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J Biol Chem, Vol. 274, Issue 53, 37845-37854, December 31, 1999

Regulation of Epithelial Na⁺ Channels by Actin in Planar Lipid Bilayers and in the Xenopus Oocyte Expression System^*

Biljana Jovov, Albert Tousson^§, Hong-Long Ji, Deborah Keeton, Vadim Shlyonsky^¶, Pierre-Jean Ripoll, Catherine M. Fuller, and Dale J. Benos^**

From the  Department of Physiology and Biophysics and the ^§ Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na⁺ channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na⁺ versus K⁺ permselectivity, and a substantial increase in mean open and closed times of wild-type -rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the  subunit was deleted (_R613X-rENaC). 2) When _R613X-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either _R613X-rENaC or wild-type -rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that -rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The physiological importance of amiloride-sensitive sodium channels is reflected by the abundance of regulatory mechanisms that impinge upon these channels (1-3). Several systems have been used to study the functional consequences of specific amino acid mutations and/or biochemical modifications of the cloned epithelial sodium channels (ENaC),¹ namely heterologous expression in Xenopus oocytes or planar lipid bilayer reconstitution studies (4-9). The properties of ENaC in the apical membrane of sodium reabsorbing epithelial cells has been established by Hamilton and Eaton (10) and Palmer and Frindt (11, 12) using patch clamp methodologies. These properties include a low single channel conductance of 4-6 pS, when conducting sodium, a high Na⁺ to K⁺ permeability ratio (P_Na⁺/P_K⁺) (>50), and opened and closed times on the order of seconds. In contrast, upon incorporation into planar lipid bilayers, -ENaC, either from in vitro translation in the presence of dog pancreatic microsomes or from a crude microsomal membrane fraction of Xenopus oocytes, induced the appearance of amiloride-sensitive sodium channel activity with a single channel conductance of 13 pS, a P_Na⁺/P_K⁺ of 10, and relatively short opened and closed times. Thus, the use of planar lipid bilayers to study ENaC appears problematic because the reconstituted channels do not exhibit the signature biophysical properties of the channel expressed in native epithelia (13).

The recent elucidation of the structure of epithelial sodium channels at both the biochemical and molecular levels has facilitated the characterization of regulatory and cytoskeletal proteins associated with the channels (14, 15). By using antibodies generated against a purified bovine renal epithelial sodium channel (16), our laboratory observed that epithelial sodium channels co-localized to the apical membrane with actin and apically associated isoforms of ankyrin and spectrin in sodium reabsorbing renal epithelial cells (17). Moreover, native sodium channels are tightly associated with the detergent-insoluble, actin-rich cytoskeleton (17). Spectrin and ankyrin also co-purify with partially purified renal epithelial sodium channels as assessed by immunoblotting with antibodies specific for ankyrin and spectrin and by direct ¹²⁵I-labeled ankyrin binding (17). In other experiments  spectrin and ankyrin could be co-immunoprecipitated with -ENaC from sucrose gradient fractions of A6 cell extracts enriched in epithelial sodium channels using an antibody against the Xenopus homolog of -ENaC, thereby further establishing that ENaC interacts with the spectrin-based membrane cytoskeleton in vivo (15). Measurements of the lateral mobility of sodium channels in filter-grown A6 cells labeled with rhodamine-conjugated Fab fragments of anti-sodium channel antibodies reveal that >80% of the sodium channels were immobile (18). These biochemical and lateral diffusion data indicate that epithelial sodium channels are linked to the spectrin-based cytoskeleton, and these cytoskeletal elements serve to restrict the lateral mobility of the sodium channels. Data provided by Rotin and colleagues (19) have established a direct interaction between -ENaC and  spectrin. The C-terminal domain of -rENaC contains two proline-rich sequences (amino acids 666-674 and amino acids 681-691) that resemble the SH3 binding motifs of signal transduction proteins. SH3 domains are conserved sequences found in several signal transduction and cytoskeletal proteins, including nonerythroid  spectrin, which mediate protein-protein interactions to their binding to proline-rich motifs (20-22). In light of our data demonstrating that nonerythroid  spectrin co-purifies with this renal epithelial sodium channel (17), Rotin and co-workers (19) presented several lines of evidence demonstrating that the proline-rich motif mediates binding of -rENaC to the SH3 domain of  spectrin. Based upon these observations, Rotin et al. (19) concluded that the interaction of -rENaC with -spectrin is involved in maintaining the polarized distribution of the channel to the apical membrane. Interestingly, a proline-rich C-terminal motif is also present in both the  and  subunits of ENaC (23), but these proteins do not have classical SH3 binding domains. In a subsequent study this same group demonstrated that this proline-rich region of - and -rENaC interacts with WW domains of rNedd4, a protein implicated in protein degradation (24).

Previous work from our laboratory has demonstrated a direct functional effect of the cytoskeletal protein actin with ENaC (9). When short actin filaments were present on the presumptive cytoplasmic surface of the channel, the single channel conductance of ENaC was decreased by half from 13 to 6 pS, with a concomitant increase in single channel open probability. Moreover, P_Na⁺/P_K⁺, as determined from reversal potentials measured under bionic salt conditions, revealed that actin increased P_Na⁺/P_K⁺ for ENaC from 10:1 to 54:1 (25). These effects of actin were seen only from the cytoplasmic side of the bilayer and suggest that actin plays a major role in determining the biophysical characteristics of these channels.

The issue of why the properties of ENaC in bilayers seemingly differ so much from ENaC expressed in native cells and viewed by patch clamp is the subject of this report. The hypothesis that we tested is that the explanation for these apparent differences in biophysical properties result from the environment in which the channel finds itself. Our laboratory has provided strong evidence that the biophysical characteristics of amiloride-sensitive Na⁺ channels are dependent upon its biochemical and physical state (26, 27). Moreover, single channel properties of amiloride-sensitive Na⁺ channels as determined using patch clamp experiments from native cells reveal a great diversity (28).

There are three specific questions that we wished to address. First, why can homomeric channels composed of -ENaC be readily recorded in planar lipid bilayers but not so by patch clamp when heterologously expressed in oocytes? Second, when reconstituting ENaC subunits into planar bilayers either from in vitro translated material or from membrane vesicles prepared from oocytes, do the subunits interact thereby forming an appropriately constructed multimeric channel? Third, what is the molecular basis for the apparent differences between the biophysical characteristics of ENaC in planar lipid bilayers versus those recorded in native cells or tissues? We have used a combination of biochemistry, immunocytochemistry, and electrophysiology (planar lipid bilayers and patch clamp of -rENaC expressed in Xenopus oocytes) to examine these questions. We used one mutant of ENaC, namely _R613X-rENaC, in which the majority of the cytoplasmic C-terminal domain of -rENaC has been eliminated resulting in an inability of actin to affect single channel properties. Our results show that -ENaC is synthesized and mostly retained in the endoplasmic reticulum of the oocyte. We show that channel subunits do interact in reconstituted vesicles. We also show that _R613X-rENaC expression in oocytes produced channels with a conductance of 12 pS and a reduced ability to discriminate among several alkali metal cations, properties identical to wild-type ENaC in the absence of actin or _R613X-rENaC in bilayers. These observations support the idea that studying ENaC in bilayers is useful not only in its simplicity but also in revealing important regulatory characteristics of the channel. In specific, interactions with associated proteins like actin strongly influence single channel properties. Thus, sodium channel-cytoskeletal interactions may represent a novel and important signal transduction mechanism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DNA Constructs-- The cDNAs encoding full-length wt -rENaC subunits (all in pSPORT1) were the kind gift of Dr. B. Rossier, University of Lausanne. Epitope tags for c-Myc (EQKLISEEDL) and HA (YPYDVPDYA) were introduced into the full-length rENaC cDNA using a series of synthetic nucleotide linkers (Life Technologies, Inc.). M2 (DYKDDDDK) -, -, and -rENaCs were the kind gift of Dr. B. Rossier at the University of Lausanne. rENaC labeled with a c-Myc tag was generated by inserting a linker composed of the two oligonucleotides 5'-gTACTTCgAACAAAAACTTATTTCTgAAgATCTggg-3' and 5'-gTACCCCAgATCTTCTTCAgAAATAAgTTTTTgTTCgAA-3' into the BsrGI site of rENaC, corresponding to residue position 186 of the rENaC open reading frame. HA-tagged -reNaC was made by inserting two oligonucleotide linkers, 5'TCgAATACCCATACgACgTCCCAgACTACgCTAgCT-3' and 5'-AgCgTAgTCTgggACgTCgTATgggTATTCgAAgCT-3', into the SacI site of -rENaC at residue position 481 of -rENaC.

Monomeric actin was purified from rabbit skeletal muscle and diluted to a final concentration of 4-10 mg/ml in a buffer containing 2 mM Tris, 0.2 mM CaCl, 0.2 mM MgATP, and 0.2 mM -mercaptoethanol (pH 8.0), and was used in the concentrations described in each figure. The purified actin was a gift from Dr. Stephen S. Rosenfeld, Department of Neurology, University of Alabama at Birmingham. The buffer alone had no effect on -rENaC properties at the dilutions used. Mouse anti-actin monoclonal antibody was purchased from Chemicon International, Inc. (Temecula, CA). Anti-HA and anti-Myc monoclonal antibodies were obtained from Roche Molecular Biochemicals, and the anti-FLAG monoclonal antibodies (M₂) were from Eastman Kodak. Anti-calreticulin and anti-calnexin antibodies were obtained from Affinity Bioreagents, Inc. Oregon Green- and Texas Red-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR).

Oocyte Stratification and Immunofluorescence-- Oocyte stratification was achieved by layering the oocytes over 30% Ficoll 400, followed by centrifugation for 1 h at 4000 × g as described by Han and Nuccitelli (29). Albino oocytes were previously injected with - or -rENaC cRNA constructs, and water-injected oocytes were used as controls. The micro-injection volume was 25 nl, and 500 ng/µl was used for each cRNA. Eggs were incubated at 18 °C for 2 days in half-strength L-15 medium supplemented with 15 mM HEPES, 5% heat-inactivated horse serum (Life Technologies, Inc.), and 1% penicillin/streptomycin/amphotericin B (Life Technologies, Inc.). Plastic dishes (35 or 60 mm in diameter) were pretreated with 1% BSA in PBS overnight to prevent eggs from sticking and ripping during processing. After centrifugation, the oocytes were fixed in 3% formaldehyde in PBS for 2 h at room temperature. After rinsing in PBS, they were post-fixed with absolute methanol at 20 °C for 30 min and again rinsed in PBS. After post-fixation, the eggs were blocked in 1% BSA in PBS for 15 min at 37 °C. The eggs were incubated with rabbit anti--bENaC in a 1:25 dilution in 1% BSA and PBS for 1 h at 37 °C or rabbit anti-calnexin (1:200 dilution) for the same length of time. Anti--bENaC antibody recognizes -rENaC as well as -bENaC but reacts negligibly with - and -ENaC. After blocking and rinsing with BSA, the eggs were then incubated with goat anti-rabbit IgG conjugated with Oregon Green (1:80) diluted in 1% BSA and PBS for an additional hour at 37 °C. For visualizing the anti-calnexin antibodies, the secondary antibody was goat anti-mouse IgG-conjugated Texas Red-X, again at a 1:80 dilution. All immunoprobes were stored in 50% glycerol at 20 °C. Non-stratified eggs were counterstained with Hoechst stain, 20 µg/ml in PBS for 4 min, to visualize the nuclei. All oocytes were examined on an Olympus IX 70 inverted epifluorescence microscope equipped with a step motor, filter wheel assembly (Ludl Electronics, Ltd., Hawthorne, NY), and filter set 83000 (Chroma Technology Corp., Brattlebroro, VT). Images were captured with a SenSys cooled CCD, high resolution, monochromatic, digital camera (Photometrics, Tucson, AZ) using IPLab spectrum software (Scanalytics, Fairfax, VA). Optical sections (40 µm thick) were deconvoluted with Power Microtome software (Vaytek, Inc., Fairfield, IA).

Fluorescence Quantitation in Stratified Xenopus Oocytes-- Quantitation of fluorescence of the endoplasmic reticulum (ER) layer was measured with IPLab Spectrum software, as described previously (30). Fluorescence measurements were standardized using the InSpeck Green (490/515) Microscope Image Intensity Calibration kit from Molecular Probes.

In Vitro Transcription and Translation-- cDNAs were transcribed and translated in vitro in the presence of canine pancreatic microsomal membranes using the TnT transcription/translation system (Promega). The circular plasmid cDNA (1.0 µg) was added to an aliquot of the TnT T7 Quick Master mix and incubated in a 50-µl reaction volume for 90 min at 30 °C. The synthesized proteins were then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography or subjected to immunoprecipitation and reconstitution into proteoliposomes. To test for protein-protein interaction between different rENaC subunits, the rENaC subunits were translated either with radioactive methionine or with nonradioactive methionine. The same amount of each in vitro translated subunit was then mixed in the following manner: 1) ³⁵S-Myc--rENaC + M₂--rENaC + HA--rENaC; 2) ³⁵S-HA--rENaC + Myc--rENaC + M₂--rENaC; 3) ³⁵S-M₂--rENaC + Myc--rENaC +HA--rENaC. To test for co-precipitation between different subunits, anti-epitope antibodies directed against nonlabeled subunits were used, and co-precipitated radioactively labeled subunits were detected using autoradiography. This protocol of mixing radioactively labeled and nonlabeled subunits was used directly after the TnT reaction or when mixing of proteoliposomes containing immunopurified rENaC subunits. To test for co-precipitation between the same subunit of rENaC, we used HA--rENaC and M₂--rENaC constructs. In vitro translated and reconstituted proteins were mixed overnight and immunoprecipitated with anti-HA or anti-M₂ antibodies. Precipitated proteins were probed with the same or different anti-epitope antibodies (Western blot) to test for co-precipitation.

Antibodies, Immunoprecipitations, Gel Electrophoresis, and Western Blots-- The anti-HA, Myc, and M₂ antibodies were used for immunopurification, co-immunoprecipitation, or Western blot detection of in vitro translated epitope-tagged rENaC subunits. Antibodies were used at the following final concentration: 5 µg/ml anti-M₂ antibody, 2 µg/ml anti-HA antibody, or 10 µg/ml anti-Myc antibody. All immunoprecipitation or co-precipitation reactions were performed in RIPA buffer. Antigen-antibody complexes were precipitated with immobilized protein A (Pierce), and precipitates were washed twice in RIPA buffer, and once in TBS buffer, following elution 100 mM glycine, pH 3, and reconstitution in proteoliposomes as described previously (8). Protein containing liposomes were mixed with different combinations of rENaC subunits as described above and incubated at 4 °C overnight. After precipitation, proteins were separated on 8% polyacrylamide gels using SDS-polyacrylamide gel electrophoresis, as described (31, 32). Radioactive proteins were detected by autoradiography. RIPA buffer consisted of 150 mM NaCl, 50 mM Tris, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS (pH 7.5). The composition of TBS buffer was 150 mM NaCl, 10 mM Tris (pH 7.5).

For Western blot analysis, oocyte membranes (1-5 µl/lane) were separated over 8% SDS-polyacrylamide electrophoresis gels under reducing conditions using 10 mM dithiothreitol. The bands were immunoblotted on Immobilon-P polyvinylidene fluoride microporous transfer membrane (Millipore, Bedford, MA) as described previously (31). Lanes were probed with either mouse monoclonal anti-actin or polyclonal (rabbit) anti-calreticulin at a 1:1000 dilution. Blots were incubated with antibody in 0.2% Tween 20/TBS (TTBS) for 1 h. After incubation with the primary antibody, blots were washed three times for 20-30 min each with TTBS. Secondary antibodies (either goat anti-mouse or donkey anti-rabbit antibodies conjugated to alkaline phosphatase) were used at a dilution of 1:5000 and added to the blot. After 1 h, the blot was washed twice with TTBS for 20-30 min each. The nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate development was used according to manufacturer's instructions (Bio-Rad). Broad Range Standards from Bio-Rad served as molecular weight standards.

Planar Lipid Bilayer Experiments-- Planar lipid bilayers were made from a phospholipid solution containing a 2:1 mixture of diphytanoyl phosphatidylethanolamine:diphytanoyl phosphatidylserine dissolved in n-octane at 25 mg/ml. Membranes were formed on a 200-µm diameter hole in a polystyrene cup. Experiments would commence when the membrane capacitance increased to 250-350 pF. The solutions bathing the bilayers were 100 mM NaCl containing 10 mM MOPS-Tris (pH 7.4). Current measurements were made with an operational amplifier connected to a 10-G feedback resistor (9). Electrical connections were provided by Ag-AgCl electrodes and 3 M KCl, 3% agar bridges. Voltage was applied to the cis chamber, and the trans chamber was held at virtual ground. Reconstituted proteoliposomes or oocyte membrane vesicles were applied to a preformed bilayer with a glass rod from the trans compartment, with the membrane potential held at 40 mV. We have found that this experimental protocol provided a specific sidedness to the incorporation of channels. In the majority of cases (>90%), the channels were oriented with the amiloride-sensitive, extracellular side facing the trans solution and the cytoplasmic side facing the cis solution. Data analysis was performed as described previously (8, 9).

Whole Cell and Single Channel Current Measurements in Oocytes-- For two microelectrode voltage clamp experiments oocytes were bathed in ND-96 solution. All experiments were performed at room temperature (22 °C). The electrical arrangement and voltage clamp protocol was identical to that already described (33).

For patch clamp experiments the vitelline membrane was removed from Stage V/VI oocytes as described. Briefly, oocytes were placed in a hypertonic solution containing (in mM) 220 potassium aspartate, 1 MgCl₂, 5 EGTA, 5 HEPES (pH 7.4). After several minutes the vitelline membrane was removed using a pair of forceps, and devitellinated oocytes were transferred to the recording chamber mounted on the stage of an inverted microscope. For cell-attached patch clamp experiments, the pipette solution contained (in mM) 100 LiCl, 1.0 CaCl₂, and 5 HEPES (pH 7.4). For inside-out patches the cytoplasmic bath solution contained (in mM) 100 KCl, 2.5 EGTA, and 5 HEPES (pH 7.4). Pipette currents were amplified with a low-pass 8-pole Bessell filter with a cut-off frequency of 200 Hz, digitized at a sample rate of 2 KHz, and stored on the hard disc of a DOS-based computer for subsequent analysis using pCLAMP software, version 6.1. Single channel currents were measured as described previously in detail.² All voltages refer to the cell interior referenced to the patch pipette. Data analysis was performed as described previously.²

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Localization of -ENaC in Endoplasmic Reticulum-- Comparisons of ENaC activity following reconstitution into bilayers with that measured by patch clamp in native tissues or in heterologously expressing cells such as oocytes reveal distinct kinetic differences. First, only two reports exist in the literature where single -rENaC currents have been measured by patch clamp (8, 35). On the other hand, many reports exist in which single channel currents of -rENaC can be recorded relatively easily with patch electrodes following heterologous expression in oocytes and other cells (for example, see Refs. 7 and 36-38). Yet, -ENaC channel activity can be readily detected in planar lipid bilayer experiments (8, 9). We hypothesized that the basis of these experimental observations results from the known differences in surface expression between -rENaC and -rENaC (30, 39), while at the same time having ample functional channel protein present in the endoplasmic reticulum (ER). We further hypothesized that the high frequency with which -ENaC is observed in bilayers as compared with that seen in patch clamp experiments is due to the fact that the oocyte membrane preparation used in the bilayer incorporation experiments contain active channels retained in the ER. To test these hypotheses, we first prepared membrane vesicles from oocytes and examined by Western blot whether these vesicles contained ER. An antibody raised against an ER-specific protein (calreticulin) was used as a probe (40). The results of these experiments are presented in Fig. 1. Calreticulin was easily detected in a crude oocyte homogenate (lane 1) and in microsomes prepared from either H₂O-injected (lane 2) or -rENaC-injected (lane 3) oocytes. These results indicate that endoplasmic reticulum is indeed present in the microsomal membranes prepared for use in bilayer reconstitution experiments.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   ER is present in the microsomal membranes prepared for use in bilayer reconstitution experiments. Oocyte membrane vesicles were analyzed by Western blot using ER-specific anti-calreticulin antibodies. Lane 1, crude oocyte homogenate (55 µg of total protein); lane 2, membrane vesicles prepared from water-injected oocytes (9 µg of total protein); lane 3, membrane vesicles prepared from -rENaC-injected oocytes (34 µg of total protein); lane 4, membrane vesicles prepared from -rENaC-injected oocytes (34 µg of total protein) and probed with secondary antibody only. This blot is representative of three such experiments.

We next wanted to visualize the distribution of -ENaC within the oocyte, specifically looking to see if heterologous expression resulted in an accumulation of -ENaC in the ER. Fig. 2 presents a photograph of whole oocytes that were stratified prior to fixation and antibody staining. This stratification procedure produced four distinct layers: lipid, ER, mitochondria, and yolk (29, 41). Epifluorescence images of -rENaC-injected oocytes probed with either anti--bENaC antibodies (Fig. 3, top left) or anti-calnexin antibodies (Fig. 3, top right) showed that staining was primarily localized to the ER layer. Anti-calnexin antibodies were used because calnexin is a chaperone protein that is restricted to the ER compartment (42). Another series of epifluorescence images of stratified -rENaC-injected (middle panel) and H₂O-injected oocytes that were stained with either anti--bENaC (left) or anti-calnexin antibodies is also shown in Fig. 3. Again, -ENaC is prominently detected in the ER layer of both the -rENaC and -rENaC-injected oocytes. There was an apparent increase in ER fluorescence intensity of both -ENaC and calnexin in -rENaC-injected oocytes as compared with -rENaC-injected oocytes. For ENaC, quantitative analysis of the green fluorescence indicated that the ER band was 1.8 times brighter in the -rENaC-injected eggs than in the -rENaC-injected oocytes. This increase in fluorescence was not due to cross-reactivity of -bENaC antibody with the  and  subunits of ENaC (30). Because calnexin is a heat-shock chaperone protein involved in the proper folding of glycoproteins (41), calnexin transiently binds to folding proteins in the ER. If proteins are exogenously overexpressed, as is the case for the -ENaC-injected oocytes where three times as much cRNA was injected, the level of free chaperone drops, in turn provoking the up-regulation of chaperone protein through a negative feedback mechanism. Thus, we hypothesize that the channel complex would be stabilized when the - and -ENaC subunits are present; even though the same quantity of -rENaC cRNA was injected, more  subunit would then be present due to its increased stability. Unfortunately, the extraction of the compartmentalized lipid layer during permeabilization interfered with channel detection at the surface plasma membrane. Nonetheless, in optical sections of non-stratified eggs, greater fluorescence was detected at the cell surface and in the immediate underlying ER layer when - and -rENaC were co-injected than when only -rENaC cRNA or water was injected (Figs. 4 and 5). The results of these experiments demonstrate that Na⁺ channels are present in the ER of -rENaC-expressing oocytes and that the  and  subunits may act at the transcriptional or post-translational level to promote the production of more  subunits or to prevent their degradation and hence stabilize the entire channel complex.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   Stratified oocytes. Three oocytes stratified by centrifugation are shown in the photograph on the right (bar = 1 mm). A schematic diagram indicating the nature of each of the layers is also included (see also Refs. 29 and 41).

View larger version (71K): [in this window] [in a new window]   Fig. 3.   Immunofluorescence reveals the presence of Na+ channels in the ER of -rENaC-expressing stratified oocytes. Oocytes were injected with cRNA for either -rENaC or -rENaC or an equivalent volume of water. Following stratification, the eggs were fixed and post-fixed with 3% formaldehyde and absolute methanol, respectively, and then probed with either anti--bENaC (1:25) or anti-calnexin (1:200) antibodies. The secondary antibodies used in these experiments were a goat anti-rabbit IgG conjugated with Oregon Green (for the Na+ channel) and goat anti-mouse IgG conjugated with Texas Red (for the calnexin). Secondary antibodies alone showed no significant staining (not shown). Bar = 180 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Confocal immunofluorescence imaging of Xenopus oocytes showing -ENaC expression at the cell surface. Oocytes were injected either with -rENaC or -rENaC cRNA and prepared for immunofluorescence confocal microscopy as described under "Experimental Procedures." Optical sections (40-µm thick) were taken every 80 µm starting at the approximate midpoint of the oocyte. Bar = 230 µm. The nuclei (blue) were visualized by Hoechst counterstaining.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Confocal immunofluorescence imaging of water-injected Xenopus oocytes for -ENaC or calreticulin. Conditions were as described under "Experimental Procedures" and legend to Fig. 4. Anti-calreticulin antibody was used as an ER marker. Bar = 230 µm.

Co-immunoprecipitation of Full-length ENaC Subunits-- We next tested the hypothesis that ENaC subunits associate into heteromeric complexes following in vitro translation of individual subunits with subsequent mixing and reconstitution into proteoliposomes. We followed the strategy of Adams et al. (43), namely to immunoprecipitate one subunit and detect the others either by autoradiography or by Western blot. The proteoliposomes were prepared in a manner identical to that done for bilayer experiments.

In the first series of experiments, -rENaC containing either an M₂-FLAG tag or an HA epitope were in vitro translated separately, the reaction products mixed and reconstituted into proteoliposomes, and immunoprecipitated (Fig. 6). When an anti-M₂ antibody was used to immunoprecipitate -rENaC from the solubilized proteoliposomes, both _M2-rENaC and _HA-rENaC could be detected by Western blot (1st 2 lanes). The same was true when the immunoprecipitation was done using the anti-HA antibody (3rd and 4th lanes). In separate experiments, anti-HA IgG could not detect _M2-rENaC (5th lane) nor could anti-M₂ antibodies recognize _HA-rENaC (6th lane), verifying the specificity of these probes. These results show that -ENaC subunits interact in a homomeric complex within proteoliposomes made from in vitro translated protein.

View larger version (118K): [in this window] [in a new window]   Fig. 6.   Western blot analysis of -rENaC subunit-subunit interactions. The -rENaC subunits containing either HA or M2 tag were used to test for protein-protein interaction between -rENaC subunits. Separately, in vitro translated and reconstituted -rENaC subunit proteins (see under "Experimental Procedures" for more details) were mixed overnight and immunoprecipitated (IP) with anti-HA or anti-M2 antibodies. Precipitated proteins were probed (Western blot) with either the same or different tagged epitope antibodies.

We also prepared proteoliposomes containing either [³⁵S]methionine-labeled -, -, or -rENaC plus the unlabeled conjugate subunit partners. As before, each subunit contained a specific epitope, namely Myc-, HA-, and M₂ for -, -, and -rENaC, respectively. The strategy was to immunoprecipitate one subunit and detect the presence of the others by autoradiography. Fig. 7 demonstrates the specificity of each of the antibodies as well as showing that each subunit migrated at two different molecular masses, presumably glycosylated and non-glycosylated forms of the protein. This pattern and the molecular masses of each subunit are comparable to those reported for rENaC (43). Fig. 8 demonstrates that anti-HA and anti-M₂ co-precipitated _Myc-rENaC, that anti-Myc and anti-M₂ co-precipitated HA-rENaC, and anti-Myc and anti-HA co-immunoprecipitated _M2-rENaC. As a control, separately in vitro translated and reconstituted _M2-rENaC and cystic fibrosis transmembrane conductance regulator were used to show that anti-M₂ antibodies only immunoprecipitated -rENaC and not cystic fibrosis transmembrane conductance regulator (not shown). These results indicate that each of the subunits can associate to form heteromeric complexes. These results complement and extend the original observations of Adams et al. (43) and Snyder et al. (44) who demonstrated ENaC subunit association in transiently transfected COS-7 cells.

View larger version (69K): [in this window] [in a new window]   Fig. 7.   Specificity of anti-tag antibodies. The ability of a given anti-tag antibody (either anti-Myc, anti-HA, or anti-M2) to immunoprecipitate (IP) 35S-labeled ENaC subunits was examined by autoradiography.

View larger version (67K): [in this window] [in a new window]   Fig. 8.   Analysis of interaction between -rENaC subunits using co-precipitation. In vitro transcription and translation of -rENaC subunits containing different epitopes were performed using either radioactive or non-radioactive methionine. Translated proteins were immunopurified, reconstituted into proteoliposomes, and mixed as described under "Experimental Procedures." To test for co-precipitation between different subunits, anti-epitope antibodies directed against non-labeled subunits were used, and the presence of co-precipitated radioactively labeled subunits was detected using autoradiography. Specificity of anti-tag antibodies was confirmed by testing each antibody for cross-reactivity with other two anti-tag antibodies.

Actin Interaction with -rENaC and _R613X-rENaC-- We have previously shown that the interaction of actin with ENaC in planar bilayers results in dramatic alterations in its single channel characteristics, essentially transforming the incorporated channel into one biophysically comparable to those seen in patch clamp studies (7, 8, 23, 45). These observations led to the hypothesis that the differences between the properties of ENaC assessed by patch clamp in native or heterologous expression systems and those recorded in planar lipid bilayers is directly referable to the presence (in patches) or absence (in bilayers) of actin. In our ongoing quest to determine the location of the actin-ENaC interaction site or sites, several subunit mutants of ENaC were prepared. The results of experiments using one such mutant, namely _R613X-rENaC, in which the majority of the cytoplasmic C terminus was removed and its interaction with actin is shown in Fig. 9. Wild-type -rENaC displayed its characteristic gating and conductance pattern following incorporation into bilayers (see Berdiev et al. (9) for a detailed analysis). The addition of 0.6 µM G-actin to the presumptive cytoplasmic bathing solution resulted in a prompt decrease in single channel conductance (from 13 ± 1 to 6 ± 1 pS, n = 4) and an increase in open and closed times. The P_Na⁺/P_K⁺ also increased from 8:1 in the absence of actin to over 50:1 in its presence (data not shown, but see Ref. 25). The biophysical and pharmacological properties of _R613X-rENaC in bilayers were indistinguishable from wild-type -rENaC. Moreover, actin was without effect on _R613X-rENaC, at least up to the final actin concentrations of 6 µM. The discovery that actin was not able to interact functionally with this particular ENaC construct provided a tool to test directly the hypothesis that the difference between ENaC in bilayers and in cells was due to actin. The prediction is that if single channel patch recordings were made of heterologously expressed _R613X-rENaC, both the single channel conductance and P_Na⁺/P_K⁺ should be higher and lower, respectively, as compared with wild-type -rENaC.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Effect of actin on the activity of either wild-type (wt) -rENaC (top left) or R613X-rENaC (top right) reconstituted into planar lipid bilayers. The holding potential was +100 mV (referenced to the trans compartment). Bathing solutions were symmetrical and contained 100 mM NaCl, 10 mM MOPS (pH 7.4). Actin was added to the cis compartment at a final concentration of 0.6 µM. Records were low pass-filtered at 100 Hz. The bottom half of the figure shows current voltage relationships of wt -rENaC and R613X-rENaC in the absence (open circles) or presence (solid circles) of actin. Each datum point represents the mean ± 1 S.D. for at least four separate experiments.

Expression of _R613X-rENaC in oocytes resulted in the appearance of amiloride-sensitive currents (Fig. 10), albeit significantly smaller than those observed for the wild-type constructs (Fig. 10, A-C). For oocytes clamped at 100 mV, amiloride-sensitive currents averaged 4.1 (± 0.8) µA (n = 6), and those for _R613X-rENaC averaged 1.2 (± 0.3) µA (Fig. 10D). These lower values of macroscopic current may have resulted from poorer channel surface expression, although we have not yet examined this possibility. Fig. 11 presents single channel current records of wild-type and mutant ENaC obtained from cell-attached patches of oocyte membranes. What is evident from these records is that channels can be recorded from _R613X-rENaC-expressing oocytes, and the single channel conductance of these mutant channels, determined from fitting the Goldman-Hodgkin-Katz equation to the data in the associated current-voltage curves, is 12.0 ± 1 pS. This conductance is significantly higher than that for wild-type ENaC (6.7 ± 1 pS). This value of conductance is comparable to what was measured in bilayers (cf. Fig. 9 and Refs. 8 and 9).

View larger version (32K): [in this window] [in a new window]   Fig. 10.   Representative example of the whole cell currents observed in wild-type -rENaC- (A) and R613X-rENaC-expressing (B) oocytes. Their corresponding steady-state current voltage curves are shown in C. These curves are difference plots, showing the current values at each potential in the presence of 10 µM amiloride subtracted from the current values measured in the absence of amiloride in the same oocyte. The bathing solution was ND-96. D summarizes the absolute values of amiloride-sensitive current measured at 100 mV (left two bars) and +40 mV (right two bars) for the wild-type (n = 3) and mutant (n = 4) channels.

View larger version (21K): [in this window] [in a new window]   Fig. 11.   Single channel records of -rENaC and R613X-rENaC expressed in oocytes. Single channel currents were obtained from cell-attached patches of ENaC-expressing oocytes. Three to six channels were typically seen from both sets of injected oocytes. Associated open state single channel current voltage curves for these channels are shown in the bottom half of the figure. Each point represents the mean value ± 1 S.E. for at least five separate experiments. The chord conductance around 40 mV was 6.7 ± 1 and 12.0 ± 1 pS for wt -rENaC and R613X-rENaC, respectively.

Fig. 12 shows single channel recordings of _R613X-rENaC in an excised inside-out patch. The single channel conductance determined under these conditions was 12.0 ± 1 pS. This figure also shows that the addition of 10 µM actin to the bath (i.e. cytoplasm) had no effect on the conductance of the channel. This experiment was repeated three times with identical results.

View larger version (16K): [in this window] [in a new window]   Fig. 12.   Lack of effect of exogenously added actin on the single channel conductance of R613X-rENaC in oocytes. Inside-out patches were isolated from oocytes expressing R613X-rENaC. The patch separated identical solutions of ND-96. Actin was applied to the bath solution at a concentration of 10 µM. The traces shown are from the same patch recorded before (top) and after (bottom) the addition of actin. This experiment was repeated three times with identical results. The holding potential was 20 mV, and the mean single channel conductance was 12.0 ± 2 pS.

In order to determine P_Na⁺/P_K⁺ for the wild-type and mutant channels, bionic reversal potential measurements were made in excised patches containing these channels. For wild-type -rENaC and mutant _R613X-rENaC, the values of the reversal potentials were 73 ± 4 and 30 ± 3 mV, respectively (n = 5 for each). These values translate into a P_Li⁺/P_K⁺ of 18.1 and 3.3 for wild-type and mutant channel, respectively. These values are in accord with those determined from the reversal potentials measured from the cell-attached experiments of Fig. 11 (P_Na⁺/P_K⁺ of 20 and 10 for wild-type and mutant ENaC, respectively, assuming a [Na]_i of 80 and 40 mM for wild-type and mutant ENaC-expressing oocytes, respectively (46). Thus, these results support the hypothesis that actin is indeed responsible for the low conductance, highly cation-discriminating properties of ENaC.

Why, then, when vesicles prepared from ENaC-expressing oocytes are incorporated into planar lipid bilayers, is actin not transferred along with the ENaC to the bilayer, thus producing low conductance ENaC in bilayers? The fact that exogenously added actin altered the single channel properties when added to bilayers but not to excised patches argues that actin is not present in sufficient quantity in the vesicles transferred to the bilayer to produce its functional effects. This is supported by the Western blot experiments shown in Fig. 13. In this experiment, a monoclonal antibody raised against mouse actin was used to probe for the presence of actin in oocyte homogenates and in membrane vesicles prepared from the homogenate. This commercially available antibody has broad specificity, reacting against actin from all animal and plant species tested. As can be seen in the blot, the antibody recognizes purified rat actin (1st lane) as well as the actin present in a crude Xenopus oocyte homogenate (2nd lane). However, in the vesicles prepared in the same way as was done for the bilayer reconstitution experiments, very little if any actin could be detected.

View larger version (46K): [in this window] [in a new window]   Fig. 13.   Western blot analysis of oocyte membrane vesicles using anti-actin monoclonal antibodies. Purified rabbit skeletal muscle actin was used as a control (1 µg loaded). The total amount of protein loaded per lane for the crude oocyte homogenate and the membrane vesicles prepared from water-injected or R613X-rENaC-injected oocytes were 10, 16.7, and 9.5 µg, respectively. This experiment was repeated three times with identical outcomes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The work presented in this paper addresses why the properties of ENaC in planar lipid bilayers seemingly differ so much from ENaC expressed in cells and recorded by patch clamp. Our results point to a possible explanation, namely that the environment of the channel determines the measured channel properties. Pure ENaC subunits in planar bilayers are devoid of any native membrane or cellular component interactions. As noted previously, interactions with associated proteins can greatly influence channel properties. Both native and cloned amiloride-sensitive Na⁺ channels have been shown to interact with cytoskeletal proteins such as actin, ankyrin, and  spectrin (17, 19). The consequences of wt -rENaC interactions with actin are shown in Fig. 9. Actin reduces single channel conductance to 6 pS (from 13 pS) and increases mean channel open time. We have previously determined that amiloride was equally effective in inhibiting the channels regardless of the presence or absence of actin. Another important feature of the effects of actin is its influence on P_Na⁺/P_K⁺ of -rENaC. In the absence of actin, the P_Na⁺/P_K⁺ of the wt channel is 8-10:1, as measured from reversal potentials under bi-ionic conditions. Following the addition of actin, the P_Na⁺/P_K⁺ increased to over 50:1, more typical of the highly selective native Na⁺ channel. These basic observations emphasize the validity of the underlying hypothesis and our conclusion, namely that interactions of amiloride-sensitive Na⁺ channels with associated proteins are essential for conferring specified biophysical properties on this channel. Moreover, the simplicity of the "one-channel" bilayer recording technique was essential to uncovering this key interaction between actin and ENaC.

There are several related issues that we have also sought to address experimentally. The first issue revolves around the inability of most investigators to record -ENaC in heterologously expressed systems. To date, only two single channel recordings of -ENaC have been made as follows: the first in a fibroblast cell line (35) and another in oocytes (8). In both cases, patch clamp recordings of cells expressing -rENaC reveal channels with a relatively large conductance of approximately 18-21 pS. Interposed among the large transitions were two additional conductance levels of 6 and 12 pS each. Except for the absolute values of the conductance states, this kinetic behavior was very similar to that observed for -rENaC in bilayers (see Ref. 8). However, in contrast to the bilayer, channel activity was infrequent. Moreover,  channels can be recorded with a high degree of fidelity and frequency in the planar lipid bilayers. This difference, in part, can be attributed to the fact that  channels do not traffic with high frequency to the plasma membrane (30, 39). Our results indicate that -ENaC is synthesized but retained in large measure in the endoplasmic reticulum (see Figs. 3-5). The microsomal membranes prepared from oocytes contain endoplasmic reticulum (Fig. 1). Hence, when these are fused to the bilayers, active channels are recorded.

The second issue has to do with protein-protein interactions between ENaC subunits. Adams et al. (43) first showed that hENaC subunits interact with each other and with themselves and that these interactions occurred early in biosynthesis prior to glycosylation. These authors also demonstrated that the interactions were strong and were not disrupted by non-ionic detergents or high concentrations of salts. The results presented here demonstrate the same thing, namely that in vitro translated  subunits can interact (Fig. 6), and correspondingly, when -, -, and -rENaC are translated separately in vitro, reconstituted into proteoliposomes, and mixed together, they interact, as demonstrated by our immunoprecipitation protocols (Fig. 8). The same interactions were found when the individual subunits were expressed in Xenopus oocytes³ or in COS-7 cells (43, 44). Thus, we confirmed the original observations of Adams et al. (43) and Snyder et al. (44) that the channel subunits interact early in biosynthesis and apparently fold, oligomerize, and assemble properly in a physiologically active form, as assayed by the subsequent fusion to planar lipid bilayers.

Thus, the data presented here provide a unifying and parsimonious interpretation concerning the interaction of actin with epithelial Na⁺ channels as an important determinant in modulating Na⁺ channel biophysical characteristics. It is our contention that the planar lipid bilayer system has been, and is, useful for reconstituting physiological activity of ENaC. The fact that the biophysical properties of ENaC in planar lipid bilayers can resemble those of ENaC as determined by patch clamp in native or heterologously expressed cells only if actin is present supports this idea. Furthermore, a mutant of ENaC (_R613X-rENaC) with which actin cannot interact, forms channels with higher conductance and lower Na⁺/K⁺ selectivity. Moreover, Rokaw et al. (25) have recently presented evidence demonstrating that -, -, and -ENaC are components of the epithelial Na⁺ channel biochemically isolated from A6 cells. Kieber-Emmons et al. (34) have shown that the monoclonal antibody RA6.3, which was used to isolate biochemically a Na⁺ channel complex from A6 cells, recognizes an amiloride-binding site on -ENaC. When these biochemically purified Na⁺ channels were incorporated into planar lipid bilayers, channel behavior (selectivity and single channel conductance) comparable to -ENaC in bilayers was observed. The addition of actin converts this channel into one indistinguishable from ENaC measured by patch clamp (28). Thus, not only is the actin and spectrin-based membrane cytoskeleton important for ENaC trafficking, retention, and clustering within the apical membrane of Na⁺ transporting epithelia, but it also plays an important role in modulating channel activity.

	ACKNOWLEDGEMENTS

We thank Jason Lockhart for excellent technical assistance, Cathy Guy for superb work in typing the manuscript, and Dr. Peter Csutora for providing us with albino oocytes. We also greatly appreciate the many discussions with Dr. Bakhrom K. Berdiev and comments on the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant DK37206.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.

^¶ Present address: Institute of Physiology and Biophysics, Uzbek Academy of Sciences, Tashkent, Uzbekistan 700095.

Present address: Dept. of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom.

^** To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., MCLM 704, Birmingham, AL 35294-0005. Tel.: 205-934-6220; Fax: 205-934-2377; E-mail: benos@phybio.bhs.uab.edu.

² H. L. Ji, C. M. Fuller, and D. J. Benos, submitted for publication.

³ B. Jovov, A. Tousson, H.-L. Ji, D. Keeton, V. Shlyonsky, P.-J. Ripoll, C. M. Fuller, and D. J. Benos, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: ENaC, epithelial Na⁺ channels; ER, endoplasmic reticulum; HA, hemagglutinin; PBS, phosphate-buffered saline; BSA, bovine serum albumin; wt, wild type; MOPS, 4-morpholinepropanesulfonic acid; pS, picosiemens; pF, picofarads.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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