 1/2

(20.9 ± 6.2 mVin control versus 16.9 ± 4.7 mV in MCD; n = 7, n.s.)(Fig. 3). Taken together, these results indicate thatprolonged MCD application increases a 4-AP-sensitivecomponent of outward current in atrial myocytes, an effectwhich appears to be due to the depletion of membranecholesterol.

Methyl-β-cyclodextrin increases Kv1.5subunit-encoded current in neonatal ratcardiomyocytes

To further determine the nature of the 4-AP-sensitivecurrent activated by MCD and because it is difficult todissect the various components of the K+ outward currentin atrial myocytes, we overexpressed the Kv1.5 subunit inneonatal cardiomyocytes which is believed to underlie alarge part of the atrial sustained current, IKur. Neonatalmyocytes were used because they are easier to transfect andto maintain in culture than adult myocytes. Moreover, theyrepresent a more physiological environment for cardiac ionchannels than heterologous expression systems. Cardio-myocytes expressing GFP-tagged Kv1.5 channels showeda large voltage-dependent, rapidly activating and slowlyinactivating outward current upon depolarizing voltagesteps (Fig. 4A). For instance, at +60 mV, a large currentactivated in transfected myocytes (183 ± 24.7 pA pF−1,n = 7, versus 8.6 ± 1.6 pA pF−1, in control, n = 6),

suggesting that roughly 30 times more functional channelswere present in the cell membrane. This current wasinhibited by 500 μm 4-AP, confirming that it resultedfrom the functional expression of GFP-Kv1.5 channels.The application of MCD caused a slow increase inthe Kv1.5-based current, which started around 7 minafter drug application (average time of 7.4 ± 0.8 min,maximum increase 33 ± 11%, n = 7) (Fig. 4B and C).The MCD-activated outward current in neonatal cardio-myocytes was inhibited by 500 μm 4-AP (n = 5; Fig. 4B).As for adult cardiomyocytes, cholesterol-loaded MCDcomplexes had no effect on Kv1.5-encoded current(n = 5) (Fig. 4C). The stimulatory effect of MCD wasassociated with a decrease of the slope factor of thesteady-state activation curve of the Kv1.5-encoded current(25.9 ± 3.7 mV in control and 15.2 ± 2.3 mV in MCD,n = 5, P < 0.05) together with a slight but statisticallysignificant shift of its V 1/2

(18.1 ± 1.5 mV in control and13.5 ± 1.2 mV in MCD; n = 5, P < 0.05) (Fig. 5). Theseresults indicate that membrane cholesterol depletioncaused by MCD is associated with increased Kv1.5subunit channel function.

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Figure 2. Pharmacological properties of the MCD-activatedcurrent in native adult atrial myocytesEffects of 50 μM (A) and 2 mM 4-AP (B), and 10 mM TEA (C), on the650 s MCD-stimulated outward current elicited as indicated by thecommand waveform at 0.2 Hz.

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1210 J. Abi-Char and others J Physiol 582.3

Kv1.5 channel complexes are not localized in caveolaeof atrial myocardium

To examine the mechanism of action of cholesterolon Kv1.5 channel properties, we next studied howthe lipid regulates channel organization in the plasmamembrane. In cardiac myocytes, cholesterol-enrichedlipid rafts (caveolae) containing caveolin-3 are abundant.Immunostaining of rat atrial cryosections revealed robustcaveolin-3 staining, seen principally at the cell peripherybut not at the level of the intercalated disc (Fig. 6B).In contrast, Kv1.5 channel labelling predominated at thelevel of the intercalated disc, with some staining at theedge of the cell periphery (Fedida et al. 2003) (Fig. 6A).We also co-immunostained cryosections with antibodiesdirected both against caveolin-3 and connexin-43 (Cx-43),which is another intercalated disc protein (Fig. 6C and D).

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Figure 3. MCD effects on electrophysiological parameters of the outward K+ current of native adultatrial myocytesCurrent traces elicited during incremental depolarizing test pulses in control (A) conditions and after MCDapplication (B). Cell capacitance: 75 pF. C, current density–voltage relationships of Ikur recorded in control conditions(•) and following MCD ( �) application. D, voltage dependence of current activation in control (•) and MCD ( �)conditions. In C and D, each point is the average of 7 cells.

Again, there was no overlay between caveolin-3 and Cx-43confirming the lack of caveolin in intercalated discs of theatrial myocardium (Fig. 6E).

We also separated detergent-resistant fromdetergent-soluble membrane proteins of rat atrialmyocardium with a step sucrose gradient (n = 4). Asexpected, caveolin-3 was found predominantly in lowdensity sucrose fractions 4–5, corresponding to lipidrafts (Fig. 7). A single or a doublet band at the expectedmolecular weight of ∼64 kDa of the Kv1.5 subunits(Dobrzynski et al. 2000) was found predominantly infractions 9–12 but also in low density fractions 4–5(Fig. 7). Interestingly, Cx-43 was also detected in fractions4–5 as a doublet corresponding to the phosphorylatedand non-phosphorylated forms of the protein. (Fig. 7).Taken together, these results indicate that Kv1.5 subunitsare mainly localized in non-lipid raft microdomains.

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J Physiol 582.3 Membrane microdomains and cardiac channel function 1211

However, a small fraction of Kv1.5 subunits are foundin cholesterol-enriched microdomains distinct fromcaveolae.

The clustering of Kv1.5 channels dependson membrane cholesterol

We then examined how plasma membrane cholesterolregulates the subcellular organization of Kv1.5 subunits.We used live neonatal rat cardiomyocytes transfected withGFP-Kv1.5 subunits. In sharp contrast with the homo-geneously distributed free GFP, GFP-tagged Kv1.5 subunitswere mainly organized in clusters concentrated at thebasal plasma membrane as shown by the Z-axis view inFig. 8A, lower panel. A similar channel distribution inclusters at the basal membrane was observed in fixed cells

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Figure 4. MCD enhances Kv1.5-encodedcurrents in neonatal cardiomyocytesA, traces of currents recorded duringincremental 10 mV step depolarizations incontrol and in Kv1.5-transfectedcardiomyocytes. B, effects of MCD on theoutward current elicited by a test pulse from−80 to +60 mV in control, and after 350, 500and 600 s MCD applications. Effect of 500 μM

4-AP on 650 s MCD-stimulated current is alsoshown. C, time course of the MCD andMCD–cholesterol complex effects on theoutward current.

immunostained with anti-Kv1.5 antibody (Fig. 8D). Cellspositive for the anti-Kv1.5 antibody were also stained withthe sarcomeric α-actinin antibody with a typical striatedpattern indicating that these cells were cardiac myocytes.The localization of Kv1.5 subunits at the basal membranecould be due to the presence of laminin on the coated dishesas observed for Kir4.1 channels (Guadagno & Moukhles,2004). The role of cholesterol in the formation of Kv1.5channel clusters was studied by incubating cells with2% MCD. After 90 min of MCD application, clusters ofGFP-Kv1.5 increased in size (25 ± 5 versus 145 ± 32 pixels;n = 20, P < 0.001) and in number (58 ± 5 versus 78 ± 6;n = 20, P < 0.05) and redistributed throughout the plasmamembrane (Fig. 8B). In order to determine the time courseof the MCD effect on clusters, the same cardiomyocyteswere visualized during the first minutes following drug

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1212 J. Abi-Char and others J Physiol 582.3

application in a single Z section in order to preventexcessive photobleaching. After 7 min of MCD application(corresponding to the time necessary for MCD to enhancethe current), there was already a significant effect on thesize (61 ± 11; n = 21 cells, P < 0.01) and a tendency toincrease in number (69 ± 6; n = 21 cells) of GFP-Kv1.5subunit clusters (Fig. 8C). The same time course ofMCD effects on Kv1.5 channel distribution was obtainedat room temperature (data not shown). These resultsindicate that cholesterol is an important determinant ofthe organization of Kv1.5 subunits in clusters and of theirdistribution into plasma membranes.

Discussion

In the present study we found that plasma membranecholesterol is an important determinant of the distribution

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Figure 5. Effect of cholesterol depletion on outward currentparameters resulting from Kv1.5 subunit overexpression inneonatal cardiomyocytesCurrent density–voltage relationships (A) and voltage dependence (B)of IKur activation under control conditions (•) and following 7 minMCD application ( �). In A and B, each point represents average datafrom 5 cells.

of Kv1.5 subunits and the properties of the correspondingcurrent IKur in atrial cardiomyocytes. A fraction of thesesubunits are located in membrane microdomains richin cholesterol but distinct from caveolae. These effectsof membrane cholesterol on Kv1.5 subunit distributionand function could have significant consequences for theregulation of cardiac excitability in both physiological andpathophysiological conditions.

The predominant effect of MCD on the outwardpotassium current of atrial cardiomyocytes is to induce aslow and progressive increase in IKur. This delayed increasein IKur appears to be related to the capacity of MCDto deplete membrane cholesterol, since it was eliminatedwhen MCD was pre-saturated with cholesterol. The effectsof MCD on current observed after several minutes ofdrug application could not be attributed to a non-specificeffect due to prolonged patch-clamp recording, asno current changes were observed over correspondingperiods in control external solution. The delay betweendrug application and increase in IKur corresponds to thetime necessary for MCD to deplete membrane cholesterol(Launikonis & Stephenson, 2001; Lam et al. 2004). Inaddition to the delayed increase in sustained current, insome cells MCD causes a fast and reversible inhibitionof the peak outward current also observed with theMCD–cholesterol complex suggesting a direct effect ofMCD on channels (Eldstrom et al. 2006).

Several arguments indicate that Kv1.5 subunit-basedchannels contributed in part to the MCD-activatedcurrent. First, Kv1.5-based channels are believed to bethe molecular basis of IKur in atrial cardiomyocytes(Fedida et al. 1993; Wang et al. 1993; Feng et al. 1997).Second, the MCD-activated current was sensitive to lowconcentrations of 4-AP, as are Kv1.5 channels (Wang et al.1993). Third, in neonatal cardiomyocytes, Kv1.5-encodedcurrent is also stimulated after prolonged (15–20 min)application of MCD, an effect no longer observed inthe presence of a saturating concentration of cholesterol.However, it is likely that other channels contribute tothe MCD-activated current as suggested by the activationof leak-type current after more than 15 min of MCDapplication.

Previous studies have demonstrated that membranecholesterol is an important regulator of ion channelproperties (Levitan et al. 2000; Martens et al. 2000,2001; Yarbrough et al. 2002; Hajdu et al. 2003; Barbutiet al. 2004; Pouvreau et al. 2004; Wong & Schlichter,2004; Xia et al. 2004; Brainard et al. 2005). As for Kv1.5subunits in the present study, cholesterol depletion causedby MCD increases the activity of maxi-K+ channels inhuman myometrium (Brainard et al. 2005), K+ channelsof Drosophila Kenyon cells (Gasque et al. 2005) or inwardlyrectifying Kir channels (Romanenko et al. 2004; Fanget al. 2006). However, the underlying mechanisms ofthis cholesterol effect on ionic channels are unclear.

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J Physiol 582.3 Membrane microdomains and cardiac channel function 1213

C

20μm20μm

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Cx43 / Cva3Cav3Cx43

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20μm

10μm 20μm

A B

Kv1.5 Cav3

Figure 6. Kv1.5 do not co-localized with caveolin-3 in adult atrial tissue.Immunolocalization of Kv1.5 subunits (A) and caveolin-3 (B) in cryosections of atrial myocardium. Double immuno-staining of connexin-43 (FITC, C) and caveolin-3 (Texas Red, D) in cryosections of atrial myocardium. E, mergedimage of the same area of the section in C and D, showing the lack of overlap between connexin-43 and caveolin-3stainings.

Single-channel studies have eliminated the possibility thatthe lipid modulates single-channel unitary conductances(Romanenko et al. 2004; Toselli et al. 2005). Rather, it seemsthat cholesterol affects the number of active channels in the

Kv1.5

Cav3

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Cx43

60 kDa

18 kDa

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5% sucrose 35% sucrose 40% sucrose

1 2 3 4 5 6 7 8 9 10 11 12

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Figure 7. Some Kv1.5 channel subunits are localized in lipid raft fractions in adult rat atrial myocytesWestern blot analysis of the distribution of Kv1.5 subunits, connexin-43 and caveolin-3 on step sucrose gradientof proteins from atrial myocardium.

plasma membrane. In our study, the relative short delaybetween MCD application and IKur increase eliminatesthe possibility of changes in Kv1.5 subunit synthesis. Thisis in agreement with the lack of variation of Kir mRNA

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1214 J. Abi-Char and others J Physiol 582.3

concentrations in endothelial cells following cholesterolaccumulation (Romanenko et al. 2004).

There are several reports suggesting that membranecholesterol can modulate the equilibrium between activeand silent forms of channels. For instance, a reduced openprobability (Po) of Kir or N-type single Ca2+ channels wasobserved following cholesterol membrane accumulation(Romanenko et al. 2004; Toselli et al. 2005). The

0

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.5 c

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ers

tota

l are

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ixel

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Control MCD - 1h 30

Free GFP

BA 10μm10μm

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Figure 8. Surface expression of Kv1.5 subunits in neonatal cardiomyocytesA, in live cardiomyocytes, transfected GFP-tagged Kv1.5 subunits are clustered at the membrane surface adjacent tothe bottom of laminin-coated glass support, as shown in the projection of Z sections in the lower panel. In contrast,GFP alone was homogeneously distributed in cardiomyocytes (inset). B, after the application of 2% MCD, clustersincreased in size and were redistributed throughout the plasma membrane. C, bar graphs summarizing changesin cluster size upon MCD exposures; data are from 21 cardiomyocytes in control, and following incubation with2% MCD for 7 min and 1 h 30 min. ∗∗P < 0.01, ∗∗∗P < 0.001. D, double immunostaining of fixed cardiomyocytesusing sarcomeric α-actinin and anti-Kv1.5 antibodies showing that Kv1.5-GFP-transfected cells are cardiomyocytes.A, B and D: scale bars represent 10 μm.

authors proposed that depletion of membrane cholesteroldecreases membrane stiffness and consequently themembrane deformation energy associated with channelopening. Such a process explains the effect of cholesterol onN-type channels, whose gating properties are modulatedby membrane tension and stiffness (Toselli et al. 2005).However, the magnitude of the effect of changes inmembrane deformation on outward current activation is

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J Physiol 582.3 Membrane microdomains and cardiac channel function 1215

unknown. Changes in cholesterol can also alter surfacecharge in the vicinity of channels. However, the lack ofmajor shift of the voltage dependence of IKur activationboth in adult and neonatal cardiac myocytes followingMCD application suggests that such a mechanism is notpredominant (Elinder et al. 1996). In the range of timeexposures of MCD (10–15 min) chosen in this study, nochange in the whole-cell capacitance was observed withthe straightforward standard technique used. However,this technique does not allow measuring the change inmembrane capacitance expected from the slight decreasein membrane thickness induced by removing cholesterol(Czub & Baginski, 2006).

In rat atrial myocardium, Kv1.5 channels are notlocalized in caveolae, the lipid rafts containing caveolin-3.First, Kv1.5 channels are detected predominantly at thelevel of the intercalated disc, but not at the cell peripherywhere caveolin-3 is seen. Cx-43, another protein localizingto intercalated discs, similarly does not co-localize withcaveolin. These data are in good agreement with publishedstudies showing that caveolin-3 is located at the peri-phery of atrial and ventricular myocytes in t-tubules andnot in intercalated discs (Yarbrough et al. 2002; Lockeet al. 2005). Moreover, caveolin-3 and Kv1.5 subunits fromrat and canine atrial myocardium fail to co-precipitateand are differentially distributed as seen using electronmicroscopy and immunogold staining (Eldstrom et al.2006). However, a small portion of Kv1.5 subunits aswell as Cx-43 can be detected in the low density sucrosefraction of the sucrose step gradient suggesting thatthere are subtypes of cholesterol-enriched membranemicrodomains distinct from caveolae. In their study,Eldstrom et al. (2006) did not detect the presence ofKv1.5 subunits in the low density fractions of step gradientsucrose. This discrepancy between the two studies couldbe explained by differences in the procedure used toextract lipid rafts which can yield different subsets of lipidrafts. This is well known for the compartmentation ofconnexins in lipid rafts that depend on the procedureused (Locke et al. 2005). Furthermore, lipid rafts areheterogeneous structures, ranging from a highly orderedcholesterol core through less-ordered regions to thedisordered structure of the bulk membrane. Proteins couldbe located in distinct regions of lipid rafts, resultingin distinct detergent solubility as reported for neuronalglycosylphosphatidylinositol-anchored (GPI) proteins(Madore et al. 1999). Proteins of the intercalated disc mayalso have a low affinity for surrounding lipids, so thatthey can be solubilized by detergents. For instance, theT cell receptor in Jurkat cells acquires resistance to Tritonextraction when cross-linked by an antibody (Janes et al.1999). However, the Eldstrom et al. (2006) study and ourown study agree that the majority of the Kv1.5 subunitsare not in the lipid raft fraction. It is possible that thelocation of a small fraction of Kv1.5 channels in lipid rafts

is transient, taking place between channel delivery to theplasma membrane and their recruitment into specializeddomains. It was reported that connexins in raft fractionsare distinct from those in gap junctions and could beunpaired hemichannels en route to junctional domains(Schubert et al. 2002; Locke et al. 2005). The compositionof membrane in cholesterol might regulate the partitionof channels in these distinct compartments, and in turntheir activity.

In live cardiomyocytes, we found that cholesterolregulates the organization of GFP-Kv1.5 subunits inclusters at the plasma membrane. Clustering appears as ageneral feature of channel organization and was reportedfor several K+ and Ca2+ channels (Ianoul et al. 2004)both in heterologous expression systems and in nativetissues such as postsynaptic sites on the soma and proximaldendrites for Kv2.1 channels (Muennich & Fyffe, 2004) orthe atrial myocardium for Kv1.5 channels using electronmicroscopy (Dobrzynski et al. 2000). Clustering maydepend on the integrity of lipid-rich membrane micro-domains, as reported for Kv2.1 in HEK-293 cells incubatedwith MCD (O’Connell & Tamkun, 2005). Overexpressionof the Kv1.5 subunits may result in the accumulationof the protein in lipid rafts. We also found that effectof cholesterol depletion on clusters of Kv1.5 subunits isfast and occurred over a similar time course to MCDeffects on IKur. This observation suggests a relationshipbetween the ability of Kv1.5 subunits to function aschannels and their spatial organization. This is reminiscentof the observations that the effects of cholesterol on theequilibrium between active and silent Kir 2.1 and 2.2channels depends on integrity of lipid rafts (Romanenkoet al. 2004; Fang et al. 2006). The physical clustering ofKcsA potassium channels determines the mode of gatingand activation of the channel (Molina et al. 2006). Ithas been shown, in HEK-293 cells, that clusters of Kv2.1channels represent a well delineated membrane domainwhere channels are delivered via trafficking vesicles andtrapped by several retention proteins within in the clusterperimeters (O’Connell et al. 2006). It is also possiblethat the increase in size and number of clusters of Kv1.5channels following MCD application is due to morechannels delivered in the plasma membrane. Thus, oneother mechanism underlying the effect of cholesteroldepletion on K+ current could be that Kv1.5 subunitclusters seen in the absence of MCD correspond to alipid-rich compartment with ready access to the plasmamembrane. When cholesterol is removed, this pool couldrelease the Kv1.5 subunits and allow them to move rapidlyinto the membrane where they can form functionalchannels.

In conclusion, membrane cholesterol content regulatesIKur, very likely by modulating the spatial organization andactivity of Kv1.5 channels. It has been recently reported thatinhibition of Kir channel function by cholesterol could

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1216 J. Abi-Char and others J Physiol 582.3

be an important mechanism underlying the endothelialdysfunction caused by hypercholesterolaemia (Fang et al.2006). There is also clinical evidence that treatmentlowering plasma cholesterol is associated with a reducedrisk of cardiac arrhythmia, notably atrial arrhythmias(Hanna et al. 2006). Thus, it would be of interest to studythe pharmacological consequences of the cholesterol effecton potassium currents.

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Acknowledgements

We thank Association Francaise contre les Myopathies,

Fondation de France, the Agence Nationale de la Recherche

(ANR-05-PCOD-006-01) and the Canadian Institutes of

Health Research for their financial support. A. Maguy was

supported by Groupe de Reflexion sur la Recherche Cardio-

vasculaire and Association Francaise contre les Myopathies, J.

Abi-Char by the Societe Francaise de Pharmacologie and the

Association Francaise contre les Myopathies, and E. Balse by

ANR-05-PCOD-006-01.

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society