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-bENaC involved
in gating and functional effects of actin
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Gating differences occur between
the 
-subunits of the bovine and rat clones of an amiloride-sensitive
epithelial Na+ channel (ENaC). Deletion of the carboxy
terminus of bovine -ENaC (-bENaC) at R567 converted the gating
properties to that of rat -ENaC (-rENaC). The equivalent
truncation in -rENaC was without effect on the gating of the rat
homologue. The addition of actin to ENaC channels composed of either
-rENaC or -bENaC alone produced a twofold reduction in
conductance and an increase in open probability. Neither -rENaC
(R613X) nor -bENaC (R567X) was responsive to actin. Using a
chimera consisting of -rENaC1-615 and
-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) -bENaC. Premature stop mutations
proximal to E685X produced channels with gating patterns like
-rENaC. Actin had no effect on the E631X truncation, whereas more
distal truncations all interacted with actin, as did wt -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.
-subunit of bovine epithelial sodium channel; planar lipid
bilayers; patch clamp; amiloride; ion channels; oocytes
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CATION CHANNELS
BELONGING to the epithelial Na+ channel family
(ENaCs) comprise multiple subunits (, 
, 
) (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
- and -subunits are coexpressed with , 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 -ENaC subunit from bovine kidney (-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. -bENaC had
a single transition step of 39 pS and exhibited bursting behavior with
long (1-5 min) closed periods between bursts (10).
Rat -ENaC (-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, -rENaC did not exhibit the
long closed times characteristic of -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
-bENaC and 630 in -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 -ENaC subunits.
Deletion of the carboxy terminus of -bENaC (R567X) converted its
gating behavior to that of -rENaC, whereas the equivalent deletion
in -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 -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 -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
-rENaC and -rENaC in the presence of PKA further increased
channel Po. Furthermore, addition of actin
enhanced the downregulation of -rENaC by cystic fibrosis
transmembrane conductance regulator (CFTR) and restored sensitivity of
the channel complex to PKA (3, 15). In contrast,
carboxy-terminally truncated -rENaC and -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 -ENaC subunits were located in the
carboxy terminus, we generated a chimeric construct consisting of
-rENaC residues 1-615 and -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 -ENaC subunits.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Subcloning and Mutagenesis
Wild-type (wt)-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 -bENaC using 5'
BspEI and 3' BglII sites. The ligation produced a
join between residue 616 in -rENaC and 568 in -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 M
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 =
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Previous studies have shown that deletion of the carboxy terminus
of -bENaC converts its gating to that of -rENaC
(10). To further pinpoint this gating switch, we made a
chimera by joining -rENaC residues 1-615 and -bENaC residues
570-650 (Fig. 1). The region of the
join is 100% conserved, with divergence starting at residue 584 in
-bENaC and 630 in -rENaC. As shown in Fig. 2, -rENaC and -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 -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 -rENaC and
-bENaC incorporated into lipid bilayers (9,
13).
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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 -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 -bENaC
and that these truncation mutants do not affect channel conductance,
ion selectivity, or amiloride sensitivity.
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We also used these chimeric constructs to examine the effect of actin
on single of -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 -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
-rENaC/-bENaC chimera or the truncation constructs, with the
exception of chimera E631X. In this case, actin had no effect on
-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 -bENaC, as would
be predicted from our earlier observations. However, whereas
chimera
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
chimera631-644 (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 -bENaC, and, because this region is
highly conserved, it is likely that this sequence performs a similar
function in -rENaC.
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To further characterize the effect of actin on the single-channel
behavior of the chimera, we expressed the -ENaC chimera together
with - and -rENaC subunits in Xenopus oocytes.
Recording in the cell-attached patch configuration, we found that
chimera-rENaC behaved very similarly to the
control (-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 -rENaC control
(Fig. 7). Replacement of chimera-rENaC with the control deletion
chimera671-684-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 
631-644-deleted -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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 -bENaC and -rENaC form triple-barrel channels, each barrel having a conductance of 13 pS. In
the case of -bENaC, the barrels gate cooperatively, so that a
single-step transition of 39 pS is observed. In the case of -rENaC,
there is an initial 13-pS subconductance state on which a 26-pS step is superimposed.
Experiments performed with a truncation mutant of -bENaC (R567X)
demonstrated that deletion of the carboxy terminus of this ENaC isoform
switched the gating pattern to that of -rENaC (10). The
corresponding deletion in -rENaC (R613X) had no effect on the
pattern of -rENaC gating. This observation leads us to suggest that
a kinetic switch located in the carboxy terminus of -bENaC (coincidentally the region that exhibits the lowest homology to -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 -bENaC from residue R568 was grafted onto -rENaC at residue position R614. This resulted in the formation of an -rENaC/-bENaC hybrid polypeptide of 696 amino acids that had an identical gating pattern to -bENaC. Insertion of several stop
mutations at intervals along the carboxy terminus of the chimera
revealed that the domain responsible for coordinating gating of
-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 -bENaC, which exhibits only 25% homology
with the corresponding portion of -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 -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, -rENaC binds to the SH3
domain of -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 -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 -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 -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 - and -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 (671-684) was
similar to the nonchimeric control. Although these findings do not
discount possible contributions to actin interaction from both - and
-rENaC subunits in the heteromeric wt channel, it does imply that
the -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 -bENaC and -rENaC (out of 14 residues, 11 are
identical between the two homologues), it is likely that this region
would serve the same function in -rENaC (Fig.
9).
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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 -ENaC is consistent with
a direct role for actin in ENaC regulation.
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ACKNOWLEDGEMENTS |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37206.
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FOOTNOTES |
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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.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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