Correspondence to: Laurent Schild, Institut de Pharmacologie et de Toxicologie, Bugnon 27, CH-1005 Lausanne, Switzerland., laurent.schild{at}ipharm.unil.ch (E-mail), Fax: 4121 692 5355; (fax)
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two , one ß, and one
homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of ß and
subunits, which precedes the second putative transmembrane
helix (M2). A residue in the
subunit (
S589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of
, ß, and
subunits (
G587, ßG529,
S541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of ßG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported
S589 mutations. We conclude that the residues
G587 to
S589 and homologous residues in the ß and
subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.
Key Words: epithelial Na+ channel, Xenopus oocyte, pore, selectivity, ion channel
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The highly selective epithelial sodium channel (ENaC)1 in the apical membrane of epithelial cells represents the predominant pathway in mediating sodium reabsorption in the distal nephron, the colon, and the lung (
ENaC belongs to a new class of channel proteins called the ENaC/DEG superfamily, which includes a variety of proteins involved in mechanotransduction and neurotransmission, and is found in nematodes, flies, snails, and mammals (for review see
ENaC is a heterotetramer, and made of two , one ß, and one
homologous subunits arranged around the channel pore in an
ß
configuration (
helix starting with a conserved Ser residue (
S589) and extending 22 residues further downstream in the defined ENaC sequence (see Figure 1 B). A pre-M2 segment can arbitrarily be defined as a sequence delineated by a conserved Gly (
G579) residue at the 5' end and
S589 at the 3' end that initiates the M2 segment. Figure 1 C shows a model of the narrow pore region of ENaC, based on this and previous work. Previous mutagenesis experiments provided evidence that the pre-M2 forms the outer pore of ENaC (
(G537) subunits decrease the affinity for the pore blocker amiloride and change single-channel conductance. In addition, a Cys substitution at the homologous position of
ENaC (
S583C) generated a high affinity Zn2+ binding site that leads to channel block by Zn2+. Recently, we have shown that mutations of the conserved Ser residue
S589 allow larger ions such as K+, Rb+, and Cs+ as well as divalent cations to pass through the channel (
S589 determines the molecular cutoff of the channel pore at its narrowest point, the selectivity filter. Thus, in the pre-M2 segment that lines the outer channel pore, the amiloride binding site precedes the selectivity filter in the sequence.
|
In this study, we have analyzed the role of conserved amino acid residues located between the amiloride binding site (corresponding to S583) and the selectivity filter (
S589) in channel permeation properties and blocking by amiloride. We have focused our interest on the conserved aromatic residues at the position corresponding to W585 in the
rENaC sequence, and on a cluster of Ser and Gly residues (
G587,
S588, and analogous amino acids in ß and
subunits; see Figure 1 B).
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Site-directed Mutagenesis
Mutations were introduced in rat ENaC cDNA as described previously (, ß,
subunit were synthesized in vitro. For binding experiments,
, ß, and
subunits that had been tagged as described by
ß
ENaC subunits at a total concentration of 100 ng/µl. For simplicity, ENaC mutants are named by the mutated subunit only, although always all three subunits (
, ß, and
) were coexpressed.
Binding Experiments
The FLAG reporter octapeptide, which had been introduced in , ß, and
subunits, is recognized by the antiFLAG M2 mouse monoclonal antibody (M2Ab) (Eastman Kodak Co.). M2Ab was iodinated as described by
counting containing 250 µl of the same solution. The samples were counted and the same oocytes were kept for subsequent measurement of the whole-cell current. Nonspecific binding was determined from parallel assays of noninjected oocytes. Theoretically, it might be possible that our mutations affect the accessibility of the FLAG epitope for the M2 antibody by changing the conformation of the extracellular loop. However, this possibility seems rather unlikely since most of the mutations did not affect 125I-M2Ab binding.
Electrophysiological Recording
Electrophysiological measurements were taken at 1620 h after injection. Two-electrode voltage-clamp recordings were obtained using a TEV-200 amplifier (Dagan Corp.). The standard bath solution contained 110 mM NaCl, 1.8 mM CaCl2, 10 mM HEPES-NaOH, pH 7.35. For selectivity measurements, Na+ was replaced by Li+, K+, Rb+, or Cs+ at the same concentration. Macroscopic amiloride-sensitive currents (I) are defined as the difference between ionic currents obtained in the presence and absence of 5 µM (or higher concentrations for some mutants, as indicated) of amiloride (Sigma Chemical Co.) in the bath. All macroscopic currents shown are amiloride-sensitive currents as defined above. Pulses for currentvoltage curves were applied, and data were acquired using a PC-based data acquisition system (Pulse; HEKA Electronik).
The cell-attached or outside-out configuration of the patch-clamp technique was used to obtain macropatch and single-channel data. Before recording, the vitelline layer of the oocyte was removed. For cell-attached patches, the oocytes were kept in a standard bath K+ solution to depolarize the membrane potential, and pipette solutions were Na+ or Li+, as described above. For outside-out patches, extracellular solutions were as described above. Changes of external solutions of outside-out patches were made using the Rapid Solution Changer RSC-200 (BioLogic International Ltd.). In this system, the perfusion solutions are driven by gravity to the rotating head containing in these experiments up to 11 glass tubes. The solution exchange is performed by a highly precise and fast rotation of the RSC head, which exposes the patch pipette to the flow of one of the tubes. Times of rotation from one tube to the adjacent one were a few milliseconds. The pipet solution contained 75 mM CsF, 17 mM N-methyl-D-glucamine, 10 mM EGTA, and 10 mM HEPES, pH 7.35. Pipettes were pulled from Borosilicate glass (World Precision Instruments, Inc.). In patch-clamp experiments, currents were recorded with a List EPC-9 patch clamp amplifier (HEKA Electronik) and filtered at 100 Hz for analysis. Data are shown as mean ± SEM, or as indicated.
Data Analysis
To analyze titration curves for inhibition of macroscopic Li+ or Na+ currents (I), the ratio I/I0 measured in the presence (I) of a particular blocker B to that in the absence of the blocker (I0) is described by the inhibition equation: I/I0 = Kin'/(Kin' + [B]n'), where Ki is the inhibitory constant of the blocker, [B] is the concentration of the blocker, and n' is a pseudoHill coefficient. For the fit of Li+ block of Na+ current through S541A ENaC (see Figure 8), n' was set equal to 1, and a nonblockable fraction of the amiloride-sensitive current (I) was introduced. This nonblockable fraction of I was set equal to the amiloride-sensitive current carried by 140 mM Li+ alone, normalized to the amiloride-sensitive current carried by 20 mM Na+ (e.g., 0.21 ± 0.01 at -150 mV, n = 6).
|
|
|
|
|
|
|
Modeling of Energy Barrier Profiles
A reaction rate theory treatment of transmembrane ionic diffusion considers ion movement as a series of discrete steps between energy minima (wells) separated by energy maxima (barriers). One may simply account for saturation or ionic block by assuming that only one ion at a time may reside in a particular energy well, which corresponds to a discrete ion-binding site in the channel, and that individual ions cannot pass each other in the channel. To fit our currentvoltage (I/V) data collected at various Na+ or Li+ concentrations to discrete-state barrier models, we have used a version of the AJUSTE program, originally developed by
The energy diagram in Figure 9 A (below) summarizes the adjustable parameters of the model. The energies of the unoccupied channel at zero voltage are defined by three peak energies (G1, G2, and G3) and two wells or site energies (U1 and U2) for Na+ and Li+. The subscripts of the parameters refer to the position with respect to the inside solution as shown in Figure 9 A. The program can deal with the simultaneous presence of three types of ions. The distances D1D6 refer to the fraction of the electric field that separates peak and well positions with the requirement that D1 + D2 + D3 + D4 + D5 + D6 = 1. In addition to the above energy and distance parameters, there is an interaction energy A(ion x, ion y), which models the effects of ionion interactions between the same or different types of ions. The reference energy state used in the model is 1 mol fraction, which corresponds to 55.5 M. Conversion to a 1-M reference state is readily accomplished by addition of 4.0 RT units to the reported peak and well energies. The translocation rate constant K for translocation from well U over peak G is related to the translocation barrier height (G = GG - GU) according to K = (kT/h) exp(-
G/RT), where k is Boltzmann's constant, T is the temperature in °K, and h is Planck's constant (kT/h = 6.2 x 1012 s-1 at 25°C).
|
For the modeling of energy barrier profiles, we used single-channel data from outside-out patches of Xenopus oocytes expressing wild-type (wt) or mutant ENaC. Data sets had been obtained at three to five different negative membrane potentials (less than -50 and more than -180 mV) and four to six different external concentrations of the permeant ion (either Na+ or Li+ at 15200 mM) per channel type, and in the absence of any permeant ions in the internal solution. As we have analyzed inward, but not outward, currents for the modeling, the part of the energy barrier profiles on the extracellular side (G2, G3, U2) are well defined, whereas the quality of the fit was relatively insensitive to changes of the energy parameters U1 and G1. The procedure for fitting single channel data consisted first in setting barrier profiles for ENaC wt with either Na+ or Li+ as the single permeant ion. The quality of fits was evaluated both visually and by SUMSQ, which is the weighted sum of squared differences between experimental and theoretical data minimized by the fitting routine. The parameter values for the electrical distance D were arbitrarily constrained by a requirement of symmetry about the central barrier located at an electrical distance of 0.5. The optimal arrangement found for wt ENaC was D1 = D6 = 0.1, D2 = D5 = 0.15, and D3 = D4 = 0.25, and this arrangement was used for fitting of all mutants. The quality of the fit was relatively insensitive to the ionion interaction parameter A. This can be readily explained by the fact that the data used for constructing the model was obtained at relatively low ion concentrations (typically <160 mM). In this concentration range, the probability of double occupancy of the channel was <0.01, even with A set to a low value (2.6). This is consistent with flux-ratio experiments indicating that ENaC forms a one-ion pore (
The model obtained for Na+ permeation of wt channels is shown in Figure 9 B (below). The model fits the conductance/ion concentration relationship well, as shown by the comparison of model prediction (solid line) and data points (symbols) in Figure 7 (below). The outer well (U2) is deeper than the inner well (U1). This difference allowed fitting of mutant data, in which the apparent channel affinity for Na+ decreased (S541A,
G587A) by just changing one well energy (one binding site) with regard to wt. This energy profile predicts an almost ohmic behavior of the channel, with outward currents <111% and >90% of inward currents over a voltage range of ±150 mV. Based on the energy profile obtained for the wt Na+ permeation, the profile best suited for Li+ permeation of wt ENaC was obtained by increasing U2 and G3 (see Figure 7 and Figure 9 A). Starting from these two models for Li+ and Na+ permeation, we attempted to fit the mutant data by changing only energy values of one well and one barrier at the extracellular side of the permeation pathway, either U2 and G2, or U2 and G3, assuming that single point mutations in the external channel pore would not affect binding sites on both sides of the highest barrier of the selectivity filter. Adjustment of at least one barrier was necessary to adjust the model to the experimental conductance values. By changing U2 and G2, we obtained better fits of our experimental data than by changing U2 and G3. For this reason, we report here models obtained by only the first method.
Finally, we used the 3B2S model to fit the block of 20 mM Na+ currents in the S541A mutant by increasing Li+ concentrations (see Figure 8). In this case, the model takes into account the simultaneous presence of Na+ and Li+ in the external solution. The use of the 3B2S model requires that macroscopic currents are converted to unitary currents. The macroscopic current data of Li+ block in macropatches were normalized at each voltage to the condition with 20 mM Na+ alone and, for each voltage, these normalized currents were multiplied by the single-channel current for 20 mM Na+ predicted by the
S541A-Na+ model. These modified data were then used to obtain a model for Li+ permeation of the
S541A channel.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The sequence alignment of the pre-M2 segment and the NH2-terminal part of M2 of ENaC subunits and homologous proteins is illustrated in Figure 1 B. Residues, which have previously been shown to be important for ion permeation and/or blocking of ENaC currents by amiloride are shown in bold on a gray background and amino acids that have been analyzed in the present study are shown in white on a dark background. S583 and the corresponding residues ßG525 and
G539 line the outer channel pore as shown in Figure 1 C at a site where amiloride plugs the channel (
S589 at the NH2 terminus of M2 is part of the narrowest region of the pore at the selectivity filter (
W585 and
V590 and their analogues in ß and
subunits, are conserved and might be important for the ion permeation properties. We have mutated the
W585, ßW527, and
W539 residues to various amino acids and did not observe any significant changes in ion selectivity, unitary conductance, or block by amiloride (Tables IIII). The level of cell surface expression of wt and mutant ENaC was determined on intact oocytes by specific binding of an iodinated monoclonal antibody to a FLAG epitope inserted in the extracellular domain of ENaC subunits (see METHODS). Some mutations resulted in a low expression of the channel at the cell surface (Table 1). Similar to Trp mutations, Ala substitutions of the conserved
V590, ßV532, and
V544 residues were without effects on the biophysical and pharmacological properties of the channel (Tables IIII). This lack of effects of mutations of the conserved Trp and Val residues suggests that these amino acids may not line the channel pore.
|
Next, we looked at the cluster of Ser and Gly residues, G587 and
S588 and the analogous amino acid residues in ß and
subunits (Figure 1 B). The
S588I mutation is known to increase single channel conductance for Na+ ions and to decrease channel affinity for blocking by amiloride (
S588 and the homologous residues ßG530 and
G542 did not change ion conductance properties or channel affinity for amiloride (Tables IIII). The effect of
S588I on channel conductance corroborates the previous observation that
S588 is close to the selectivity filter (
Mutations of G587, ßG529, and
S541 Change Ion-permeation Properties and Channel Block by Amiloride
Conserved Gly residues in the and ß subunits at positions
587 and ß529, and the unique Ser residue in the
subunit (
S541) were substituted with the polar Ser, the nonpolar residues Cys, Gly, or Ala or the negatively charged Asp. Analysis of the concentration-dependent block of macroscopic Na+ currents by amiloride (Table 1) showed that specific mutations in
and ß subunits decreased channel affinity for amiloride. The ßG529A, ßG529S, and ßG529C mutations resulted in a 40- to 130-fold increase in Ki for amiloride, and a significant sevenfold increase in amiloride Ki was measured for
G587S. Some substitutions of these residues had no effect on block by amiloride (
G587A, ßG529D, ßG529R; Table 1), suggesting that these amino acids are not part of the amiloride binding site, but rather are indirectly involved in channel block.
No INa was detected for G587D and
S541N or
S541F mutants, and only a small INa (<0.3 µA) was detected for
S541R even using high (1 mM) amiloride or benzamil concentrations. In the case of the
G587D mutant, INa was not detected despite a 5.5-fold higher antiFLAG binding signal compared with the background signal. The specific antiFLAG binding to oocytes expressing the
G587D mutant was only slightly lower than binding to oocytes expressing the functional
G587A mutant (Table 1). Similarly, specific antiFLAG antibody binding was about the same for the nonconducting
S541N and
S541F mutants and for
S541R compared with the functional
S541A mutant (Table 1). These observations indicate that
G587D,
S541N,
S541F, and
S541R mutants are expressed at the cell surface, but are nonconducting channels or channels with minuscule conductance (
S541R), consistent with the notion that mutations of
G587 and
S541 residues affect ion permeation through the channel. Thus it appears that ENaC can tolerate relatively conservative substitutions at these positions and that less conservative replacements can make the channel nonfunctional.
We tested whether G587A, ßG529A, or
S541A mutations affect channel selectivity for Li+ over Na+ ions. Representative current traces in the presence of extracellular Na+ solution at different voltages are shown in Figure 2A and Figure B, for wt and the
S541A mutant. When Li+ replaced Na+ in the external medium, the amiloride-sensitive inward current increased in oocytes expressing ENaC wt (Figure 2 A), but clearly decreased in oocytes expressing
S541A (Figure 2 B). The macroscopic currentvoltage relationships of ENaC wt and the
G587A, ßG529A, or
S541A mutants in the presence of external Na+, Li+, or K+ ions are plotted in Figure 2 C. The currents of the I/V curves are normalized to INa of each channel type measured at -100 mV. In external Na+ solution, the I/V behavior of wt and mutant channels was identical. The ILi/INa ratio was lower for the three mutants compared with wt, and followed the sequence wt >
G587A > ßG529A >
S541A. The current ratio ILi/INa at -100 mV of all the
G587, ßG529, and
S541 mutants is shown in Table 2. In addition to mutants shown in Figure 2, ßG529C also exhibited a decreased ILi/INa ratio, whereas other mutants such as ßG529D, ßG529R, or
S541G maintained ILi/INa ratios > 1, similar to ENaC wt. Thus, the different effects by different ßG529 substitutions cannot be correlated with properties of the amino acid side chain. The fact that substitution of ßG529 by the much larger Arg did not change ion permeation indicates that the side chain may point away from the channel pore (see DISCUSSION).
|
The I/V relationship in external K+ did not provide evidence for a detectable K+ permeability for the G587A, ßG529A, and
S541A mutants even at hyperpolarized membrane potentials (Figure 2 C). However, ßG529S generated a significant amiloride-sensitive inward K+ current as illustrated by the currentvoltage behavior in Figure 3. In the presence of external Na+ the I/V relationship of the ßG529S mutant was identical to wt. When K+ replaced Na+ ions in the external medium, ßG529S channels exhibited measurable inward currents at negative membrane potentials. At -100 mV, IK represented 22% of INa (IK/INa = 0.22 ± 0.2, Table II). The ßG529S mutant allows to a lesser extent Rb+ ions to pass through the channel (IRb/INa = 0.04 ± 0.01, n = 8), but not larger ions like Cs+ (ICs/INa < 0.01, n = 8). The permeability of the ßG529S mutant to K+ and Rb+ suggests that this mutation increased the molecular cutoff of the channel. Similarly, the ßG529C mutant was also slightly permeant to larger ions with an IK/INa ratio of 0.06 ± 0.0 (Table 2) and an IRb/INa ratio of 0.03 ± 0.01 (n = 8). The ßG529D mutant, which exhibited a IK/INa ratio of 0.19 had a very low INa expression level and was not analyzed further. None of the
G587 or
S541 mutants showed significant K+ permeability (Table 2). We conclude from the analysis of these macroscopic current data that (a) the residues
G587 and ßG529 in the pre-M2 segment play a role in channel block by amiloride, (b)
G587, ßG529, and
S541 are involved in defining channel Li+/Na+ permeability ratio, and (c) ßG529 codetermines the molecular cutoff of the channel that normally prevents K+ ions from going through the channel.
Unitary Currents from G587, ßG529, and
S541 Mutants
Measurements of channel unitary currents are necessary to ultimately demonstrate changes in ion permeation through the channel. Our single-channel analysis was focused mainly on channel mutants that exhibit important changes in macroscopic current properties. Many of the single-channel recordings were performed in the excised outside-out configuration to allow the determination of the channel sensitivity to amiloride to ascertain that unitary currents detected in the patch indeed represented the activity of ENaC mutants. Representative recordings are shown in Figure 4. As anticipated from macroscopic currents, G587A and ßG529A showed reduced unitary currents with Li+ ions as charge carrier, and no obvious changes in unitary Na+ current (iNa) or channel gating were observed. The corresponding
S541A mutation decreased iNa, and the unitary Li+ current (iLi) was considerably reduced so that transitions between open and closed states could not be precisely determined under our recording conditions (Figure 4). Single-channel conductance values, which are summarized in Table 3, show that the unitary Li+ conductance (gLi) for the mutants follows the order wt >
G587A > ßG529A >
S541A. The gLi/gNa ratios agree with the Li+/Na+ permeability ratios measured for macroscopic amiloride-sensitive currents (Table 2). The higher Li+/Na+ permeability of the ßG529R and the
S541G mutants compared with wt measured by the macroscopic ILi/INa ratio (ILi/INa = 2.37 and 3.11, respectively, compared with 1.4 for wt), could not be confirmed by changes in either unitary currents for Na+ or Li+ ions, indicating that ßG529R and
S541G mutations do not increase channel permeability to Li+ relative to Na+ ions.
|
Comparison with other members of the ENaC gene family (Figure 1 B) shows that at the position homologous to G587 all known genes have a Gly residue, except for S541 in the ENaC
subunit. Substitution of the
S541 by Gly did not change the conductance or the ion selectivity of the channel. We also tested whether Gly and Ser residues are interchangeable at the homologous positions in
and ß subunits. The
G587S mutant showed a decreased unitary Na+ and Li+ conductance, but the gLi/gNa ratio remained unchanged (Table 3). The most dramatic effect was observed with ßG529S. As shown before (Figure 3), this mutation makes the channel permeable to K+ and preserves a macroscopic ILi/INa ratio >1. At the single-channel level, ßG529S decreased unitary Na+ and Li+ current amplitudes to such an extent that transitions between open and closed states could not be detected. As shown in Figure 5 in a representative experiment, channel openings or closures could not be detected under our conditions in excised outside-out patches containing channels with the ßG529S mutation and exhibiting amiloride-sensitive Na+ and Li+ currents. This indicates that gLi and gNa of the ßG529S mutant are more than likely <1 pS. Thus the analysis of the single-channel currents of the
G587, ßG529, and
S541 mutants indicate that substitution of these residues by Ala, Ser, or Cys change the ion permeation properties by decreasing channel conductances for either Li+ ions alone or for both Li+ and Na+.
Apparent Affinities for the Permeating Na+ and Li+ Ions of Mutants of the G587, ßG529, and
S541 Residues
The decrease in unitary Li+ current in the mutants reflects a slower movement of Li+ ions along the pore of ENaC mutants. Strong binding interactions between the permeant ion and the residues lining the channel pore can account for a slow ion permeation through the pore. Alternatively, high energy barriers for ion translocation along the conduction pore are also expected to slow ion permeation. We addressed these possibilities by measuring the changes in unitary current iNa and iLi with increasing concentrations of the permeant ion, as illustrated in typical recordings in Figure 6. From iNa and iLi measurements at different holding potentials, values for single-channel conductance (gNa and gLi) were obtained for ion concentrations ranging from 10 to 200 mM. Unitary conductances are plotted versus the concentration of the permeant ion (Na+ or Li+) in Figure 7. ENaC wt shows saturation of gNa around 5 pS for Na+ concentrations >100 mM, whereas gLi does not saturate even at concentrations >100 mM. Values of ion concentration for half-maximal unitary conductance (KM) were determined from fits of the conductanceion concentration relationship to the Michaelis-Menten equation and are given in Table 4. In the case of ENaC wt, KM values were 38 mM for Na+ and 118 mM for Li+. The gNa and gLi saturation curves of wt ENaC suggest that the difference in Li+ versus Na+ permeability is due to differences in channel affinity for the two ions, implying that the ILi/INa ratio strongly depends on the absolute concentration of Na+ and Li+.
|
In the G587A mutant, which is equally permeant to Na+ and Li+, the apparent affinity for Na+ is slightly lower and the apparent affinity for Li+ ions is increased (Figure 7). The KM of 114 mM for Na+ ions is close to that for Li+ ions (90 mM) (Table 4). The
G587S mutant is characterized by a dramatic decrease in maximal conductances for Na+ and Li+ ions and a lower apparent KM for Li+. The unitary Na+ conductance of
G587S saturates at concentrations <100 mM. Due to the low gNa, the KM for Na+ permeation was not determined more precisely. The ßG529A mutant shows a gLi that saturates between 2 and 3 pS with an apparent affinity for Li+ ions around 40 mM, thus the apparent affinity is increased threefold compared with wt. It is remarkable that the change in apparent affinity and maximal conductance of the ßG529A mutant affects only Li+ ions, whereas the KM for Na+, and maximal gNa, remained basically unchanged (Table 3 and Table 4). Interestingly the ßG529R mutation has no apparent effects on the permeation properties of Na+ and Li+ ions (Figure 7). The barely detectable single-channel Li+ currents of the
S541A mutant (Figure 4) made the determination of gLi impossible. The
S541A mutant showed a slight decrease in gNa, with a slightly lower apparent affinity for Na+ ions compared with ENaC wt. Thus mutations of
G587 and ßG529 change the apparent affinity for Li+ and/or Na+ ions, resulting in alterations in the single channel conductance and the microscopic conductance ratio gLi/gNa.
What is the affinity of the S541A mutant for Li+ ions? In a few excised outside-out macropatches, we measured macroscopic Li+ conductance of the
S541A mutant at five different Li+ concentrations, and obtained an apparent KM for Li+ as low as 11 ± 1 mM (n = 6). From this high apparent channel affinity for Li+ ions and low Li+ permeability of the
S541A mutant, we expected that Li+ ions should block Na+ currents. The experiment illustrated in Figure 8 shows that this is indeed the case. Macroscopic, amiloride-sensitive inward currents were measured in 20 mM external Na+ and increasing Li+ concentrations from excised outside-out macropatches from oocytes expressing the
S541A mutant. 80 mM Li+ blocked 60% of the Na+ current in the presence of 20 mM external Na+, a Na+ concentration below the KM for Na+ of the
S541A mutant. The inward amiloride-sensitive current is not completely blocked by Li+ because Li+ still carries current in the
S541A channel. From the fit of the Li+ inhibition curve of the INa to a simple inhibition scheme that takes into account a nonblockable fraction (see METHODS), we obtained an apparent Li+ affinity for INa inhibition of 28 mM. In contrast to the situation in the
S541A mutant addition of 80 mM Li+ to the extracellular solution increased in the ENaC wt the amiloride-sensitive current by 3.1 ± 0.1-fold (n = 5) at -150 mV. We conclude that the low unitary conductance of the
S541A mutant is related to a higher apparent channel affinity for Li+ ions.
Model of Na+ and Li+ Permeation in wt and Mutant ENaC
To interpret the changes in the maximal open state conductance and apparent affinity for Na+ and Li+ ions induced by G587, ßG529, or
S541 mutations, we have used a simple model of ion permeation through ENaC, based on reaction rate theory of ion permeation (
The use of Eyring reaction rate theory has several limitations, among them the fact that it describes ion movements over well-to-well distances of fractions of Angstroms, whereas in our 3B2S model the ion translocation over the three energy barriers takes place over a distance of several Angstroms. In discrete-state permeation models, the calculation of the barrier energies depends on the use of a prefactor, kT/h according to the Eyring absolute reaction rate theory (G587A, ßG529A, and
S541A mutants relative to wt. These relative changes are much less model dependent. The small changes in the Na+ permeation through these channel mutants are simply due to a parallel positive shift in G2 and U2 (Figure 9 C), corresponding to a slight decrease in channel affinity for Na+ ions but a conserved maximal gNa. The gradual decrease in Li+ permeability and the changes in the gLi[Li+] relationship observed in
G587A,
G587S, and ßG529A mutants compared with wt are essentially due to a substantial increase in the peak energy of the middle barrier (G2) relative to wt and only small changes in the energy of the outer well (U2) (Figure 9B and Figure C, and Table 4). This results in a higher energy (
G) required for Li+ ions to hop over G2 from an outer binding site U2 to the inner binding site U1, and a consequently slower translocation rate of Li+ ions from U2 to U1.
We have used the macroscopic current data of Na+ current block by external Li+ (Figure 8) to obtain an energy profile for Li+ permeation through S541A ENaC, as described in METHODS. The prediction of the Li+ block by the model is shown as a dotted line in Figure 8, and the energy parameters for the fit are listed in Table 4 and shown in Figure 9 B. As for the
G587A and ßG529A mutants, the corresponding energy profile predicted for Li+ permeation through
S541A shows a higher peak energy of the middle energy barrier that increases the energy required for translocation of Li+ ions from U2 to U1 (Figure 9B and Figure C). Thus, when Li+ occupies the pore of the
S541A in the single filing region, it slows the movement of Na+ ions.
Is the Pre-M2 Segment the Specific Target Region for Amiloride Block and Na+/Li+ Selectivity?
The question remains as to whether amiloride binds to a unique site in the external pore, and whether the ion selectivity filter is confined to the region we identified in the pre-M2 segment. Recently, ENaC as a putative amiloride-binding domain. Later, it was reported that conserved Lys residues at positions 550 and 561 in
ENaC are critical for channel sensitivity to amiloride and for ion selectivity (
ENaC subunits reconstituted in lipid bilayers, we tested the relevance of these mutations for the pharmacological and functional properties of ENaC composed of the three types of subunits,
, ß, and
.
Following expression of an mutant with deletion of the WYRFHY sequence together with ß and
subunits, no INa could be measured and no ENaC cell surface expression could be detected using antiFLAG antibodies directed against ß and
subunits. The point mutation within this sequence,
H282D, reported to be responsible for ENaC insensitivity to amiloride did not result in changes in amiloride sensitivity or macroscopic ionic selectivity of channels made of
H282Dß
(Table 2 and Table 3). The
K550E and the
K561E mutants, when coexpressed with ß and
subunits, exhibited the same sensitivity to block by amiloride as ENaC wt and showed normal macroscopic Na+/Li+ selectivity and unitary currents (Tables IIII). From these results, we conclude that the putative amiloride-binding sequence described by
, ß, and
subunits.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our mutagenesis experiments have identified amino acid residues at homologous positions in , ß, and
ENaC subunits, located two residues upstream of the predicted NH2 terminus of the second transmembrane
helix, that play an important role in ion conduction. Mutations of these residues
G587, ßG529, and
S541 resulted in the following changes in ion permeation and channel ionic selectivity. First, the mutations
G587D,
S541N,
S541F, and
S541R resulted in channels, which were expressed at the cell surface at normal densities, but did not conduct ionic current or showed only a minuscule ionic current (
S541R). Second,
G587A, ßG529A, ßG529C, and
S541A mutations decreased Li+ permeability with relatively small changes in Na+ permeability. Third, introduction of a Ser residue in the
(
G587S) and ß subunits (ßG529S) resulted in a decrease in both Na+ and Li+ unitary currents with no apparent changes in the Li+/Na+ permeability ratio. Fourth, the ßG529S (and similarly ßG529C and ßG529D) mutant was permeant to K+ ions and to a lesser extend to Rb+ ions. Finally, the mutants
G587S, ßG529A, ßG529C, and ßG529S with major changes in ion permeation or selectivity also showed resistance to amiloride block. These observations indicate that
G587, ßG529, and
S541 interact closely with the permeant Na+ and Li+ ions, and participate at least in part in ion discrimination at the selectivity filter. The selectivity filter appears in close vicinity of the amiloride binding site.
The amiloride-sensitive ENaC, target of aldosterone action in the distal nephron and colon, is made of homologous ß
subunits. This channel is highly selective for Na+ over K+ ions and has the unique characteristic among the other members of the ENaC/DEG gene family of being more permeant to Li+ than to Na+ ions. The higher permeability of ENaC for Li+ compared with Na+ ions was evident from early macroscopic current measurements in flat epithelia like frog skin or toad urinary bladder. In these preparations, it was generally not possible to detect currents through ENaC carried by K+ ions. The only ion other than Na+ and Li+ allowed to pass through the channel was H+. The selectivity profile H+ > Li+ > Na+ of ENaC suggested that the pore discriminates among cations mainly on the basis of the size of the dehydrated ion allowing small cations to pass through the channel. Larger cations such as K+, Rb+, or Cs+ go only part-way along the ion conduction pathway, and block the channel pore at high concentrations, but are not able to pass the most constricted region of the pore, the selectivity filter (
In support of this hypothesis for ion selectivity in ENaC is our recent finding that mutation of the highly conserved S589 residue alters the molecular sieving properties of ENaC (
S589 mutants exhibit different IK/INa ratios. In particular, the
S589D mutant with the highest K+/Na+ permeability ratio showed a permeability profile for alkali metal cations following the order K+ > Rb+ > Cs+, indicating that
S589 mutations result in an increase in the molecular cutoff of ENaC. It was concluded that
S589 is part of the selectivity filter, which acts as a molecular sieve to discriminate between small Na+ or Li+ ions and larger K+ ions.
In the present study, the mutants that show a significant K+ permeability are ßG529S, ßG529C, and ßG529D; the permeability relative to Na+ of these mutants was higher for K+ ions than for Rb+, and no current carried by Cs+ could be detected. The increase in the channel molecular cutoff by the ßG529 mutations is consistent with the notion that, like the S589 mutation, they enlarge the pore diameter at the selectivity filter and alter the molecular sieving properties of the channel (
S589 or ßG529 residues. It suggests that these two residues are important for the steric selectivity of the channel by maintaining the proper pore geometry to tightly accommodate the permeating ion and exclude larger ions.
Beside the changes in ion selectivity of the ßG529S, ßG529C, and ßG529D mutants, mutations of the G587, ßG529, and
S541 residues resulted in important changes in the permeability properties of the channel for Na+ and Li+ ions. These effects involve both the changes in the maximal open state conductance (Figure 7) and/or in the apparent channel affinity for Na+ and Li+ ions as determined by KM values for channel conductance. Recordings of individual channels in the native tissue have shown that the open state conductance of ENaC saturates with increasing concentrations of Na+ and Li+ ions, indicating that Na+ and Li+ ions bind to specific sites in the ion permeation pathway. This saturation process arises when the bindingunbinding steps of the ion conduction at specific binding sites along the pore become rate limiting. At high ion concentrations, the channel pore is occupied most of the time and the rate of ion translocation is determined by the maximal rate at which the permeant ions dissociate from their binding site and the ion flux approaches saturation. For ENaC wt, the dependence of open state conductance on the concentration of the conducting ion gives an apparent affinity (KM) of 38 mM for Na+ and 118 mM for Li+. This difference in channel affinity for Na+ and Li+ ions can account for the higher Li+ over Na+ permeability usually observed at an ion concentration around 100 mM since the flux of Na+ ions already reaches saturation at these concentrations (see Figure 7). Specific binding sites for Na+ have been postulated on the basis of competitive interactions between Na+ and amiloride or K+ ions that inhibit Na+ current through the channel in a voltage-dependent manner. In addition, the lack of voltage dependence of channel occupancy by Na+ ions was consistent with the presence of multiple ion binding sites along the channel pore. Reasonable models for Na+ permeation have been proposed that are consistent with these experimental observations. These models involve at least two common binding sites for Na+, Li+, K+, and amiloride in the outer mouth of the channel (
How can the decrease in Li+ permeability relative to Na+ of the G587A, ßG529A, or
S541A mutants be explained in molecular terms? High affinity binding of ions in the channel pore tends to slow ion permeation provided that the channel is occupied by a single ion at a time. A low Li+ permeability was observed in
G587A, ßG529A, or
S541A mutants characterized by a lower maximal gLi and an apparent higher affinity for Li+ compared with wt. Alternatively, the mutations might result in steric changes within the narrow region of the pore, making it more difficult for Li+ ions to pass through. For the interpretation of the changes in ion permeation in ENaC mutants, we need to consider ion conduction through ENaC consisting basically of three fundamental processes: (a) diffusion of an ion up to the outer entrance of the channel pore, (b) dehydration and solvation of the ion by polar chemical groups lining the pore, and (c) translocation through the selectivity filter. Models of ion permeation in terms of energy profile and Eyring rate theory can formulate these processes (
G587A, ßG529A, or
S541A mutants can be related to particular permeation mechanisms. In the ENaC wt, the energy barrier profile obtained for Li+ permeation compared with Na+ is characterized by a higher outer energy well consistent with a lower affinity for Li+ at an external binding site (Figure 9 and Table 4), accounting for the differences in Li+ versus Na+ permeability. In our models for Li+ permeation through the
G587A, ßG529A, or
S541A mutants, the predominant changes in Li+ versus Na+ conductance are due to an increase in the middle energy barrier, with little changes in the outer energy well. It suggests that translocation of Li+ ions in the single filing region at the central energy barrier requires more energy in mutants compared with wt. Thus, the
G587, ßG529, or
S541 residues can be assigned to a region of the pore that represents a significant barrier for ion translocation. It is possible that ßG529 and
S541 have preferential interactions with Li+ ions since mutations of these residues did not affect Na+ permeation much.
We observed that the ßG529S, ßG529C, and ßG529D mutations change the channel molecular cutoff allowing K+ and Rb+ ions to pass through the channel. Thus, ßG529 plays a role in defining the molecular cutoff of the channel and is at the same time important for the Li+/Na+ selectivity. The dual role of ßG529 underlines that the discrimination between Li+ and Na+, and between Na+ and larger ions, occurs at overlying sites in the selectivity filter. Our data suggest that G587A, ßG529A, and
S541A mutations result in steric changes at the selectivity filter that impair translocation of Li+ ions through the narrowest part of the channel pore. In moving from the outer binding site U2 into the narrowest region of the pore, the permeant ion likely has to lose a few more molecules of its hydration shell. This loss of the hydration shell raises the energy of the permeant ion. Our data are consistent with the hypothesis that the energy cost for Li+ dehydration at the U2
G2 translocation is raised in the
G587A, ßG529A, and
S541A mutants because it cannot be sufficiently compensated by the interactions between the permeant ion and the polar groups lining the pore. Mutations such as
G587S and ßG529S decrease unitary conductance for both Na+ and Li+ ions, although the low unitary currents made it difficult for
G587S and impossible for ßG529S to fit the I/V data to our 3B2S model. The energy profile of
G587S for Li+ conduction predicts an increased peak energy of the middle barrier, consistent with what we observed for the other mutants discussed above. The conductance data of ßG529S are also compatible with a mutation resulting in a considerable increase in the peak energy of the middle barrier for Na+ and Li+ permeation, but a decrease in the energy barrier for K+ conduction. The binding site U2 cannot be assigned yet to a molecular structure, but is likely to be in the close vicinity of the selectivity filter.
It is somewhat surprising that the effects of substitutions of G587, ßG529, and
S541 did not correlate in a predictable way with general physicalchemical properties of the substituting amino acid residues. The fact that ion permeation is not altered by the ßG529R mutation indicates that charges do not play a crucial role in ion coordination at the selectivity filter, and basically excludes the possibility that the ßG529 side chain faces the lumen of the channel pore. As already suggested for
S589 mutations (
G587, ßG529, and
S541 in place and maintaining the proper conformation of the pore at the selectivity filter. In ENaC wt, Gly, and Ser, amino acid residues with short side chains make these interactions, and the positive charge in the ßG529R mutant (four carbon atoms between the
-carbon atom and NH3 group) might be too far away to disrupt this interaction. In mutant channels that are permeable to K+ (
S589 and some ßG529 mutants), the mutated side chain might push parts of the pre-M2/M2 domains of the subunits away from each other or tilt them, resulting in a widening of the pore.
In summary, our mutagenesis experiments indicate that G587, ßG529,
S541, and
S589 residues are part of the narrow region of the pore that constitutes the selectivity filter of ENaC (Figure 1 C). It seems that the important steps of ion conduction that involve ion dehydration, solvation, and translocation occur in a very restricted region of the ion permeation pathway that encompasses the
G587 and analogues and
S589 residues. For comparison, the crystal structure of the KcsA channel reveals that the narrow selectivity filter lined by the backbone of VGYG residues is only 12-Å long, whereas the overall length of the pore is 45 Å (
Finally, the mutations G587S, ßG529A, and ßG529S that change ion selectivity and ion permeation properties also decrease channel affinity for amiloride, whereas the ßG529 mutation without consequences on ion permeation properties did not change channel blocking by amiloride. It is unlikely that
G587 and ßG529 are involved in specific binding interactions with amiloride since nonconserved substitutions have no effect on channel affinity for amiloride. The
G587A, ßG529A, or
S541A mutations may indirectly impair the binding interaction of amiloride with the channel pore by steric changes that result in alterations in the pore geometry. According to its size, amiloride is not supposed to penetrate deep into the narrowest part of the selectivity filter. We propose that
G587 and ßG529 are located at the external entrance of the narrow selectivity filter where steric alterations of the pore have consequences on amiloride binding in the channel outer vestibule.
Figure 1 C, showing the ion pore of ENaC, illustrates some relevant experimental observations regarding the structures involved in ion conduction. The Gly 525 and 537 residues of ß and subunits and to a lesser extent the corresponding
S583 residue form the amiloride binding site in the pore (
S583 residues in the ENaC tetramer to Cys creates a high-affinity binding site for Zn2+ (
subunits are most likely on opposite sides of the channel pore (
ß
subunits. We suggest that, in analogy to the KcsA channel, the cytoplasmic part of the pore is lined by residues from the second transmembrane
helix.
![]() |
Footnotes |
---|
Portions of this work were previously published in abstract form (Kellenberger, S., N. Hoffmann-Pochon, E. Schneeberger, I. Gautschi, and L. Schild. 1998. J. Am. Soc. Nephrol. 9: A0193).
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank B.C. Rossier, J.-D. Horisberger, and P. Greasley for helpful comments on the manuscript
This work was supported by grant 31-49654.96 from the Swiss National Foundation for Scientific Research (L. Schild).
Submitted: March 5, 1999; Revised: April 23, 1999; Accepted: April 30, 1999.
1used in this paper: 3B2S, three-barriertwo-site; ENaC, epithelial Na+ channel; I/V, currentvoltage; KM, ion concentration for half-maximal unitary conductance; wt, wild type
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alvarez, O., Villarroel, A., Eisenman, G. (1992) Calculation of ion currents from energy profiles and energy profiles from ion currents in multibarrier, multisite, multioccupancy channel models. Methods Enzymol. 207:816-854[Medline].
Andersen, O.S. (1989) Kinetics of ion movement mediated by carriers and channels. Methods Enzymol. 171:62-112[Medline].
Andersen, O.S., Koeppe, R.E., II. (1992) Molecular determinants of channel function. Physiol. Rev. 72:S89-S158[Medline].
Canessa, C.M., Merillat, A.-M., Rossier, B.C. (1994a) Membrane topology of the epithelial sodium channel in intact cells. Am. J. Physiol. 267:C1682-C1690
Canessa, C.M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J.-D., Rossier, B.C. (1994b) Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature. 367:463-467[Medline].
Doyle, D.A., Cabral, J.M., Pfuetzner, R.A., Kuo, A.L., Gulbis, J.M., Cohen, S.L., Chait, B.T., MacKinnon, R. (1998) The structure of the potassium channelmolecular basis of K+ conduction and selectivity. Science. 280:69-77
Firsov, D., Gautschi, I., Merillat, A.M., Rossier, B.C., Schild, L. (1998) The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO (Eur. Mol. Biol. Organ.) J. 17:344-352
Firsov, D., Schild, L., Gautschi, I., Mérillat, A.-M., Schneeberger, E., Rossier, B.C. (1996) Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc. Natl. Acad. Sci. USA. 93:15370-15375
French, R.J., Worley, J.F., III, Wonderlin, W.F., Shavantha Kularatna, A., Krueger, B.K. (1994) Ion permeation, divalent ion block, and chemical modification of single sodium channels. Description by single- and double-occupancy rate-theory models. J. Gen. Physiol. 103:447-470[Abstract].
Fuller, C.M., Berdiev, B.K., Shlyonsky, V.G., Ismailov, I.I., Benos, D.J. (1997) Point mutations in -bENaC regulate channel gating, ion selectivity, and sensitivity to amiloride. Biophys. J. 72:1622-1632[Abstract].
Garty, H., Palmer, L.G. (1997) Epithelial sodium channelsfunction, structure, and regulation. Physiol. Rev. 77:359-396
Hille, B. (1992) Ionic Channels of Excitable Membranes. 2nd ed Sunderland, MA, Sinauer Associates, Inc.
Ismailov, I.I., Kieberemmons, T., Lin, C.M., Berdiev, B.K., Shlyonsky, V.G., Patton, H.K., Fuller, C.M., Worrell, R., Zuckerman, J.B., Sun, W. et al. (1997) Identification of an amiloride binding domain within the alpha-subunit of the epithelial Na+ channel. J. Biol. Chem. 272:21075-21083
Kellenberger, S., Gautschi, I., Schild, L. (1999) A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving. Proc. Natl. Acad. Sci. USA. 96:4170-4175
Krovetz, H.S., VanDongen, H.M.A., VanDongen, A.M.J. (1997) Atomic distance estimates from disulfides and high-affinity metal-binding sites in a K+ channel pore. Biophys. J. 72:117-126[Abstract].
Lingueglia, E., Champigny, G., Lazdunski, M., Barbry, P. (1995) Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel. Nature. 378:730-733[Medline].
Palmer, L.G. (1982) Na+ transport and flux ratio through apical Na+ channels in toad bladder. Nature. 297:688-690[Medline].
Palmer, L.G. (1990) Epithelial Na channels: the nature of the conducting pore. Renal Physiol. Biochem. 13:51-58[Medline].
Palmer, L.G., Andersen, O.S. (1989) Interactions of amiloride and small monovalent cations with the epithelial sodium channel. Inferences about the nature of the channel pore. Biophys. J. 55:779-787[Abstract].
Renard, S., Lingueglia, E., Voilley, N., Lazdunski, M., Barbry, P. (1994) Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel. J. Biol. Chem. 269:12981-12986
Schild, L., Schneeberger, E., Gautschi, I., Firsov, D. (1997) Identification of amino acid residues in the , ß,
subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. J. Gen. Physiol. 109:15-26
Tavernarakis, N., Driscoll, M. (1997) Molecular modeling of mechanotransduction in the nematode Caenorhabditis elegans. Annu. Rev. Physiol. 59:659-689[Medline].
Waldmann, R., Champigny, G., Bassilana, F., Voilley, N., Lazdunski, M. (1995a) Molecular cloning and functional expression of a novel amiloride-sensitive Na+ channel. J. Biol. Chem. 270:27411-27414
Waldmann, R., Champigny, G., Lazdunski, M. (1995b) Functional degenerin-containing chimeras identify residues essential for amiloride-sensitive Na+ channel function. J. Biol. Chem. 270:11735-11737
Waldmann, R., Lazdunski, M. (1998) H+-gated cation channelsneuronal acid sensors in the ENaC/DEG family of ion channels. Curr. Opin. Neurobiol. 8:418-424[Medline].