Laboratory of Physiology and Physiopathology, Université Libre de Bruxelles, 1070 Brussels, Belgium
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ABSTRACT |
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The activity of epithelial Na+ selective channels is modulated by various factors, with growing evidence that membrane lipids also participate in the regulation. In the present study, Triton X-100 extracts of whole cells and of apical membrane-enriched preparations from cultured A6 renal epithelial cells were floated on continuous-sucrose-density gradients. Na+ channel protein, probed by immunostaining of Western blots, was detected in the high-density fractions of the gradients (between 18 and 30% sucrose), which contain the detergent-soluble material but also in the lighter, detergent-resistant 16% sucrose fraction. Single amiloride-sensitive Na+ channel activity, recorded after incorporation of reconstituted proteoliposomes into lipid bilayers, was exclusively localized in the 16% sucrose fraction. In accordance with other studies, high- and low-density fractions of sucrose gradients likely represent membrane domains with different lipid contents. However, exposure of the cells to cholesterol-depleting or sphingomyelin-depleting agents did not affect transepithelial Na+ current, single-Na+ channel activity, or the expression of Na+ channel protein. This is the first reconstitution study of native epithelial Na+ channels, which suggests that functional channels are compartmentalized in discrete domains within the plane of the apical cell membrane.
sodium reabsorption; A6 cells; amiloride; lipid bilayers
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INTRODUCTION |
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AMILORIDE-SENSITIVE NA+ CHANNELS mediate vectorial transport of Na+ across reabsorbing epithelia including renal distal and collecting tubules, distal colon, and lungs. Their function is essential in salt and water homeostasis, including the regulation of blood volume and pressure. These channels, located in the apical cell membrane of polarized epithelia, consist of heterooligomeric complexes comprising several proteins required for full activity and hormone responsiveness (1, 8, 12, 24, 32). A number of intracellular factors and signaling pathways regulate these channels, but the influence of the composition and biophysical properties of the cell membrane itself on the number and location of functional native channel complexes remains unclear.
The heterogeneous distribution of lipid in the cell membranes of epithelia, 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 the composition 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, we report the reconstitution of functional amiloride-sensitive Na+ channels obtained from cultured renal epithelial cells (A6) into artificial planar lipid bilayer membranes. When apical membrane-enriched extracts are subjected to sucrose density centrifugation, active Na+ channels float in a low-density, detergent-insoluble fraction whereas channel protein found in the detergent-soluble fractions is inactive, indicating that the association with native lipids directly contributes to the regulation of Na+ movement through the channel.
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EXPERIMENTAL PROCEDURES |
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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 a 20% dilution with distilled water and supplemented with 5% FBS (HiClone). For biochemical work, cells were plated on 100-cm2 homemade structures with porous supports (HAWP, Millipore) and harvested after 10 days in culture. Maximum and stable values of transepithelial Na+ transport and electrical parameters are observed at this time, indicating that apical Na+ channels are functional (27).
Transepithelial measurements of voltage and resistance were performed on 0.33-cm2 structures (Costar) using an EVOM volt-ohmmeter (World Precision Instruments). The corresponding amiloride-inhibitable Na+ current was calculated from these values.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. Samples were allowed to solubilize for 1 h on ice in the presence of 1% Triton X-100. Sucrose was then added to a final concentration of 40%.
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 at 28,000 g for 1 h. The apical membrane-enriched pellets (cf. Ref. 27) were recovered, resuspended in MBS, and solubilized for 1 h on ice in the presence of 1% Triton X-100.
Floatation on sucrose density gradients. Samples were placed at the bottom of linear 5-30% sucrose gradients 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 and snap-frozen. Typically, ~20 fractions were recovered, in which sucrose concentration was measured by refractometry.
SDS-PAGE and immunoblotting.
Aliquots of sucrose gradient fractions were subjected to 7.5% SDS-PAGE
under 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 probed
with a polyclonal rabbit antibody at a final concentration of 4.5 µg/ml. The antibody (a gift from Dr. T. Kleyman) was raised against a
portion of the extracellular loop of the -subunit of the cloned
epithelial Na+ channel from A6 cells and was previously
shown to recognize native Na+ channels in A6 cells
(35). Reactive proteins were detected using a 1:5,000
dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG and
the Renaissance chemiluminescence reagent (NEN). Caveolin was probed
using a commercial antibody raised against human caveolin-1 (Santa Cruz
Biotechnology, Santa Cruz, CA), at a 1:200 dilution.
Proteoliposome reconstitution. For functional studies, aliquots (100 µl) from each sucrose gradient fraction were added to 100 µg of dried palmitoyl-oleoyl-phosphatidylcholine (POPC; Avanti Polar Lipids). Detergent was removed by incubating the samples overnight at 4°C with BioBeads equilibrated with MBS without Triton X-100. Proteoliposomes were recovered by decanting. Before their reconstitution into liposomes, the fractions obtained from membrane material were concentrated six times.
Planar lipid bilayer experiments and analysis.
Bilayer membranes were formed at room temperature by passing a bubble
from a pipette tip prewettted with a membrane-forming solution of POPC
(25 mg/ml in n-octane) over a 150-µm-diameter aperture
drilled in a 50-µm-thick wall of a delrin cup containing symmetrical
200 mM Na-gluconate solutions. Currents were measured using a
conventional current-to-voltage converter based on an OPA-101
(Burr-Brown, Tucson, AZ) operational amplifier with a 1-G feedback
resistor. 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. Membrane
formation was monitored by the increase in capacitive current to
triangle pulses from a function generator. Only membranes with a
capacitance of 150-200 pF and a basal conductance of <10 pS were
considered satisfactory for experimentation. The trans side
was held at
40 mV until the appearance of channel activity. The
incorporation protocol consisted of consecutive additions of 1-2
µl of proteoliposome suspension to the trans chamber,
under constant stirring, to a maximum of 5 µl. Twenty minutes of
stirring were allowed between the additions. Fusion events occurred
after 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 a
TL-1 DMA interface and Axotape 1.2 software (Axon Instruments).
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RESULTS |
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As a first approach, we used whole cell, Triton X-100-treated
samples to look for the presence of Na+ channel protein and
for single Na+ channel activity recovered from each of the
sucrose density gradient fractions. With the use of an antibody shown
previously to recognize native Na+ channel complexes in A6
cells (15), protein was detected by immunoblotting in
gradient fractions corresponding to sucrose concentrations of 16 and
18-28.5% (Fig. 1A).
Channel protein was not detected in the lighter fractions (5-15%
sucrose). Identical volumes of each fraction were used, and it is
obvious that channel protein becomes more abundant toward the bottom of
the gradient, which contains the detergent-soluble material. In
functional studies, we used bathing solutions consisting simply of
buffered Na-gluconate, so we would detect only Na+
channels. In agreement with the pattern of protein distribution, Na+ channel activity from reconstituted proteoliposomes
into lipid bilayers was not detected in fractions with <16% sucrose.
The 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 of
POPC 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 of
proteoliposomes from a single fraction (16% sucrose) resulted in the
appearance of Na+ channel activity typical of native in
situ as well as reconstituted channels from A6 cells (Fig.
1B) (9, 29).
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Based on these data on whole cell extracts, we proceeded with the study
of apical membrane-containing fractions from A6 cells. These membrane
preparations have been extensively studied previously. Although they
contain only 3% of the total cell protein, they are enriched 10-fold
in apical membrane markers; active Na+ channels are also
exclusively located in them (27). Similar to the
observations in whole cell extracts, Na+ channel protein
was detected by immunoblot analysis in fractions of sucrose densities
of 16% and 18.5-30% and was more abundant in the heavy-density
pellet, which contains the solubilized protein. Figure
2 shows the specific protein
pattern obtained by immunoblotting of the 16% sucrose
membrane-enriched fraction. The 97-kDa protein was again detected,
along with a very faint band of 150 kDa relative mass
(Mr). Similar Mr
polypeptides were previously identified as components of the native
Na+ channel (3, 15, 28, 35) and as related to
its Na+ transport function (13, 26). We also
observed a 50-kDa band, previously identified with this anti-X.
laevis epithelial Na+ channel (ENaC) antibody
(15).
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Functionally, only the 16% sucrose fraction contained active
amiloride-sensitive Na+ channels. In particular, similar to
the observation in whole cell extracts, no amiloride-sensitive channel
activity was found in the pellet. This confirms previous observations
that once Na+ channel proteins are solubilized in Triton
X-100, they lose their amiloride-binding and
Na+-transporting properties (27). A
representative example of the amiloride-sensitive Na+
channel activity from the 16% sucrose fraction is shown in the top trace of Fig. 3. Of nine
successful incorporations of protein from 16% sucrose fractions, five
bilayers contained single Na+ channels, and the remainder
contained multiple channels. These channels had linear current voltage
(I/V) relationships, with the slope conductances
averaging 10.1 ± 0.8 pS in the 200 mM Na-gluconate solution (Fig.
3B, open symbols, n = 9). Open times varied
from tens of milliseconds to several seconds. The nature of the
observed channels was confirmed by their inhibition by amiloride, which reduced the channel open probability by 90% at a concentration of 1 µM (Fig. 3A, bottom trace, n = 7). Amiloride-sensitive Na+ channels were the only channels
observed in the 16% sucrose fraction in Na-gluconate buffer. We
examined ion selectivity for two channels under bi-ionic conditions.
Ion selectivity was calculated from the values of the reversal
potentials (23.1 and 25.9 mV) that yielded a
PNa+/PK+ selectivity coefficient of ~2.5
(Fig. 3B, closed symbols). This low selectivity was reported
previously for the native unsolubilized Na+ channel from A6
cells incorporated into planar lipid bilayers (29).1
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To test the influence of lipid environment on channel function, we tried to reconstitute Na+ channels into bilayers composed of PC-cholesterol-sphingomyelin (1:1:1 molar ratio), a lipid composition resembling that found in membrane microdomains shown in other systems to contain active proteins (see DISCUSSION). We did not observe the appearance of channel activity from membrane-enriched fractions in bilayers of this composition. However, this kind of highly packed bilayer may limit the fusion of proteoliposomes.
Agents that modify the lipid composition of membrane microdomains
have been shown to alter the function of associated proteins (21). To begin characterizing the lipid surroundings of
the active channels, we tested the effects of fumonisin, an inhibitor of the biosynthetic pathway of sphingomyelin (16), and of
cyclodextrin, an agent used to deplete the cells of cholesterol
(4), on Na+ channel expression,
transepithelial amiloride-sensitive Na+ currents, and
single Na+ channel activity. A6 cells were exposed to 25 µM fumonisin and 5 mM 2-hydroxypropyl--cyclodextrin before
membrane preparation and sucrose floatation.
Membrane lipid modification was assessed by the expression of caveolin,
a glycolsylphosphatidylinositol (GPI)-anchored protein present in
microdomains enriched in cholesterol and sphygomyelin (5).
As shown in Fig. 4A, caveolin
was detected in control membranes but not in membranes prepared from
cells exposed to the lipid-modifying agents. By contrast,
Na+ channel protein was still detected in fractions of 16 and 18-30% sucrose density (Fig. 4B).
Amiloride-sensitive transepithelial Na+ currents were not
affected by treatment of the cells with either fumonisin (25 µM) or
cyclodextrin (5 mM) (Fig. 5A).
When both agents were added together, currents dropped after 2 h
of incubation. However, this drop could be attributed to a 30.3 ± 6.5% drop in transepithelial resistance, indicating an effect of these
drugs 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 was
found only in the 16% sucrose density fraction containing membrane
material prepared from cells exposed to fumonisin and cyclodextrin,
with conductance and open probability similar to control channels (Fig.
5B). These data suggest that active Na+ channels
cluster in membrane microdomains that do not depend on cholesterol and
sphingomyelin.
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DISCUSSION |
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The present study combines biochemical and functional analyses of
native Na+ channel protein in Triton X-100-soluble and
-insoluble fractions obtained from membranes of A6 renal epithelial
cells in culture and shows that amiloride-sensitive Na+
channels, recorded in artificial bilayer membranes, are restricted to
Triton X-100-resistant membrane microdomains. Resistance of membrane
domains to solubilization in nonionic detergent conferred by an
enriched lipid content results in a high buoyancy (5), which is consistent with our observation of channel protein in the
low-density region of the sucrose gradient. The preservation of native
protein-lipid interactions is important for the biological activity of
the extracted proteins. In this respect, we previously found that
solubilization of native A6 Na+ channels with cationic,
anionic, or nonionic detergents destroys transport activity, while
extracts obtained with a zwitterionic detergent contained functional
Na+ channels (27). The zwitterionic
detergents, which are the most efficient detergents in extracting
active protein, have been shown to produce the highest solubilized
lipid/protein ratios (2), while all hydrophobic detergents
such as Tritons extract little protein-associated lipid, and their use
results in poorly active (2) or inactive protein
(11). Our data are consistent with the idea that native
Na+ channels must be closely associated with native lipids
in the membrane to sustain functional activity. Because this activity was observed with the cloned -ENaC subunit alone as well as with all
combinations of
- with
- and
-subunits (22), the
absence of channel activity in heavier gradient fractions can be
attributed to disruption of this association by Triton rather than to a
change 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 be
associated.2
Although protein-lipid interactions are potentially important for regulating native Na+ channel function (18, 33), the results obtained with cloned ENaCs are variable. For example, expression of ENaC was not observed in membrane fractions resistant 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 be transformed from a Triton X-100-soluble form in the endoplasmic reticulum to a Triton X-100-insoluble form during trafficking to the cell surface (23). Because these cells presumably transport Na+, these are apparently contradictory findings. However, Na+ channel function was not evaluated in either of those studies.
The influence of the lipid environment on the function of membrane proteins was highlighted by the discovery of lipid rafts, which are particular membrane microdomains enriched in cholesterol and sphingomyelin. These liquid-ordered regions are insoluble in nonionic detergent (17). Such membrane regions have proven important for clustering of active proteins (25, 30) but also ion channels. In this regard, targeting of a functional isoform of the voltage-gated K+ channel Kv1.5 to distinct lipid rafts of transfected mouse L-cells was shown (20, 21). In that study, the soluble material represented mostly overexpressed intracellular channels. This is consistent with our observations that Na+ channel protein is abundantly present at the bottom of the gradients obtained with whole cell extracts. Because the amount of this soluble material is greatly reduced in the apical membrane-enriched preparations, it is likely to represent intracellular proteins, possibly in an immature form (15, 32).
In the apical membrane extracts, the amount of channel protein in detergent-resistant fractions represents only a small part of the total membrane pool of these proteins. Quantitative analysis of transepithelial current vs. the number of protein molecules at the membrane led Firsov et al. (6) to suggest the presence of two pools of conducting channels at the apical membrane, a large pool of merely silent channels and a small pool of activated channels. Our results could provide a mechanistic interpretation for these observations. Compartmentalization of active Na+ channels within discrete regions of the apical cell membrane could explain the very low probability of finding channel activity in native or cultured cells using the patch-clamp method (7, 9) and could also reconcile the diverging biophysical properties of this channel in different tissues, species, or artificial membranes.
Our negative results with agents known to modify the amount of cholesterol and sphingomyelin in the cell membrane indicate that the microdomains surrounding active Na+ channels in epithelia may be different from the classic rafts that consist of these molecules. In support of this conclusion, it was recently shown that some proteins cluster in membrane microdomains that do not depend on cholesterol and sphingomyelin content (34). In this regard, it was shown that at ambient temperature saturated PC alone can form lipid domains that are Triton insoluble (17).
Regardless of whether specialized membrane regions or simply a close protein-lipid association is required for functional activity, future studies will attempt to define the lipid composition of the membrane fraction containing active Na+ channels as well as the effect of different lipid environments on native Na+ channel behavior to characterize further the regulatory role of the native membrane environment on Na+ transport function.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. R. Kleyman for the generous gift of
the anti-xENaC antibody and Nancy Leclercq for technical assistance.
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FOOTNOTES |
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V. G. Shlyonsky was the recipient of a "Subside à savant" from the Université Libre de Bruxelles. This research was supported by CER funds from Université Libre de Bruxelles (to S. Sariban-Sohraby).
1 The low selectivity of reconstituted channels that we observed differs from the higher selectivity of endogenous channels found in epithelia studied by the path-clamp technique (7, 9). Recently, Jovov et al. (14) addressed these discrepancies, showing that the addition of the cytoskeletal protein actin to reconstituted rat ENaC activity resulted in the increase in Na+/K+ ion selectivity of the channel. We cannot exclude that Triton X-100 treatment and/or differential centrifigation affect cytoskeletal proteins from the channel complex, which, in turn, could result in the decrease in channel ion selectivity.
2
Recently, Hill WG, An B, and Johnson JP
(Originally published August 6, 200210.1074/jbc.C200309200.
J Biol Chem 277: 33541-33544, 2002) have shown the
discontinuous sucrose gradient centrifugation fractionation pattern of
Na+ channels endogenously expressed in A6 cells in the
absence of Triton X-100. -,
-, and
-subunits of the native
channels were found to localize in cholesterol-enriched regions of high
buoyancy and to migrate in the sucrose gradient similarly to caveolin, suggesting raft localization. However, function was not tested in these fractions.
Address for reprint requests and other correspondence: S. Sariban-Sohraby, Campus Erasme, CP 604, 808, route de Lennik, 1070 Brussels, Belgium (E-mail: sohraby{at}ulb.ac.be).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
September 11, 2002;10.1152/ajprenal.00216.2002
Received 6 June 2002; accepted in final form 4 September 2002.
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