Epithelial sodium channel activity in detergent-resistantmembrane microdomains - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Vadim G.Shlyonsky, FrédériqueMies,  SarahSariban-Sohraby作者单位：Laboratory of Physiology and Physiopathology,Université Libre de Bruxelles, 1070 Brussels,Belgium ) q' Z5 P* [: H2 z5 M. q# a+ C


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      【摘要】( C$ o6 l0 F# M% P3 B
   The activity of epithelialNa selective channels is modulated by various factors,with growing evidence that membrane lipids also participate in theregulation. In the present study, Triton X-100 extracts of whole cellsand of apical membrane-enriched preparations from cultured A6 renalepithelial cells were floated on continuous-sucrose-density gradients.Na channel protein, probed by immunostaining of Westernblots, was detected in the high-density fractions of the gradients(between 18 and 30% sucrose), which contain the detergent-solublematerial but also in the lighter, detergent-resistant 16% sucrosefraction. Single amiloride-sensitive Na channel activity,recorded after incorporation of reconstituted proteoliposomes intolipid bilayers, was exclusively localized in the 16% sucrose fraction.In accordance with other studies, high- and low-density fractions ofsucrose gradients likely represent membrane domains with differentlipid contents. However, exposure of the cells to cholesterol-depletingor sphingomyelin-depleting agents did not affect transepithelialNa current, single-Na channel activity, orthe expression of Na channel protein. This is the firstreconstitution study of native epithelial Na channels,which suggests that functional channels are compartmentalized indiscrete domains within the plane of the apical cell membrane.
      【关键词】 sodium reabsorption A cells amiloride lipid bilayers
   INTRODUCTION' q  A9 B/ O# n. T

AMILORIDE-SENSITIVE NA CHANNELS mediate vectorial transport of Na across reabsorbingepithelia including renal distal and collecting tubules, distal colon,and lungs. Their function is essential in salt and waterhomeostasis, including the regulation of blood volume and pressure.These channels, located in the apical cell membrane of polarizedepithelia, consist of heterooligomeric complexes comprising severalproteins required for full activity and hormone responsiveness( 1, 8, 12, 24, 32 ). A number of intracellular factors andsignaling pathways regulate these channels, but the influence of thecomposition and biophysical properties of the cell membrane itself onthe number and location of functional native channel complexes remains unclear.

The heterogeneous distribution of lipid in the cell membranes ofepithelia, first described by Simons and Van Meer ( 31 ), leads to the formation of lipid microdomains that resist detergent solubilization. A number of studies have been done to characterize thecomposition of microdomains and reveal that specific proteins, including ion channels, tend to localize in them in a functional way( 17, 21 ). Furthermore, it was recently demonstrated that lipid modifications of microdomains alone are sufficient to confer specific sublocalization of active proteins ( 34 ). Here, wereport the reconstitution of functional amiloride-sensitiveNa channels obtained from cultured renal epithelial cells(A6) into artificial planar lipid bilayer membranes. When apicalmembrane-enriched extracts are subjected to sucrose densitycentrifugation, active Na channels float in alow-density, detergent-insoluble fraction whereas channelprotein found in the detergent-soluble fractions is inactive,indicating that the association with native lipids directly contributesto the regulation of Na movement through the channel.

EXPERIMENTAL PROCEDURES5 v4 w& L/ A" z" J9 @8 A  R
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Cell culture. A6 cells (American Type Culture Collection derived originally from Xenopus laevis ) were maintained in culture on plastic flasks in DMEM-F-12 growth medium, adapted for amphibian tissue culture by a20% dilution with distilled water and supplemented with 5% FBS(HiClone). For biochemical work, cells were plated on100-cm 2 homemade structures with porous supports (HAWP,Millipore) and harvested after 10 days in culture. Maximum and stablevalues of transepithelial Na transport and electricalparameters are observed at this time, indicating that apicalNa channels are functional ( 27 ).6 V0 R: T, L0 x- L
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Transepithelial measurements of voltage and resistance were performedon 0.33-cm 2 structures (Costar) using an EVOM volt-ohmmeter(World Precision Instruments). The corresponding amiloride-inhibitableNa current was calculated from these values.# V* `* J8 i8 a6 ~1 x9 H3 `
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Whole cell Triton X-100 extracts. Cells were scraped from porous supports in MOPS-buffered saline (MBS;25 mM MES, 150 mM NaCl, 1 mM PMSF, pH 6.5) and homogenized. Sampleswere allowed to solubilize for 1 h on ice in the presence of 1% Triton X-100. Sucrose was then added to a finalconcentration of 40%.' L1 u* a- C! b0 X& Y- L1 _; u
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Apical membrane-enriched Triton X-100 extracts. Cells were scraped in MBS, homogenized, and centrifuged at low speed(5,500 g ) for 10 min. Supernatants were then centrifuged at28,000 g for 1 h. The apical membrane-enriched pellets(cf. Ref. 27 ) were recovered, resuspended in MBS, andsolubilized for 1 h on ice in the presence of 1% Triton X-100.+ L* A/ k2 ~* p' K0 p

Floatation on sucrose density gradients. Samples were placed at the bottom of linear 5-30% sucrosegradients prepared in MBS without Triton X-100 and centrifuged to equilibrium in a Beckman SW41 rotor at 39,000 rpm for 18 h at 4°C. Gradient fractions of 600 µl were collected from the top andsnap-frozen. Typically, ~20 fractions were recovered, in which sucrose concentration was measured by refractometry.
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SDS-PAGE and immunoblotting. Aliquots of sucrose gradient fractions were subjected to 7.5% SDS-PAGEunder reducing conditions and transferred to nitrocellulose. Immunoblotting was performed in Tris-buffered saline with 5% powered low-fat milk and 0.1% Tween 20. Na channels were probedwith a polyclonal rabbit antibody at a final concentration of 4.5 µg/ml. The antibody (a gift from Dr. T. Kleyman) was raised against aportion of the extracellular loop of the -subunit of the clonedepithelial Na channel from A6 cells and was previouslyshown to recognize native Na channels in A6 cells( 35 ). Reactive proteins were detected using a 1:5,000dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG andthe Renaissance chemiluminescence reagent (NEN). Caveolin was probedusing a commercial antibody raised against human caveolin-1 (Santa CruzBiotechnology, Santa Cruz, CA), at a 1:200 dilution.

Proteoliposome reconstitution. For functional studies, aliquots (100 µl) from each sucrose gradientfraction were added to 100 µg of driedpalmitoyl-oleoyl-phosphatidylcholine (POPC; Avanti Polar Lipids).Detergent was removed by incubating the samples overnight at 4°C withBioBeads equilibrated with MBS without Triton X-100. Proteoliposomeswere recovered by decanting. Before their reconstitution intoliposomes, the fractions obtained from membrane material wereconcentrated six times.+ R8 m" G, R; I$ l1 n
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Planar lipid bilayer experiments and analysis. Bilayer membranes were formed at room temperature by passing a bubblefrom a pipette tip prewettted with a membrane-forming solution of POPC(25 mg/ml in n -octane) over a 150-µm-diameter aperturedrilled in a 50-µm-thick wall of a delrin cup containing symmetrical200 mM Na-gluconate solutions. Currents were measured using aconventional current-to-voltage converter based on an OPA-101(Burr-Brown, Tucson, AZ) operational amplifier with a 1-G feedbackresistor. The current-to-voltage converter was connected to the trans compartment (0.8 ml) of a bilayer chamber using an Ag-AgCl electrode and 3 M KCl-3% agar bridge. Thus the trans side was a virtual ground. The cis compartment (0.6 ml) was connected to a voltage source. Membraneformation was monitored by the increase in capacitive current totriangle pulses from a function generator. Only membranes with acapacitance of 150-200 pF and a basal conductance of trans sidewas held at 40 mV until the appearance of channel activity. Theincorporation protocol consisted of consecutive additions of 1-2µl of proteoliposome suspension to the trans chamber,under constant stirring, to a maximum of 5 µl. Twenty minutes ofstirring were allowed between the additions. Fusion events occurredafter 20-60 min. Currents were monitored on an oscilloscope and/or a computer screen. Current records were low-pass filtered at 200 Hz through an 8-pole Bessel filter (900 LPF, Frequency Devices, Haverhill, MA) before acquisition at the rate of 1 kHz using aTL-1 DMA interface and Axotape 1.2 software (Axon Instruments).

The unitary currents were determined from current amplitude histogramsconstructed from events lists generated by WinASCD software(Laboratorium voor Fysiologie, KU Leuven, Belgium). The relativeNa /K permeability was calculated from thereversal potentials measured after switching of solutions to bi-ionicconditions, using the Goldman-Hodgkin-Katz equation.
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RESULTS

As a first approach, we used whole cell, Triton X-100-treatedsamples to look for the presence of Na channel protein andfor single Na channel activity recovered from each of thesucrose density gradient fractions. With the use of an antibody shownpreviously to recognize native Na channel complexes in A6cells ( 15 ), protein was detected by immunoblotting ingradient fractions corresponding to sucrose concentrations of 16 and18-28.5% (Fig. 1 A ).Channel protein was not detected in the lighter fractions (5-15%sucrose). Identical volumes of each fraction were used, and it isobvious that channel protein becomes more abundant toward the bottom ofthe gradient, which contains the detergent-soluble material. Infunctional studies, we used bathing solutions consisting simply ofbuffered Na-gluconate, so we would detect only Na channels. In agreement with the pattern of protein distribution, Na channel activity from reconstituted proteoliposomesinto lipid bilayers was not detected in fractions with fusion of reconstituted liposomes from higher density gradient fractions (from 18 to 28.5% sucrose) into planar lipid bilayers resulted in the appearance of big integral currents. To discern single-channel events, each fraction was diluted with liposomes made ofPOPC in MBS plus 7.2% sucrose and subjected to freeze-thawing on ice.However, no amiloride-sensitive Na current was recorded( n = 15, data not shown). Bilayer incorporation ofproteoliposomes from a single fraction (16% sucrose) resulted in theappearance of Na channel activity typical of native insitu as well as reconstituted channels from A6 cells (Fig. 1 B ) ( 9, 29 ).
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Fig. 1. A : distribution of A6 Na channelprotein from whole cell extracts in sucrose density gradient fractions.Samples were treated with 1% Triton X-100, floated on a 5-30%linear sucrose gradient, and separated on 7.5% polyacrylamide SDSgels. Samples (25 µl; i.e., 4% vol/vol of each fraction) were loadedper lane. Immunoblotting with an anti- - Xenopus laevis epithelial Na channel (ENaC) antibody shows thedistribution of A6 Na channel protein in the differentsucrose density fractions. In these preliminary experiments, the blotswere cut and only the region where part of the channel protein was mostlikely to be found was probed, i.e., the 90- to 100-kDa region( 35 ). B : single-channel records of ENaCactivity from the 16% sucrose fraction of whole cell Triton X-100extract. Top trace : bilayers were bathed with symmetricalsolutions containing 200 mM Na-gluconate, 10 mM HEPES-Tris, pH 7.4. Bottom trace : 1 µM amiloride was added to bothcompartments of the bilayer chamber. Records were digitally filtered at100 Hz using pCLAMP software. Horizontal markers at the right indicate closed state of the channel. Holdingpotential was 100 mV.
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Based on these data on whole cell extracts, we proceeded with the studyof apical membrane-containing fractions from A6 cells. These membranepreparations have been extensively studied previously. Although theycontain only 3% of the total cell protein, they are enriched 10-foldin apical membrane markers; active Na channels are alsoexclusively located in them ( 27 ). Similar to theobservations in whole cell extracts, Na channel proteinwas detected by immunoblot analysis in fractions of sucrose densitiesof 16% and 18.5-30% and was more abundant in the heavy-densitypellet, which contains the solubilized protein. Figure 2 shows the specific proteinpattern obtained by immunoblotting of the 16% sucrosemembrane-enriched fraction. The 97-kDa protein was again detected,along with a very faint band of 150 kDa relative mass( M r ). Similar M r polypeptides were previously identified as components of the nativeNa channel ( 3, 15, 28, 35 ) and as related toits Na transport function ( 13, 26 ). We alsoobserved a 50-kDa band, previously identified with this anti- X.laevis epithelial Na channel (ENaC) antibody( 15 ).3 j" d* ^) o* _: l" }7 v
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Fig. 2. A6 Na protein from apical membrane extract.Immunoblotting of the 16% sucrose gradient fraction withanti- - X. laevis ENaC antibody shows specific bands at M r of 150 (very faint), 97, and 50 kDa. Thesebands were not detected when the blots were incubated with nonimmuneserum in place of the primary antibody (data not shown). Due to lossduring the membrane preparation and despite concentration of thesamples on microconcentrators (Microcon, Amicon), protein concentrationis 4.6 times less than in the corresponding lane of Fig. 1.9 [4 k. w- g$ j0 u
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Functionally, only the 16% sucrose fraction contained activeamiloride-sensitive Na channels. In particular, similar tothe observation in whole cell extracts, no amiloride-sensitive channelactivity was found in the pellet. This confirms previous observationsthat once Na channel proteins are solubilized in TritonX-100, they lose their amiloride-binding andNa -transporting properties ( 27 ). Arepresentative example of the amiloride-sensitive Na channel activity from the 16% sucrose fraction is shown in the top trace of Fig. 3. Of ninesuccessful incorporations of protein from 16% sucrose fractions, fivebilayers contained single Na channels, and the remaindercontained multiple channels. These channels had linear current voltage( I / V ) relationships, with the slope conductancesaveraging 10.1 ± 0.8 pS in the 200 mM Na-gluconate solution (Fig. 3 B, open symbols, n = 9). Open times variedfrom tens of milliseconds to several seconds. The nature of theobserved channels was confirmed by their inhibition by amiloride, which reduced the channel open probability by 90% at a concentration of 1 µM (Fig. 3 A, bottom trace, n = 7). Amiloride-sensitive Na channels were the only channelsobserved in the 16% sucrose fraction in Na-gluconate buffer. Weexamined ion selectivity for two channels under bi-ionic conditions.Ion selectivity was calculated from the values of the reversalpotentials (23.1 and 25.9 mV) that yielded aP Na  /P K  selectivity coefficient of ~2.5(Fig. 3 B, closed symbols). This low selectivity was reportedpreviously for the native unsolubilized Na channel from A6cells incorporated into planar lipid bilayers ( 29 ). 1& z' ~; m, }# K0 }) k! q
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Fig. 3. Single-channel records of epithelialNa channel activity from the 16% sucrose fraction ofapical membrane preparations. A : bilayers were bathed withsymmetrical solutions containing 200 mM Na-gluconate, 10 mM HEPES-Tris,pH 7.4 ( top trace ), and 1 µM amiloride was added to bothcompartments of the bilayer chamber ( bottom trace ). Recordswere digitally filtered at 100 Hz using pCLAMP software. Horizontalmarkers at the right indicate closed state of the channel.Holding potential was  100 mV. B : current-voltage( I / V ) relationships in symmetrical and bi-ionicconditions. Data points represent single-channel current amplitudes insymmetrical solutions (200 mM Na-gluconate, open symbols, n = 9) and after the switch to bi-ionic conditions (200 mM Na-gluconate/200 mM K-gluconate, trans/cis, closedsymbols, n = 2). In symmetrical solutions, the linerepresents linear regression of the data. In bi-ionic conditions,approximation to the Goldman-Hodgkin-Katz current equation was drawnusing SigmaPlot software.

To test the influence of lipid environment on channel function, wetried to reconstitute Na channels into bilayers composedof PC-cholesterol-sphingomyelin (1:1:1 molar ratio), a lipidcomposition resembling that found in membrane microdomains shown inother systems to contain active proteins (see DISCUSSION ).We did not observe the appearance of channel activity frommembrane-enriched fractions in bilayers of this composition. However,this kind of highly packed bilayer may limit the fusion of proteoliposomes.% d! \' d7 F1 l0 o: D) L9 [% n/ \$ ^
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Agents that modify the lipid composition of membrane microdomainshave been shown to alter the function of associated proteins ( 21 ). To begin characterizing the lipid surroundings ofthe active channels, we tested the effects of fumonisin, an inhibitor of the biosynthetic pathway of sphingomyelin ( 16 ), and ofcyclodextrin, an agent used to deplete the cells of cholesterol( 4 ), on Na channel expression,transepithelial amiloride-sensitive Na currents, andsingle Na channel activity. A6 cells were exposed to 25 µM fumonisin and 5 mM 2-hydroxypropyl- -cyclodextrin beforemembrane preparation and sucrose floatation.
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Membrane lipid modification was assessed by the expression of caveolin,a glycolsylphosphatidylinositol (GPI)-anchored protein present inmicrodomains enriched in cholesterol and sphygomyelin ( 5 ).As shown in Fig. 4 A, caveolinwas detected in control membranes but not in membranes prepared fromcells exposed to the lipid-modifying agents. By contrast,Na channel protein was still detected in fractions of 16 and 18-30% sucrose density (Fig. 4 B ).Amiloride-sensitive transepithelial Na currents were notaffected by treatment of the cells with either fumonisin (25 µM) orcyclodextrin (5 mM) (Fig. 5 A ).When both agents were added together, currents dropped after 2 hof incubation. However, this drop could be attributed to a 30.3 ± 6.5% drop in transepithelial resistance, indicating an effect of thesedrugs on the tight junctions rather than on the Na channels. This was confirmed by reconstitution of single-channel activity in lipid bilayers. Amiloride-sensitive channel activity wasfound only in the 16% sucrose density fraction containing membranematerial prepared from cells exposed to fumonisin and cyclodextrin,with conductance and open probability similar to control channels (Fig. 5 B ). These data suggest that active Na channelscluster in membrane microdomains that do not depend on cholesterol andsphingomyelin.

Fig. 4. Effects of lipid-modifying agents on membrane proteins.A6 cells were exposed to 25 µM fumonisin for 68 h, together with5 mM cyclodextrin in the last 2 h before membrane preparation. A : immunoblot of caveolin control (100 µg protein; lane 1 ) and treated membranes (125 µg protein; lane2 ). B : immunoblot of Na channel proteinisolated from membranes of treated A6 cells and floated on a5-30% sucrose gradient. The 90- to 100-kDa region is shown.Sucrose densities are indicated at the bottom." i# a/ _" X9 |/ C' ^

Fig. 5. Effects of lipid-modifying agents on Na transport function. A : amiloride-sensitive transepithelialmeasurements of Na currents on A6 cells. Confluentmonolayers were incubated with 5 mM cyclodextrin in water, 25 µMfumonisin in DMSO, or a combination of both for the times indicated.Control wells received both vehicles. Normalized data of short-ciruitcurrent ( I SC ) calculated with respect to controlvalues (5.33 ± 0.21 µA/cm 2 ) are shown.* P = 0.002 ( n = 3, Student's t -test). Cyclodextrin (10 mM) was also tested as well asfumonisin for up to 68 h without further change (data not shown). B : single-channel I / V relationships in symmetrical 200 mM Na-gluconate. Data points representsingle-channel current amplitudes., Control ( = 10.1 ± 0.8 pS);, experimental ( = 9.3 ± 0.9) ( P = 0.2, NS).

DISCUSSION

The present study combines biochemical and functional analyses ofnative Na channel protein in Triton X-100-soluble and-insoluble fractions obtained from membranes of A6 renal epithelialcells in culture and shows that amiloride-sensitive Na channels, recorded in artificial bilayer membranes, are restricted toTriton X-100-resistant membrane microdomains. Resistance of membranedomains to solubilization in nonionic detergent conferred by anenriched lipid content results in a high buoyancy ( 5 ), which is consistent with our observation of channel protein in thelow-density region of the sucrose gradient. The preservation of nativeprotein-lipid interactions is important for the biological activity ofthe extracted proteins. In this respect, we previously found thatsolubilization of native A6 Na channels with cationic,anionic, or nonionic detergents destroys transport activity, whileextracts obtained with a zwitterionic detergent contained functionalNa channels ( 27 ). The zwitterionicdetergents, which are the most efficient detergents in extractingactive protein, have been shown to produce the highest solubilizedlipid/protein ratios ( 2 ), while all hydrophobic detergentssuch as Tritons extract little protein-associated lipid, and their useresults in poorly active ( 2 ) or inactive protein( 11 ). Our data are consistent with the idea that nativeNa channels must be closely associated with native lipidsin the membrane to sustain functional activity. Because this activity was observed with the cloned -ENaC subunit alone as well as with allcombinations of - with - and -subunits ( 22 ), theabsence of channel activity in heavier gradient fractions can beattributed to disruption of this association by Triton rather than to achange in subunit composition. In this regard, it was recently reported that in sucrose gradient fractions from A6 cells, -, -, and -subunits of native channels were found to beassociated. 2( z- d- D6 s( s& T' j0 i/ |

Although protein-lipid interactions are potentially important forregulating native Na channel function ( 18, 33 ), the results obtained with cloned ENaCs are variable. Forexample, expression of ENaC was not observed in membrane fractionsresistant to Triton X-100 solubilization in MDCK cells( 10 ), which contain microdomains ( 19 ),whereas in transfected COS-7 and HEK-293 cells, ENaC was shown to betransformed from a Triton X-100-soluble form in the endoplasmicreticulum to a Triton X-100-insoluble form during trafficking to thecell surface ( 23 ). Because these cells presumablytransport Na  , these are apparently contradictory findings.However, Na channel function was not evaluated in eitherof those studies.% Q( g6 ?: T; }  J; l$ L3 l( U

The influence of the lipid environment on the function of membraneproteins was highlighted by the discovery of lipid rafts, which areparticular membrane microdomains enriched in cholesterol andsphingomyelin. These liquid-ordered regions are insoluble in nonionicdetergent ( 17 ). Such membrane regions have proven important for clustering of active proteins ( 25, 30 ) butalso ion channels. In this regard, targeting of a functional isoform ofthe voltage-gated K channel Kv1.5 to distinct lipid raftsof transfected mouse L-cells was shown ( 20, 21 ). In thatstudy, the soluble material represented mostly overexpressedintracellular channels. This is consistent with our observations thatNa channel protein is abundantly present at the bottom ofthe gradients obtained with whole cell extracts. Because the amount ofthis soluble material is greatly reduced in the apicalmembrane-enriched preparations, it is likely to represent intracellularproteins, possibly in an immature form ( 15, 32 ).% y( o5 W0 G, N
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In the apical membrane extracts, the amount of channel protein indetergent-resistant fractions represents only a small part of the totalmembrane pool of these proteins. Quantitative analysis oftransepithelial current vs. the number of protein molecules at themembrane led Firsov et al. ( 6 ) to suggest the presence oftwo pools of conducting channels at the apical membrane, a large poolof merely silent channels and a small pool of activated channels. Ourresults could provide a mechanistic interpretation for theseobservations. Compartmentalization of active Na channelswithin discrete regions of the apical cell membrane could explain thevery low probability of finding channel activity in native or culturedcells using the patch-clamp method ( 7, 9 ) and could alsoreconcile the diverging biophysical properties of this channel indifferent tissues, species, or artificial membranes.

Our negative results with agents known to modify the amount ofcholesterol and sphingomyelin in the cell membrane indicate that themicrodomains surrounding active Na channels in epitheliamay be different from the classic rafts that consist of thesemolecules. In support of this conclusion, it was recently shown thatsome proteins cluster in membrane microdomains that do not depend oncholesterol and sphingomyelin content ( 34 ). In thisregard, it was shown that at ambient temperature saturated PC alone canform lipid domains that are Triton insoluble ( 17 ).

Regardless of whether specialized membrane regions or simply a closeprotein-lipid association is required for functional activity, futurestudies will attempt to define the lipid composition of the membranefraction containing active Na channels as well as theeffect of different lipid environments on native Na channel behavior to characterize further the regulatory role of thenative membrane environment on Na transport function.; f/ \: m9 j) M2 D0 C* G$ @

ACKNOWLEDGEMENTS' m. r* K( m9 I8 \

We thank Dr. T. R. Kleyman for the generous gift ofthe anti- xENaC antibody and Nancy Leclercq for technical assistance.
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