Regions in the carboxy terminus of alpha -bENaC involved in gating and functional effects of actin

Susan J. Copeland, Bakhrom K. Berdiev, Hong-Long Ji, Jason Lockhart, Suzanne Parker, Catherine M. Fuller, and Dale J. Benos

Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gating differences occur between the alpha -subunits of the bovine and rat clones of an amiloride-sensitive epithelial Na+ channel (ENaC). Deletion of the carboxy terminus of bovine alpha -ENaC (alpha -bENaC) at R567 converted the gating properties to that of rat alpha -ENaC (alpha -rENaC). The equivalent truncation in alpha -rENaC was without effect on the gating of the rat homologue. The addition of actin to ENaC channels composed of either alpha -rENaC or alpha -bENaC alone produced a twofold reduction in conductance and an increase in open probability. Neither alpha -rENaC (R613X) nor alpha -bENaC (R567X) was responsive to actin. Using a chimera consisting of alpha -rENaC1-615 and alpha -bENaC570-650, we examined several different carboxy-terminal truncation mutants plus and minus actin. When incorporated into planar bilayers, the gating pattern of this construct was identical to wild-type (wt) alpha -bENaC. Premature stop mutations proximal to E685X produced channels with gating patterns like alpha -rENaC. Actin had no effect on the E631X truncation, whereas more distal truncations all interacted with actin, as did wt alpha -bENaC. Key findings were confirmed using channels expressed in Xenopus oocytes and studied by cell-attached patch-clamp recording. Our results suggest that the site of actin regulation at the carboxy terminus of the chimera is located between residues 631 and 644.

alpha -subunit of bovine epithelial sodium channel; planar lipid bilayers; patch clamp; amiloride; ion channels; oocytes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CATION CHANNELS BELONGING to the epithelial Na+ channel family (ENaCs) comprise multiple subunits (alpha , beta , gamma ) (6, 7, 21, 35). The subunit, when expressed in Xenopus oocytes and incorporated into planar lipid bilayers, can form an amiloride-sensitive Na+ channel (13). When the beta - and gamma -subunits are coexpressed with alpha , however, the current increases due to increased surface expression of the channel complex at the plasma membrane (1). ENaCs have been found in epithelial and nonepithelial cells from multiple species, including human, rat, and mouse (2). Our laboratory previously cloned the alpha -ENaC subunit from bovine kidney (alpha -bENaC) (8). Functional comparison of this bovine homologue with the subunit cloned from rat kidney in a planar lipid bilayer assay system revealed distinct differences in gating patterns. alpha -bENaC had a single transition step of 39 pS and exhibited bursting behavior with long (1-5 min) closed periods between bursts (10). Rat alpha -ENaC (alpha -rENaC), however, had a nearly constitutively open 13-pS conductance with an additional step of 26 pS to a final conductance level of 39 pS. Additionally, alpha -rENaC did not exhibit the long closed times characteristic of alpha -bENaC (13). Sequence analysis of the two proteins showed identical domain organization, similar size, and high homology (83%) at the amino acid level over most of their lengths. However, the amino acid homology diverges at the carboxy termini, starting at amino acid 584 in alpha -bENaC and 630 in alpha -rENaC. To test the hypothesis that the carboxy terminal region was responsible for the difference in gating, we previously made carboxy-terminal deletions in both alpha -ENaC subunits. Deletion of the carboxy terminus of alpha -bENaC (R567X) converted its gating behavior to that of alpha -rENaC, whereas the equivalent deletion in alpha -rENaC (R613X) had no effect on gating (10).

We have also used these truncated constructs to identify a potential site of interaction for actin with alpha -ENaC subunits. The primary function of the ENaC in the mammalian kidney is the reabsorption of sodium from the cortical collecting duct, which is stimulated by the cAMP-coupled agonist vasopressin. However, direct activation of the heterologously expressed channel by protein kinase A (PKA) has been difficult to demonstrate, leading to the suggestion that intermediary proteins may be required for signal transduction. We recently showed that one such protein could be actin. Addition of actin to alpha -rENaC incorporated into planar lipid bilayers was associated with a reduction of the single-channel conductance and an increase in open probability (Po) (3, 15). Addition of actin to alpha -rENaC and alpha beta gamma -rENaC in the presence of PKA further increased channel Po. Furthermore, addition of actin enhanced the downregulation of alpha beta gamma -rENaC by cystic fibrosis transmembrane conductance regulator (CFTR) and restored sensitivity of the channel complex to PKA (3, 15). In contrast, carboxy-terminally truncated alpha -rENaC and alpha -bENaC were unaffected by the addition of actin in terms of either their gating behavior or sensitivity to PKA (11, 19).

To test the hypothesis that both the gating differences and the site of actin interaction with the alpha -ENaC subunits were located in the carboxy terminus, we generated a chimeric construct consisting of alpha -rENaC residues 1-615 and alpha -bENaC residues 570-650, resulting in a chimeric polypeptide of 695 amino acids. A series of mutants that inserted premature stop codons at intervals along the length of the carboxy-terminal region of this chimera were generated to further isolate regions responsible for the differences in gating behavior and effects of actin on alpha -ENaC subunits.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subcloning and Mutagenesis

Wild-type (wt) alpha -rENaC was subcloned into the vector pSP70 (Promega) using 5' and 3' BglII sites. The carboxy-terminal tail was replaced with the carboxy terminus of alpha -bENaC using 5' BspEI and 3' BglII sites. The ligation produced a join between residue 616 in alpha -rENaC and 568 in alpha -bENaC, producing a duplication of three amino acids (RFR), forming the amino acid sequence MLLRRFRRFRSRYWSP. These additional three amino acids were removed with the Chameleon double-stranded mutagenesis kit (Stratagene) and specific primers, resulting in MLLRRFRSRYWSP. Premature stop mutations and deletion mutants were made in the chimera with the Chameleon kit and appropriately designed primers (Life Technologies). All constructs were screened by restriction digest and dideoxy sequencing (Iowa State University Sequencing Facility). ClaI was used for linearization to transcribe cRNA using mMessage mMachine (Ambion) with T7 RNA polymerase, and the integrity of the transcribed cRNAs was verified by electrophoresis through denaturing 1% agarose-formaldehyde gels.

Planar Lipid Bilayers

Stage V or VI oocytes taken from Xenopus laevis (Xenopus I, Ann Arbor, MI) were defolliculated in Ringer solution (82.5 mM NaCl, 2.4 mM KCl, 5 mM MgCl2, and 5 mM HEPES, pH 7.4) containing 1 mg/ml type 1A collagenase (320 U/mg; Sigma) for 2 h. Incubation for 24 h was done in L-15 medium containing 15 mM HEPES and 2% of a 10,000 U/ml penicillin-streptomycin solution at 18°C. Fifty nanoliters of RNase-free water with or without 25 ng of the appropriate cRNA was injected into each oocyte. Vesicles were prepared 48 h after injection using a discontinuous sucrose gradient (28). Vesicles were fused to a lipid bilayer membrane composed of diphytanoyl phosphatidylethanolamine-diphytanoyl phosphatidylserine-oxidized cholesterol (20 mg/ml) in a 2:1:2 ratio. Incorporation was done at -40 mV, and recording was done in a symmetrical solution of 100 mM NaCl and 10 mM MOPS-Tris (pH 7.4). For amiloride sensitivity experiments, amiloride was added to the trans (extracellular) side of the bilayer. Monomeric actin was the kind gift of Dr. S. Rosenfeld (Dept. of Medicine, University of Alabama at Birmingham). It was diluted before use to a final concentration of 4-10 mg/ml in buffer [2 mM Tris (pH 8.0), 0.2 mM CaCl2, 0.2 mM MgATP, and 0.2 mM 2-mercaptoethanol]. The final concentration of actin used in the experiments was 0.6 µM.

Cell-Attached Channel Recording in Xenopus Oocytes

Xenopus oocytes were obtained and injected with appropriate cRNAs as described above. The vitelline layer was removed by hand dissection after the oocytes were placed in hyperosmotic medium as previously described (18). Cell-attached single-channel currents were recorded using an Axopatch 1B amplifier (Axon Instruments) (26). The patch pipettes were pulled from fire-polished, filamented borosilicate glass (WPI) with a multistepped micropipette puller (model M97, Flaming/Brown). The electrode tips were fire polished. The resistance of the electrode was 2-10 MOmega when filled with 100 mM LiCl, 10 mM HEPES, and 2 mM CaCl2 (at pH 7.4). Currents were collected using the Clampex 7.0 feature of pCLAMP at a sampling interval of 500 s. The current traces were filtered with the 0.1 kHz built-in low-pass filter of Clampex 7.0 and digitized by DigiData 1200 (Axon).

Data Analysis

Bilayers. All data analysis was performed in bilayers containing a single channel and analyzed using pCLAMP software. Records were filtered at 300 Hz with an eight-pole Bessel filter and acquired at 1 ms/point. Steady-state single-channel current-voltage (I-V) curves were measured after channel incorporation by applying a known voltage and measuring individual channel current (i). Single-channel Po was calculated from the equation Po = I/Ni, where I is the mean current, N is the number of active channels, and i is the unitary current. In cases where substates were observed, i was the maximum total current recorded. N and i were estimated from all-points current amplitude histograms produced by pCLAMP. All bilayer experiments were repeated at least three times, and the duration of any individual recording was 3-5 min.

Patch clamp. Patch-clamp analysis was performed using Fetchan and pSTAT programs of Clampex software version 7.0 (Axon Instruments). The polarity of the single-channel currents was reversed and the baseline was corrected. The single-channel Po was calculated using the following formula:

Po = <A><AC>I</AC><AC>&cjs1171;</AC></A>/iN, where N is the total number of channels (1), <A><AC>I</AC><AC>&cjs1171;</AC></A> is the mean current over the period of observations, and i is the unitary current determined from all-points current amplitude histograms produced by Fetchan. <A><AC>I</AC><AC>&cjs1171;</AC></A> was calculated using the events list of files generated by Fetchan software.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have shown that deletion of the carboxy terminus of alpha -bENaC converts its gating to that of alpha -rENaC (10). To further pinpoint this gating switch, we made a chimera by joining alpha -rENaC residues 1-615 and alpha -bENaC residues 570-650 (Fig. 1). The region of the join is 100% conserved, with divergence starting at residue 584 in alpha -bENaC and 630 in alpha -rENaC. As shown in Fig. 2, alpha -rENaC and alpha -bENaC exhibit characteristic gating patterns when incorporated into planar lipid bilayers, and both can be inhibited by the K+-sparing diuretic amiloride. The chimera, when expressed in Xenopus oocytes and fused to planar lipid bilayers, had a phenotype identical to alpha -bENaC (Fig. 3A), exhibiting a 39-pS main state conductance. The characteristic sensitivity of ENaC to amiloride was not affected in the chimera, which exhibited an apparent inhibition constant of 190 nmol/l (Fig. 3B), comparable to that exhibited by both alpha -rENaC and alpha -bENaC incorporated into lipid bilayers (9, 13).


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Fig. 1.   Sequence alignment of the COOH termini of alpha -subunit of rat (alpha -rENaC) and bovine (alpha -bENaC) epithelial Na+ channels. A: the chimera was made by joining alpha -rENaC residues 1-615 (black) and alpha -bENaC residues 570-650 (blue). The region of the join is 100% conserved, with divergence starting at residue 584 in alpha -bENaC and 630 in alpha -rENaC. B: location of the premature stop mutations inserted in the alpha -rENaC/alpha -bENaC chimera. The portion of the sequence corresponding to alpha -rENaC is shown in black, whereas that corresponding to alpha -bENaC is shown in red. The residues converted to stop mutations by site-directed mutagenesis are given in blue.



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Fig. 2.   Single-channel recordings of alpha -rENaC and alpha -bENaC in planar lipid bilayers. When incorporated into planar lipid bilayers, each alpha -ENaC homologue exhibited a distinctive gating pattern. In the case of alpha -rENaC, this comprised a nearly constitutively open 13-pS subconductance state on top of which was superimposed a 26-pS transition. In contrast, in the case of alpha -bENaC, a single 39-pS transition was observed. Both alpha -ENaC homologues were sensitive to amiloride. The zero current level is marked by the dashed line, and records were obtained at +100 mV. wt, Wild type; Po, open probability.



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Fig. 3.   Single-channel recordings of the chimeric alpha -ENaC construct incorporated into planar lipid bilayers. A: the full-length chimera gated identically to alpha -bENaC and exhibited a 39-S main-state conductance. In contrast, all the premature stop constructs (2 of which are illustrated) gated with an alpha -rENaC-like pattern. The characteristic sensitivity of alpha -ENaC to amiloride was not affected in the chimera or in the premature stop constructs, all of which exhibited an apparent inhibition constant (Ki) of 190 nmol/l. All records were obtained at +100 mV, and the zero current level is marked by a dashed line. B: dose-response curve illustrating the effect of amiloride on channel Po for the full-length chimera and 2 truncated constructs, C645X and E685X. All constructs had identical Ki values.

To further identify the location of the gating switch, a series of premature stop mutations were made in the chimera (Figs. 1B and 3). All four premature stop mutations, E631X, C645X, Y671X, and E685X, were examined in bilayers. All of these constructs had a nearly always open 13-pS conductance state, with an additional 26-pS transition step to 39 pS (shown in Fig. 3 for two constructs, C645X and E685X). This gating pattern was identical to wt alpha -rENaC. All premature stop mutations retained amiloride sensitivity, and had identical I-V curves and Na:K permeability ratios (PNa/PK) as illustrated in Figs. 3 and 4 and shown in Table 1. The PNa/PKs for the full-length chimera and constructs E631X, C645X, Y671X, and E685X were all ~10:1 as determined from the respective reversal potential measurements made under biionic conditions (Fig. 4A, Table 1). There was no significant difference in the reversal potentials of either the 13-pS or 26-pS states under biionic conditions for any of the constructs studied (Fig. 4B). These results indicated that the gating difference was located in the last 12 amino acids of alpha -bENaC and that these truncation mutants do not affect channel conductance, ion selectivity, or amiloride sensitivity.


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Fig. 4.   Current-voltage (I-V) curves for full-length and E685X chimeric constructs under symmetrical and biionic conditions. A: under symmetrical or biionic conditions, both the full-length chimera and the truncated construct had identical I-V relationships. The shift in reversal potential illustrated under biionic conditions is consistent with a Na:K permeability (PNa/PK) ratio of ~10:1 for the maximum conductance of 39 pS. B: illustrates the I-V relationship for the 13-pS and 26-pS subconductance states of E685X as revealed by truncation of the COOH terminus. There was no significant difference in the reversal potentials for the subconductance states compared with that for the maximum conductance of 39 pS. i, unitary current.


                              
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Table 1.   Single-channel characteristics of different chimeric constructs in the absence and presence of actin in planar lipid bilayers

We also used these chimeric constructs to examine the effect of actin on single of alpha -ENaC channels. In these experiments, actin was added to the cis compartment of the bilayer chamber (putative intracellular side). The characteristic effect of this maneuver, when the bilayer membrane contains alpha -rENaC, is a reduction in single-channel conductance and increases in channel Po and PNa/PK, with no change in amiloride sensitivity (3, 19). This observation was faithfully replicated when the bilayer membrane contained the alpha -rENaC/alpha -bENaC chimera or the truncation constructs, with the exception of chimera E631X. In this case, actin had no effect on alpha -rENaC (Fig. 5, Table 1), suggesting that the site of interaction between actin and the chimera lay between residues E631 and C645. To test this hypothesis, we made two additional deletion constructs, one that deleted the 14 amino acids between E631 and F644 and a corresponding control deletion that removed the residues between Y671 and A684. When incorporated into the bilayer, both deletions had a gating pattern identical to that of alpha -bENaC, as would be predicted from our earlier observations. However, whereas chimeraDelta 671-684 exhibited a reduction in conductance, an increase in Po, and an increased PNa/PK ratio in the presence of 0.6 µM actin, actin had no effect on chimeraDelta 631-644 (Fig. 6, Table 1). These results suggest that the residues between E631 and F644 form a site for actin interaction within the carboxy terminus of alpha -bENaC, and, because this region is highly conserved, it is likely that this sequence performs a similar function in alpha -rENaC.


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Fig. 5.   Effect of actin on full-length and E631X chimeric constructs incorporated into planar lipid bilayers. A: the addition of 0.6 µM actin to the full-length chimera both reduced the single-channel conductance and doubled the single-channel Po. In contrast in the E631X truncation construct, actin was without effect on either parameter. Zero current level is shown by the dashed line, and records were obtained at +100 mV. B: I-V curves for the full-length and E631X truncated construct under biionic conditions and in the presence of actin. Whereas the full-length chimera exhibited a marked shift in reversal potential (Erev) in the presence of actin corresponding to an increase in the PNa/PK, the reversal potential for E631X was not significantly different to that seen in the absence of actin. C: amiloride dose-response curve for the full-length and E631X truncated constructs in the presence and absence of actin. Addition of actin to the bilayer chamber was without effect on the apparent Ki for amiloride for either construct.



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Fig. 6.   Effect of actin on the chimera containing deletions in the putative actin binding site. A: deleting 14 amino acids between residues E631 and F644 in the chimera completely abrogated any effect of actin on either conductance or channel Po. In contrast, in a construct where 14 amino acids in a region distal to F644 were deleted (Y671 to A644), actin both reduced conductance and increased channel Po. Dashed line indicates the zero current level; all records were obtained at +100 mV. B: I-V relationship for alpha -rENaCDelta 631-644 and alpha -rENaCDelta 671-684 under biionic conditions ± actin. In the absence of actin, both constructs had identical I-V relationships. However, in the presence of actin, alpha -rENaCDelta 671-684 exhibited a shift in Erev consistent with a PNa/PK of 55:1, whereas the PNa/PK for alpha -rENaCDelta 631-644 remained at 10:1. C: amiloride dose-response curve for alpha -rENaCDelta 631-644 and alpha -rENaCDelta 671-684 in the presence and absence of actin. In contrast to the results obtained for Po and PNa/PK, deletion of residues 631-644 had no consequences for amiloride block of the channel in the presence of actin.

To further characterize the effect of actin on the single-channel behavior of the chimera, we expressed the alpha -ENaC chimera together with beta - and gamma -rENaC subunits in Xenopus oocytes. Recording in the cell-attached patch configuration, we found that alpha chimerabeta gamma -rENaC behaved very similarly to the control (alpha beta gamma -rENaC), exhibiting long open and closed times (Fig. 7), although the single-channel conductance was slightly increased (6.5 ± 0.0003 pS, n = 6) compared with a unitary conductance of 4.6 ± 0.0002 pS (n = 3) for the wt alpha beta gamma -rENaC control (Fig. 7). Replacement of alpha chimerabeta gamma -rENaC with the control deletion alpha chimeraDelta 671-684beta gamma -rENaC did not affect either the Po (0.41 ± 0.01) or the single-channel conductance (6.97 ± 0.0004 pS, n = 4). However, when we substituted the Delta 631-644-deleted alpha -subunit in place of the intact chimera, we found that the characteristic long open and closed times were replaced by more frequent although briefer channel openings (Po 0.29 ± 0.03) and that the conductance had increased to 9.42 ± 0.0002 pS (n = 4) (Figs. 7 and 8).


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Fig. 7.   Cell-attached patch-clamp recordings of alpha beta gamma -rENaC and chimeric constructs expressed in Xenopus oocytes. Single-channel recordings of alpha beta gamma -rENaC and chimeric alpha -ENaC subunits coexpressed with beta gamma -rENaC are compared. Although the wild-type (wt) alpha beta gamma -rENaC, alpha chimerabeta gamma -rENaC, and alpha chimeraDelta 671-684beta gamma -rENaC all exhibited similar characteristics in terms of kinetics and conductance, alpha chimeraDelta 631-644beta gamma -rENaC activity was characterized by a higher conductance and more frequent transitions between open and closed states.



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Fig. 8.   I-V relationships alpha beta gamma -rENaC and chimeric constructs expressed in Xenopus oocytes recorded by cell-attached patch clamp. When coexpressed with beta gamma -rENaC subunits, both the full-length chimera and the construct containing the Delta 671-684 deletion had identical I-V relationships and only a slightly higher conductance (6.5 ± 0.0003 pS, n = 6) than that exhibited by alpha beta gamma -rENaC channels (4.6 ± 0.0002 pS, n = 3) as calculated from the I-V curves. In contrast, the conductance of alpha chimeraDelta 631-644beta gamma -rENaC (9.42 ± 0.0002 pS, n = 4) was considerably greater than either the wt rENaC channel or the chimeric control (6.97 ± 0.0004 pS, n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that single ENaC composed purely of the subunits of either bENaC or rENaC exhibit distinct gating behaviors when incorporated into planar lipid bilayers (10, 13). The data support a model in which alpha -bENaC and alpha -rENaC form triple-barrel channels, each barrel having a conductance of 13 pS. In the case of alpha -bENaC, the barrels gate cooperatively, so that a single-step transition of 39 pS is observed. In the case of alpha -rENaC, there is an initial 13-pS subconductance state on which a 26-pS step is superimposed.

Experiments performed with a truncation mutant of alpha -bENaC (R567X) demonstrated that deletion of the carboxy terminus of this ENaC isoform switched the gating pattern to that of alpha -rENaC (10). The corresponding deletion in alpha -rENaC (R613X) had no effect on the pattern of alpha -rENaC gating. This observation leads us to suggest that a kinetic switch located in the carboxy terminus of alpha -bENaC (coincidentally the region that exhibits the lowest homology to alpha -rENaC) was responsible for the apparent difference in gating patterns (10).

To test this hypothesis further, we constructed a chimera in which the carboxy terminus of alpha -bENaC from residue R568 was grafted onto alpha -rENaC at residue position R614. This resulted in the formation of an alpha -rENaC/alpha -bENaC hybrid polypeptide of 696 amino acids that had an identical gating pattern to alpha -bENaC. Insertion of several stop mutations at intervals along the carboxy terminus of the chimera revealed that the domain responsible for coordinating gating of alpha -bENaC was located in the last 12 amino acids. No other properties of the channel (amiloride sensitivity, ion selectivity, or I-V relationship) were affected by the deletion of these residues. This region in alpha -bENaC, which exhibits only 25% homology with the corresponding portion of alpha -rENaC, contains several charged residues that are not conserved between the two isoforms and that could potentially influence gating (Fig. 1).

We also exploited these chimeric constructs to analyze further the role of actin in ENaC gating. We previously showed that actin has a profound effect on ENaC incorporated into planar lipid bilayers, not only reducing single-channel conductance and increasing Po, but also making the channel susceptible to regulation by PKA and CFTR (3, 14). In contrast, a carboxy terminus-deleted construct of alpha -rENaC, R613X, could not be regulated by actin (19). Actin is an integral component of the cytoskeleton, and considerable precedent has implicated this structural protein in the regulation of a number of membrane ion channels, (5, 12, 22-25, 27, 29-31, 33, 36-38).

A large number of studies have examined the interaction of the cytoskeleton with Na+ channels expressed in epithelia. ENaC has been shown to colocalize with ankyrin and spectrin (34, 39), and in particular, alpha -rENaC binds to the SH3 domain of alpha -spectrin via a carboxy-terminal interaction (32). In inside-out patch-clamp studies of A6 renal epithelial cells, a cell line derived from Xenopus kidney that endogenously expresses ENaC, Cantiello and coworkers (5) demonstrated that short actin filaments activated an ENaC when added to the recording bath, although the identity of this channel could not be established at the time the experiments were done. Furthermore, actin was found to confer sensitivity to PKA phosphorylation under these conditions (29). Both of these findings have been replicated by investigators using heterologously expressed rENaC incorporated into planar lipid bilayers (3, 15). Importantly, bilayer studies of alpha beta gamma -rENaC in which actin was included in the bath yielded identical results to those obtained by cell-attached patch-clamp recordings of Xenopus oocytes heterologously expressing alpha beta gamma -rENaC (19).

We have extended these observations in the present study by using site-directed mutagenesis to identify a region in the carboxy terminus of alpha -bENaC that can interact with actin or an actin-binding intermediary protein. Deletion of a short stretch of amino acids from E631 to F644 in the chimeric construct completely abrogated all effects of actin on the chimeric channel as determined by planar lipid bilayer experiments. Using cell-attached patch-clamp recording, we repeated these experiments in the mutated chimeric constructs coexpressed in Xenopus oocytes with beta - and gamma -rENaC subunits. We found that expression of the E631-F644 deletion was associated with a marked increase in conductance and disruption of the characteristic gating pattern of ENaC, whereas the gating and conductance of the corresponding control deletion (Delta 671-684) was similar to the nonchimeric control. Although these findings do not discount possible contributions to actin interaction from both beta - and gamma -rENaC subunits in the heteromeric wt channel, it does imply that the alpha -ENaC subunit plays a key role in the regulation of the channel by actin. As this portion of the chimeric sequence is well conserved between both alpha -bENaC and alpha -rENaC (out of 14 residues, 11 are identical between the two homologues), it is likely that this region would serve the same function in alpha -rENaC (Fig. 9).


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Fig. 9.   Proposed actin binding site in alpha -bENaC and alpha -rENaC. The region deleted in the chimeric COOH-terminal tail is shown, as is the corresponding sequence in the COOH-terminal tail of alpha -rENaC. Given the close homology between the two sequences, it is likely that the similar sequence in alpha -rENaC is also involved in regulation of the channel by actin. Vertical dashes indicate amino acid identity, and dots represent degrees of similarity between residues.

It has been proposed that interactions between membrane polypeptides and the cytoskeleton are important in the maintenance of a polarized distribution of proteins, and thus the cytoskeleton contributes to the vectorial nature of electrolyte and fluid transport in epithelia. Additionally, the cytoskeleton may serve as a mechanotransducer, linking signals resulting from cell volume changes to the ion channels responsible for volume correction (4). In the case of ENaC, actin clearly has a direct effect on fundamental channel properties (PNa/PK, conductance), such that many of the characteristics attributed to ENaC in native cells can be closely replicated even in a cell-free system (planar lipid bilayers) upon the addition of actin. Previous studies from both our own laboratory and those of others showed that filament length partly determines the effect of actin on ENaC (3, 14, 29). These observations suggest that the effects of actin on membrane ion channels that bring about cell volume change may be regulated at the level of filament length as opposed to mechanical stretch of the cytoskeleton, leading to membrane distension and mechanical stretching of the channel. However, a direct effect of mechanical stretch on channel activity cannot be discounted (16, 17, 20). The identification in the present study of a potential binding site for actin (or an intermediary bridging protein that binds actin and ENaC) in the carboxy terminus of alpha -ENaC is consistent with a direct role for actin in ENaC regulation.


    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37206.


    FOOTNOTES

Address for reprint requests and other correspondence: C. M. Fuller, Univ. of Alabama at Birmingham, Dept. of Physiology and Biophysics, MCLM 830 1918 Univ. Blvd., Birmingham, AL 35294 (E-mail: fuller{at}physiology.uab.edu).

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.

Received 16 November 2000; accepted in final form 5 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Awayda, MS, Tousson A, and Benos DJ. Regulation of a cloned epithelial Na+ channel by its beta - and gamma -subunits. Am J Physiol Cell Physiol 273: C1889-C1899, 1997[Abstract/Free Full Text].

2.   Benos, DJ, and Stanton BA. Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels. J Physiol (Lond) 520: 631-644, 1999[Abstract/Free Full Text].

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