Point mutations in the post-M2 region of human alpha -ENaC regulate cation selectivity

Hong-Long Ji, Suzanne Parker, Anne Lynn B. Langloh, Catherine M. Fuller, and Dale J. Benos

Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha -ENaC (alpha -hENaC) led to a decrease and increase in the macroscopic conductance of alpha R586E,R587Ebeta gamma - and alpha R589E,R591Ebeta gamma -hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alpha R586E,R587Ebeta gamma - and alpha R589E,R591Ebeta gamma -hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alpha R586E,R587Ebeta gamma -hENaC and increased for alpha R589E,R591Ebeta gamma -hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alpha R586E,R587Ebeta gamma -hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.

ion permeability; oocyte; voltage clamp; site-directed mutagenesis; patch clamp; sodium channels; amiloride


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MUTAGENESIS has become an experimental mainstay in structure-function analyses of membrane proteins, including ion channels. In cloned epithelial Na+ channels (ENaC), mutagenesis studies have revealed that amino acid residues located in hydrophobic regions (H2) preceding the second transmembrane domain (M2) may constitute the cation selectivity filter of the channel (16, 17, 31, 33 35, 37). A functional ENaC consists of three homologous subunits: alpha , beta , and gamma  (5, 8, 19, 34), and all three are thought to participate in forming both the selectivity filter and pore of the channel (16, 33, 35).

The two membrane-spanning domains of alpha -ENaC, which by itself can form a functional, homomeric amiloride-sensitive Na+-selective channel, contain several highly conserved, negatively charged amino acids located on the putative pore-lining surface of an alpha -helix, as predicted by alpha -helical wheel analysis (21). In alpha -hENaC, changing the charge of amino acid 108 (in M1) from the negative aspartate (D) to the positive arginine (R) had no effect on channel properties. However, altering any of the three negatively charged amino acids in M2, i.e., E568, E571, or D575, to arginines dramatically decreased single-channel conductance but produced no change in Na+ vs. K+ permeability (PNa/PK) or amiloride sensitivity. We have identified a sequence of positively charged amino acids between residues 586 and 597 of alpha -hENaC that is identical in the five mammalian alpha -ENaC subunits cloned to date (rat, mouse, bovine, human, and guinea pig) and is arginine rich (RRFRSRYWSPGR). This region is also conserved in the human delta -ENaC homolog (RRLRRAWFSWPR; Ref. 37) but is not present in any of the beta - or gamma -ENaC subunits or in the ENaC-related Aplysia Na+ channel that is gated by FMRF-amide (Phe-Met-Arg-Phe-NH2) (23). This arginine-rich amino acid domain is located just distal to the M2 region in the cytoplasmically located COOH-terminal tail of the alpha -ENaC protein. Hence, we hypothesized that this region of concentrated positive charge may constitute part of the inner mouth of the ENaC pore and thereby influence the conduction and/or selectivity properties of the channel. We tested this hypothesis in whole cell and single-channel patch-clamp experiments of alpha beta gamma -hENaC heterologously expressed in Xenopus oocytes using double point mutants. Double point mutations of arginine residues in the post-M2 region of alpha -hENaC resulted in an increase in the alpha R589E,R591Ebeta gamma -hENaC- and a decrease in the alpha R586E,R587Ebeta gamma -hENaC-associated chord conductance but left the single-channel Li+ conductance unaltered. However, both the apparent equilibrium dissociation constant for Na+ (K<UP><SUB>m</SUB><SUP>Na</SUP></UP>) and the maximal amiloride-sensitive Na+ current (I<UP><SUB>max</SUB><SUP>Na</SUP></UP>) were significantly decreased. Moreover, the relative permeabilities of Li+ and K+ vs. Na+ (PLi/PNa and PK/PNa) were increased 11.25- to 27.57-fold in the mutant of alpha R586E,R587Ebeta gamma -hENaC. Radii of channel pore analyses revealed that the diameter of the alpha R586E,R587Ebeta gamma -hENaC channel is smaller, and the diameter of the alpha R589E,R591Ebeta gamma -hENaC channel is larger, than that of the wild-type channels. These results are consistent with the hypothesis that this region of positive charge is important for the ion permeation properties of the channel and may, in fact, form part of the pore structure itself.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Construction of site-directed point mutations. Full-length alpha beta gamma -hENaC cDNAs were a gift of Dr. Michael J. Welsh (University of Iowa) (25, 26). Single point mutations in the alpha -subunit were constructed using the Quick Change mutagenesis kit (Stratagene, La Jolla, CA). Primers were constructed to be complementary to the sense and antisense strands of a given region of alpha -hENaC. Each set of primers contained the appropriate base changes required to code for two glutamates instead of the wild-type arginine residues. Plasmid DNA was subjected to the following PCR protocol, using one set of mutagenic primers: 1 cycle at 95°C (1 min) and then 15 cycles at 95°C (30 s), 55°C (1 min), and 68°C (12 min). Nonmutated DNA was removed from the PCR reactions by digestion with DpnI at 37°C (1 h). This enzyme is specific for methylated DNA and will not digest the nonmethylated PCR products. Each product was then transformed into supercompetent Escherichia coli (XL1 BLUE, Stratagene) and grown on agar plates containing 50 µg/ml ampicillin overnight at 37°C. The DNA was isolated from the colonies according to a standard alkaline lysis miniprep procedure. Products containing the specific mutations were confirmed by dideoxy sequence analysis.

In vitro transcription. DNA samples were transcribed in vitro with the use of either SP6 or T7 mMessage mMachine kits (Ambion, Austin, TX), as appropriate. The quality and size of the cRNA were confirmed by denaturing formaldehyde-agarose gel electrophoresis. RNA concentration was estimated using ultraviolet spectrophotometry at a wavelength of 260 nm.

Oocyte preparation. Oocytes were surgically removed from appropriately anesthetized adult female Xenopus laevis (Xenopus Express, Beverly Hills, FL) by standard techniques (14). Follicle cells were removed in OR-2 calcium-free medium (in mM: 82.5 NaCl, 2.4 KCl, 1.0 MgCl2, and 5.0 HEPES-Na, pH 7.5) with the addition of collagenase. Defolliculated oocytes were washed in OR-2 medium (in mM: 82.5 NaCl, 2.4 KCl, 1.0 MgCl2, 5.0 CaCl2, and 5.0 HEPES, pH 7.4) and allowed to recover overnight in half-strength Liebovitz medium at 18°C. Groups of stage VI oocytes were injected with 50 nl (25 ng of the appropriate alpha -, beta -, and gamma -hENaC cRNAs; all subunit mixtures were 1:1:1). Two-electrode voltage-clamp and/or single-channel measurements were made 24-48 h postinjection.

Oocyte confocal microscopy. Biotin-streptavidin-Texas red surface labeling of images from Xenopus oocytes previously injected with wild-type or mutant alpha -hENaC plus green fluorescent protein (GFP)-tagged beta - and gamma -rENaC (rat ENaC) was performed as previously described (21). Images were acquired with an Olympus FluoroView BX50 upright confocal laser scanning microscope. The 488-nm argon laser and the 568-nm krypton laser line excited enhanced GFP (EGFP) and Texas red, respectively. EGFP fluorescence was collected through the 510- and 550-nm barrier filters, and Texas red fluorescence was collected through the 610-nm filter. X-Y scans were obtained at 12-bit resolution at approximately the midsection of each oocyte. Acquired images were imported into Adobe Photoshop 5.0 for processing. Quantitation was done as previously described (13).

Dual-electrode voltage clamp. Whole cell membrane currents were measured in oocytes expressing hENaC using dual-electrode voltage clamp as described previously (15). Oocytes were impaled with two 3 M KCl-filled electrodes, having resistances of 0.5-2 MOmega . A Dagan TEV-200 voltage-clamp amplifier was used to clamp oocytes with concomitant recording of currents. Two reference electrodes were connected to the bath by 3 M KCl-3% agar bridges. The continuously perfused bathing solution was ND96 (in mM: 96 NaCl, 1 MgCl2, 1.8 CaCl2, 2 KCl, and 5 HEPES, pH 7.4). Equimolar concentrations of N-methyl-D-glucamine chloride (NMDG-Cl) were used to replace NaCl in those experiments where the [Na+] was varied. Experiments were controlled by pCLAMP 6.04 software (Axon Instruments, Burlingame, CA), and current at -100 and +60 mV was continuously monitored with an interval of 2 s by using a strip chart recorder. Oocytes were clamped at a holding potential of 0 mV. The current-voltage (I-V) relationships were acquired by stepping the holding potential in 10-mV increments from -160 to +60 mV. I-V data were recorded after the monitoring currents were stable, before and after the application of 10 µM amiloride to the bath. Data were sampled at the rate of 1 kHz and filtered at 500 kHz.

Patch clamp. Oocytes were shrunk in a hypertonic medium, and the vitelline membrane was removed before patch clamping (15). The inside-out configuration was used to record single-channel currents with an Axopatch 1B amplifier (Axon Instruments) (10, 27). The patch pipettes were pulled from fire-polished, filamented borosilicate glass (WPI, Sarasota, FL) using 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 µs. 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). The inside-out patches were formed in low-Ca2+ bath solution. Li+ was substituted with K+ and Na+ for investigating ion selectivity in the bath. Variable testing potentials were applied depending on the prominent internal cations.

Data analysis. All macroscopic currents presented here are amiloride-sensitive currents that were derived by subtracting, using Clampfit, the amiloride-resistant current measured in the presence of 10 µM amiloride from the total current amplitude measured in the absence of the drug. Amiloride-sensitive currents measured between 200 and 400 ms after application of the test clamp were averaged.

The chord conductance (G) carried by Na+, Li+, K+, and NMDG+ for each construct was computed according to Ohm's law as
G=<FR><NU>I<SUB>amil</SUB></NU><DE><IT>E</IT><SUB>test</SUB><IT>−E</IT><SUB>rev</SUB></DE></FR> (1)
where Iamil is the amiloride-sensitive current, Etest represents the test potential, and Erev represents the reversal potential.

To analyze the steady-state transport kinetics for external cations, perfusates containing different concentrations of cations (ranging from 0 to 96 mM) were switched into the chamber. Km and Imax were retrieved by fitting the amiloride-sensitive current curves against the concentration of extracellular cations with the Michaelis-Menten equation
I=I<SUB>max</SUB><IT>·</IT><FR><NU><IT>X</IT></NU><DE>(<IT>K</IT><SUB>m</SUB><IT>+X</IT>)</DE></FR> (2)
where Km is the concentration required for activating half of the maximal current, Imax is the maximal current, I is the measured amiloride-sensitive current, and X represents the extracellular concentration of cation.

The voltage dependencies of the external cation transport kinetics were further analyzed using the formulation of Woodhull (38)
K<SUB>m</SUB>(<IT>E</IT><SUB>test</SUB>)<IT>=K</IT><SUB>m</SUB>(<IT>0</IT>)<IT>·</IT>exp<FENCE><IT>&dgr;·</IT><FR><NU><IT>z·F·E</IT><SUB>test</SUB></NU><DE><IT>R·T</IT></DE></FR></FENCE> (3)
where Km(Etest) is an association constant at a given holding potential (Etest), Km(0) is the association constant at 0 mV, delta  is the fraction of the electric field between the binding site for the blocking cation and the channel surface, z is the valence of the test cation, and, R, T, and F have their usual meanings.

The absolute permeabilities for Na+ (PNa), K+ (PK), and Li+ (PLi) and intraoocyte concentration were retrieved by fitting the macroscopic I-V curves with the Goldman-Hodgkin-Katz equation (4)
<FR><NU>I<SUP><IT>X</IT></SUP><SUB>amil</SUB></NU><DE><IT>A</IT><SUB>cell</SUB></DE></FR><IT>=P<SUB>X</SUB>·z·E</IT><SUB>test</SUB><IT>·F<SUP>2</SUP> </IT><FR><NU>[<IT>X</IT><SUP>+</SUP>]<SUB>out</SUB><IT>−</IT>[<IT>X</IT><SUP>+</SUP>]<SUB>in</SUB><IT>·e</IT><SUP><IT>F·E</IT><SUB>test</SUB><IT>/R·T</IT></SUP></NU><DE><IT>R·T</IT></DE></FR> (4)

<IT>·</IT>(<IT>1−e</IT><SUP><IT>F·E</IT><SUB>test</SUB><IT>/R·T</IT></SUP>)
where R, T, and F have their usual meanings, z is the valence of the cation, I<UP><SUB>amil</SUB><SUP><IT>X</IT></SUP></UP> represents the amiloride-sensitive current carried by the cation X+, PX is the absolute permeability value for the cation X+, and [X+]out and [X+]in are the extra- and intraoocyte concentrations, respectively. Acell is the measured surface area of an oocyte, assuming that the oocyte is a spheroid with a diameter of 1.0 mm. The ratio of permeabilities was calculated by dividing the values of PLi and PK with PNa.

The absolute cation permeability coefficients from single-channel recordings were computed by fitting the bi-ionic I-V curves with the modified Goldman-Hodgkin-Katz current equation
i<SUB>Li</SUB><IT>+i<SUB>X</SUB>=</IT>(<IT>P</IT><SUB>Li</SUB><IT>·</IT>[Li<SUP>+</SUP>]<SUB>pipette</SUB><IT>+P<SUB>X</SUB>·</IT>[<IT>X</IT><SUP>+</SUP>]<SUB>bath</SUB>) (5)

<IT>·</IT><FR><NU><IT>z·E</IT><SUB>test</SUB><IT>·F<SUP>2</SUP></IT></NU><DE><IT>RT</IT></DE></FR><IT>·</IT><FR><NU><IT>e</IT><SUP><IT>E</IT><SUB>test</SUB><IT>·F/R·T</IT></SUP></NU><DE><IT>1−e</IT><SUP><IT>E</IT><SUB>test</SUB><IT>·F/R·T</IT></SUP></DE></FR>
where iLi and iX stand for the unitary current carried by Li+ in the pipette solution ([Li+]pipette) and X+ in the bath solution ([X+]bath), respectively. The corresponding permeabilities were expressed as PLi and PX.

The reversal potentials for both macroscopic and microscopic currents were calculated by applying the retrieved parameters, including the absolute permeabilities and intraoocyte cation concentration, to the following equation (11)
E<SUB>rev</SUB><IT>=</IT><FR><NU><IT>RT</IT></NU><DE><IT>F</IT></DE></FR> ln <FR><NU><IT>P</IT><SUB>A</SUB><IT>·</IT>[<IT>A</IT>]<SUB>out</SUB></NU><DE><IT>P</IT><SUB>B</SUB><IT>·</IT>[<IT>B</IT>]<SUB>in</SUB></DE></FR> (6)
where A and B represent extracellular and intracellular cation concentration, respectively.

The apparent molecular radii of the channel permeability pathway formed by wild-type and double point mutants were compared by fitting the plot of the relative permeabilities to Na+ as a function of the diameter of the tested cations (1)
P<SUB>X</SUB>/P<SUB>Na</SUB><IT>=f·</IT><FENCE><IT>1−</IT><FR><NU><IT>d/2</IT></NU><DE><IT>r</IT></DE></FR></FENCE><SUP><IT>2</IT></SUP> (7)
In this space-filling model, d stands for the Pauling ionic diameter of the alkali metal cations, r is the radius of the cylindrical hole (in Å), and f is the slope of the fitting curve.

Analysis of single-channel data was performed with the Fetchan and pSTAT programs of Clampex 7.0 software (Axon Instruments) and with the program Gauss (3) as previously described (15). The activity coefficients of the cations were taken from standard tables of molal activity coefficients (22) and recalculated into molar activity.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of alpha -hENaC arginine mutants. Figure 1 presents the linear amino acid sequence alignments in the immediate post-M2 region of alpha -, beta -, and gamma -hENaC. This concentration of arginine residues in the alpha -ENaC subunit is highly conserved among the known members of the degenerin/ENaC superfamily but not among the beta - and gamma -ENaC subunits. Because alpha -ENaCs by themselves form bona fide amiloride-sensitive, highly Na+-selective cation channels, we reasoned that the alpha -ENaC subunit is essential in forming the conduction/selective pathway of the heteromeric channel. We employed site-directed mutagenesis to alter two pairs of arginines (R586, R587 and R589, R591) to glutamates (E). Thus these positively charged amino acids were changed to negative residues.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   Amino acid alignment of the post-M2 regions of alpha -, beta -, and gamma -subunits of human epithelial Na+ channels (hENaC) and double point mutants of alpha -subunit. The 4 arginines in alpha -hENaC that were replaced by the negatively charged glutamic acids are highlighted in different colors. Numbers at the left and right ends of each wild-type subunit indicate the cDNA amino acid position. Solid red lines indicate the amino acids contained in the most distal end of the second hydrophobic domain (M2).

Macroscopic currents in Xenopus oocytes. Whole cell amiloride-sensitive Na+ currents after the expression of wild-type alpha beta gamma -hENaC, alpha R586E,R587Ebeta gamma -hENaC, and alpha R589E,R591Ebeta gamma -hENaC in oocytes are shown in Fig. 2. Currents induced by wild-type alpha beta gamma -hENaC were typically -1,300 nA at -100 mV (Fig. 2A). Macroscopic amiloride-sensitive Na+ currents produced by the double alpha -hENaC mutant alpha R586E,R587Ebeta gamma -hENaC were significantly lower than those of the wild-type channel (P < 0.05, Fig. 2B), while the magnitude of the currents induced by the alpha R589E,R591Ebeta gamma -hENaC mutant was 30% higher than that of the wild type (P < 0.05, Fig. 2C). The corresponding chord conductances carried by 96 mM external Na+ at -120 mV were 19.05 ± 0.28 (n = 21), 13.67 ± 1.41 (n = 29), and 25.41 ± 0.9 µS (n = 23) for wild-type, alpha R586E,R587Ebeta gamma -hENaC, and alpha R589E,R591Ebeta gamma -hENaC, respectively. On average, there were significant differences among the amiloride-sensitive currents of all three constructs (Fig. 2D). For all three channels, the amiloride-sensitive Li+ currents were ~1.4-fold greater than the amiloride-sensitive Na+ current.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2.   Macroscopic amiloride-sensitive Na+ and Li+ currents of wild-type alpha beta gamma -hENaC and double point mutants expressed in Xenopus oocytes. Representative whole cell, amiloride-sensitive Na+ currents are presented in oocytes expressing wild-type alpha beta gamma - (A), alpha R586E,R587Ebeta gamma - (B), and alpha R589E,R591Ebeta gamma -hENaC (C). The eggs were voltage clamped from -160 to + 60 mV in 10-mV increments, with each clamp potential maintained for 500 ms. Dotted lines indicate zero current level. D: mean amiloride-sensitive Na+ (INa) and amiloride-sensitive Li+ currents (ILi) of hENaCs at a holding potential of -120 mV, digitized from 100 to 400 ms. The numbers of oocytes used per condition are indicated on each bar graph. AS, amiloride sensitive. *P < 0.05 compared with wild-type hENaC.

We next investigated any potential alteration in cation selectivity or conductance due to these double mutations, specifically because we hypothesized that these positively charged amino acids may constitute part of the inner mouth of the ion conduction pathway. As a prelude, we determined the effects of these mutations on macroscopic currents at reduced [Na+]. I-V curves at different extracellular [Na+] (using NMDG-Cl as replacement) are shown for wild-type alpha beta gamma -hENaC (Fig. 3A) and the two double mutants (Fig. 3, B and C). The reversal potentials shifted leftward for all three constructs when the extracellular cation (Na+ or Li+) concentration was switched from 96 mM to 0 mM (Fig. 4), in good agreement with what is predicated by the Nernst equation for a cation-selective channel. As before, the inward currents for alpha R586E,R587Ebeta gamma -hENaC and alpha R589E,R591Ebeta gamma -hENaC were smaller and greater, respectively, than those for the wild-type channel.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   Current-voltage (I-V) relationships of wild-type alpha beta gamma -hENaC and two double point mutants at different extracellular [Na+]. The Na+ concentrations were adjusted by replacement of equimolar N-methyl-D-glucamine (NMDG). Data are from 6 separate experiments and are presented as means ± SE. The protocol for voltage clamping the oocytes is detailed in METHODS AND MATERIALS and in the legend to Fig. 2.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Measured reversal potential (Erev) from whole cell recordings plotted against external Na+ ([Na+]o) and Li+ concentrations ([Li+]o). A: computed Erev values plotted as a function of [Li+]o. The Erev of alpha beta gamma -, alpha R586E,R587Ebeta gamma -, and alpha R589E,R591Ebeta gamma -hENaC shifted to the right by 48.63 ± 1.54, 50.26 ± 9.37, and 58.47 ± 5.54 mV per decade, respectively. B: Erev plotted as a function of [Na+]o. The Erev of alpha beta gamma -, alpha R586E,R587Ebeta gamma -, and alpha R589E,R591Ebeta gamma -hENaC shifted to the right by 72.65 ± 5.09, 63.85 ± 6.88, and 72.62 ± 6.25 mV per decade, respectively. Linear lines were created by linear regression for alpha beta gamma - (solid), alpha R586E,R587Ebeta gamma - (dashed), and alpha R589E,R591Ebeta gamma -hENaC (dotted).

Several investigators have utilized the Goldman-Hodgkin-Katz current equation to fit amiloride-sensitive Na+ I-V relationships (9, 18, 20, 28, 30). We likewise fitted the I-V data presented in Fig. 3 to this equation to compute permeability coefficients for Na+ (PNa), Li+ (PLi), K+ (PK), and NMDG+ (PNMDG) at different external cation concentrations. Table 1 summarizes the calculated values of PNa, PLi, PK, and PNMDG for wild-type alpha beta gamma -hENaC, alpha R586E,R587Ebeta gamma -hENaC, and alpha R589E,R591Ebeta gamma -hENaC. For the wild-type channel, PLi/PNa was 1.97 and PNa/PK was 51, in good agreement with previously reported values estimated from reversal potential measurements in oocytes (15). PLi/PNa was significantly increased from 1.97 to 22.2 for the double point mutant alpha R586E,R587Ebeta gamma -hENaC and was virtually unchanged for alpha R589E,R591Ebeta gamma -hENaC. However, for alpha R586E,R587Ebeta gamma -hENaC and alpha R589E,R591E beta gamma -hENaC, PNa/PK decreased to 1.8 and 10.0, respectively. Likewise, PLi/PK values for alpha R586E,R587Ebeta gamma -hENaC and alpha R589E,R591Ebeta gamma -hENaC were lower (41 and 14.9, respectively) than that for the wild-type channel (100.5) (Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   PNa, PLi, PK, and PNMDG as determined from macroscopic, amiloride-sensitive currents of hENaC expressed in oocytes

The effects of variations in the [Na+] of the oocyte bathing solution on amiloride-sensitive Na+ current at 0-mV clamp potential for all three constructs are shown in Fig. 5A. The data were recast as Eadie plots (Fig. 5B) to allow calculation of the kinetic parameters Km and Imax. The apparent equilibrium dissociation constants (Km) for Na+ were 32.6 ± 2.0 (n = 20), 16.3 ± 4.4 (n = 20), and 15.3 ± 3.1 mM (n = 20) for wild-type, alpha R586E,R587Ebeta gamma -hENaC, and alpha R589E,R591Ebeta gamma -hENaC, respectively. The normalized maximal current at saturating [Na+] (i.e., INa) was different for alpha R586E,R587Ebeta gamma -hENaC and alpha R589E,R591Ebeta gamma -hENaC compared with wild type (497 ± 36 and 1,422 ± 80 nA, respectively, vs. 986 ± 12 nA for the wild-type channel). These results indicate that pronounced change in the steady-state kinetic parameters of hENaC occurs with these post-M2 double arginine mutations in the alpha -subunit.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Kinetic analyses of extracellular Na+ transport by hENaCs expressed in oocytes. A: normalized macroscopic amiloride-sensitive Na+ currents at a holding potential of 0 mV at 0-96 mM Na+ in the bath solution ([Na+]o). Data were fitted with the Michaelis-Menten equation for wild-type alpha beta gamma - (solid line) alpha R586E,R587Ebeta gamma - (dotted line), and alpha R589E,R591Ebeta gamma -hENaC (dashed line). B: Eadie plot for recalculation of kinetic parameters.

Because these analyses were done at a voltage-clamp potential of 0 mV, we repeated the measurements of Km and Imax at different voltage-clamp potentials using both Na+ and Li+ as charge carriers to examine any voltage dependence of these parameters (Fig. 6). Figure 6, B and D, presents the Km and Imax for Na+ for all three constructs expressed in oocytes as a function of holding potential. The Km for all constructs was relatively insensitive to membrane voltage in the range from -160 to -50 mV. However, at more depolarizing potentials there was a steep increase in Km (a decrease in affinity) with no tendency toward saturation, at least to +50 mV. The increase in Km with voltage was nearly twice as great for the wild-type channel compared with the two mutant constructs, i.e., there was a 1.96, 1.23, and 1.00 mM change in Km per e-fold change in potential for the wild type, alpha R586E,R587Ebeta gamma -hENaC, and alpha R589E,R591Ebeta gamma -hENaC, respectively, in the voltage range from 0 to +50 mV. Essentially similar results were found when Li+ was the major extracellular cation (Fig. 6, A and C). It should be noted that values from voltages more negative than -30 mV are due primarily to influx of Na+ or Li+ and that values from voltages more positive than +40 mV are due primarily to efflux of Na+ or Li+. However, at all holding potentials and for all three constructs, both the Km and Imax for Li+ were greater than those for Na+.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6.   Voltage dependence of the apparent equilibrium dissociation constants (Km) and the maximal current for Na+ and Li+ transport through hENaC. Data for Km, computed by the Michaelis-Menten formula for Li+ (A) and Na+ (B), are plotted as a function of the membrane potential (V) (-160 to + 60 mV). C: maximal ILi-V curves for wild-type alpha beta gamma -, alpha R586E,R587Ebeta gamma -, and alpha R589E,R591Ebeta gamma -hENaC carried by external Li+. D: maximal INa-V curves for wild-type alpha beta gamma -, alpha R586E,R587Ebeta gamma -, and alpha R589E,R591Ebeta gamma -hENaC carried by external Na+.

To analyze the voltage dependence of the kinetics of external cation binding, we fit the data shown in Fig. 6, A and B, with the Woodhull equation. Identical to the measured macroscopic currents, the calculated maximal amiloride-sensitive Li+ currents (I<UP><SUB>max</SUB><SUP>Li</SUP></UP>) in Fig. 6C associated with alpha R586E,R587Ebeta gamma -hENaC and alpha R589E,R591Ebeta gamma -hENaC were less than and greater than wild type, respectively. Similar changes in the maximal amiloride-sensitive Na+ currents (I<UP><SUB>max</SUB><SUP>Na</SUP></UP>) were also found for these three constructs (Fig. 6D). The retrieved parameters, including the fractional electrical distance from the outside (delta Na for Na+ and delta Li for Li+) and the values of Km at the membrane potential of 0 mV [K<UP><SUB>m</SUB><SUP>Na</SUP></UP>(0) and K<UP><SUB>m</SUB><SUP>Li</SUP></UP>(0)] are summarized in Table 2. Consistent with the steepness of Km shown in Fig. 6, A and B, the electrical distance (delta ) of the binding site for Na+ from the outside was the same for both double point mutants and no different from that of the wild-type channel, but the delta  for Li+ was significantly reduced for both double point mutants. In other words, the double arginine mutations in the alpha -subunit effectively lengthened the fractional electrical distance from the outermost binding site [assuming Km is primarily determined by interaction of external Na+ (or Li+) with this site] to the inner mouth of the pore (1 - delta ), at least for Li+. Our results support the idea that the ENaC contains at least two interaction sites for cations: the first one located near the outer or external entrance of the pore, and the second one closer to the cytosolic face of the channel.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Parameters retrieved from the fitting of voltage-dependence of Km with the Woodhull function

Confocal imaging. The most parsimonious explanation for the observation that the Imax of the alpha R589E,R591Ebeta gamma -hENaC construct was higher, and the Imax of the alpha R586E,R587Ebeta gamma -hENaC construct was lower, than the Imax for the wild-type channel (Fig. 2D and Fig. 6, C and D) is that there was a variation in the number of functional channels at the surface of the membrane. To test this hypothesis, we visualized the cellular localization of the ENaC constructs by using GFP and confocal laser microscopy. As a control for background fluorescence in oocytes (data not shown), some were injected with 50 nl of water, surface-labeled with the Texas red conjugate (as described in MATERIALS AND METHODS), and injected at midsection. The biotinylation protocol labeled proteins in the plasma membrane of the eggs with Texas red. As shown in Fig. 7, the membrane of the cells was clearly defined by a distinct banding of Texas red fluorescence. The compact band of GFP fluorescence (Fig. 7, middle) in the hENaC-injected eggs, represents the localization of the ENaC protein. For all water-injected oocytes, there was no GFP fluorescence (21) (data not shown). Figure 7, right, is an overlay of the Texas red and GFP images and shows colocalization of the GFP and Texas red fluorescence, which appears as yellow fluorescence. When the wild-type yellow fluorescence is normalized to 100, the fluorescence of the alpha R589E,R591Ebeta gamma -hENaC-injected oocytes averages 157 ± 10 (±SE; n = 10), a value significantly different from that of the wild type (P < 0.05). These results confirm that the degree of yellow fluorescence is much greater in the alpha R589E,R591Ebeta gamma -hENaC-injected oocytes than in either the wild type or alpha R586E,R587Ebeta gamma -hENaC-injected oocytes. In contrast, the fluorescence of the alpha R586E,R587Ebeta gamma -hENaC-injected oocytes was not significantly lower than that of the wild type (102 ± 5, ±SE; n = 10; P > 0.1).


View larger version (120K):
[in this window]
[in a new window]
 
Fig. 7.   Laser scanning confocal images of Xenopus oocytes expressing green fluorescent protein (GFP)-labeled hENaC. Each set of 3 images (A-C) depicts the same laser scanning section of the identical oocyte: left, oocyte surface delineated by Texas red fluorescence; middle, distribution and channel density of hENaCs tagged with enhanced GFP (EGFP)-ENaC fluorescence; right, overlay of first 2 images. The colocalization of red and green fluorescence (shown as yellow at right) indicates the relative density of channel proteins at the plasma membrane. WT, wild type. Scale bar, 40 µm. Results are representative of 10 identical experiments.

Single-channel measurements. Single-channel current measurements were made for all three constructs to determine the effects of these double mutations on the single-channel conductance and selectivity properties and compare them with those deduced from the macroscopic measurements. Figure 8 presents inside-out single-channel records for each construct at -60 mV in symmetrical salt solutions (Li+). All patches contained multiple channels, but the probability of finding multiple channels was much greater for the alpha R589E,R591Ebeta gamma -hENaC construct than for the other two (see Table 3). Their associated I-V curves are presented in Fig. 9. The single-channel Li+ conductance in symmetrical 100 mM LiCl for wild-type alpha beta gamma -hENaC was not significantly different for either alpha R586E,R587Ebeta gamma -hENaC or alpha R589E,R591Ebeta gamma -hENaC (Table 3). However, calculated values of single-channel open probability (Po) revealed significant differences between the wild-type and mutant channels (Table 3).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8.   Single-channel traces of hENaCs in inside-out patches excised from oocytes. Oocytes expressing wild-type alpha beta gamma - (top) alpha R586E,R587Ebeta gamma - (middle), and alpha R589E,R591Ebeta gamma -hENaC (bottom) 48 h after cRNA injection were used for patch clamping. Symmetrical Li+ (100 mM) was in the pipette and bath solutions. Dotted lines represent the zero current level. The holding potential was -60 mV.


                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Single-channel characteristics of hENaCs



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 9.   Single-channel unitary current (i)-voltage (i-V) curves obtained under bi-ionic conditions. i-V curves are plotted from inside-out patches containing wild-type alpha beta gamma - (A), alpha R586E,R587Ebeta gamma - (B), and alpha R589E,R591Ebeta gamma -hENaC (C) channels. Li+ (100 mM) was in the pipette solution, and equimolar Li+ (), Na+ (), and K+ (black-triangle) were in the bath solution. The test potentials for measuring the current carried by Li+ were +100, +80, +60, +40, -40, -60, -80, and -100 mV. For K+, the voltage potential of the pipette ranged from +40 to -100 mV in 20-mV increments. Vm, membrane potential. Data were fit using Eq. 5.

To determine the channel permeability characteristics from the single-channel recordings, bi-ionic potential measurements (K+, Na+, and Li+) were made using inside-out patches of membrane from oocytes expressing these hENaC constructs. The reversal potential of the three constructs measured with symmetrical Li+ solutions was approximately 0 mV. The reversal potential shifted to around +20 mV for wild type and alpha R589E,R591Ebeta gamma -hENaC and to +60 mV for alpha R586E,R587Ebeta gamma -hENaC when the bath solution was replaced with equimolar Na+ and to more than +100 mV when equimolar K+ was substituted for the Li+. The absolute permeability coefficients and the corresponding permeability ratios (PLi/PNa and PK/PNa) retrieved by fitting the bi-ionic I-V curves (Fig. 9) with the Goldman-Hodgkin-Katz current equation were essentially identical to the values derived from the aforementioned macroscopic results presented in Table 1 (see Table 4 for comparisons). For example, from the single-channel experiments, the absolute permeability coefficients of PNa 10-6 cm/s) were 1.21, 0.08, and 1.44 for wild-type alpha beta gamma -, alpha R586E,R587Ebeta gamma -, and alpha R589E,R591Ebeta gamma -hENaC, respectively. Also, excellent agreement was achieved in computing permeability ratios from either macroscopic or single-channel curve fitting procedures or from measuring single-channel reversal potentials under bi-ionic conditions (see Table 4).

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Comparison of permeability ratios retrieved from the fitting of macroscopic I-V curves and unitary i-V curve and calculated from the reversal potentials of the i-V curves

Figure 10 presents the ratio of the permeability of Li+ and K+ relative to Na+ as a function of the Pauling crystal diameter of each of these three alkali metal cations. The PNa/PK permeability ratios appeared to be constant in both mutant constructs. However, the PLi/PK permeability ratio was higher for the alpha R586E,R587Ebeta gamma -hENaC than for either the wild type or the alpha R589E,R591Ebeta gamma -hENaC. From these data, the radius of the conduction pathway can be calculated to be 1.53 Å for the wild-type alpha beta gamma -hENaC, 1.17 Å for the alpha R586E,R587Ebeta gamma -hENaC, and 1.83 Å for the alpha R589E,R591Ebeta gamma -hENaC (Table 3). The conduction pathway radius for wild-type hENaC compares well with that for wild-type rENaC derived by Kellenberger et al. (17) by using a similar analysis after recalculating the data of the authors. We conclude that these double point mutations of arginine in the post-M2 region of alpha -hENaC have differential effects on pore size.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 10.   Mean molecular diameters of the hENaC channel pore estimated from space-filling models. A: apparent diameters (in Å) of the channel pore formed by wild-type alpha beta gamma - (solid line), alpha R586E,R587Ebeta gamma - (dashed line), and alpha R589E,R591Ebeta gamma -hENaC (dotted line) were calculated by plotting the normalized permeability (PX/PNa) of Li+, Na+, and K+ against the crystal diameter of Li+, Na+, and K+, where X represents the test cation. Smooth lines were obtained by fitting the data to Eq. 7. The deduced pore diameters of wild-type alpha beta gamma -, alpha R586E,R587Ebeta gamma -, and alpha R589E,R591Ebeta gamma -hENaC are 1.53, 1.17, and 1.83 Å, respectively. B: data are linearly replotted [PX/PNa]1/2 as a function of the diameter of cations. The y-axis interpolations are the slope, and the x-axis interpolations are the diameter of the hENaC channel pore.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this paper, we present comparative data regarding the functional effects of two double point mutations in the alpha -subunit of hENaC. These mutations were generated in the post-M2 region at the beginning of the cytoplasmic COOH-terminal tail. We show significant differences in a number of steady-state kinetic and biophysical properties for these double point mutants compared with the wild-type ENaC. Altered properties include macroscopic current, Po, Km and Imax for Na+, and ion selectivity. Neither pair of mutations changed the single-channel conductance, but the whole cell chord conductance of both double point mutants was modified. Using molecular sieving theory, we calculated the apparent radius of the conduction pathway. Our calculations show that these two double point mutation constructs have pore radii different from that of the wild-type channel. These data indicate that the double point mutations in the post-M2 region have significant effects on ion transport through hENaC, indicating that these residues or amino acids may form part of the conduction/selectivity pathway itself as well as affect the normal balance of insertion and/or retrieval mechanisms that exist to set a given density of Na+ channels in the plasma membrane.

A literature comparison of the permeability coefficients for Na+ in different transport systems determined from fitting I-V curves with the Goldman-Hodgkin-Katz equation reveals that the values of PNa for alpha beta gamma -ENaC in oocytes (1 × 10-6 cm/s) is comparable to that calculated by Palmer (0.9 × 10-6 cm/s) (28, 29) in toad urinary bladder but is one order of magnitude smaller that that for native frog skin epithelium (9). These results would suggest that alpha beta gamma -ENaC is the predominant permeability pathway for Na+ in urinary bladder. Because nearly all of the Na+ influx across frog skin is amiloride inhibitable (18) and presumably also occurs through ENaC, the comparatively high PNa may indicate a different ENaC subunit composition or a different regulatory state of the channel when those measurements were made. This idea is supported by the observation that alpha -ENaC, devoid of the beta - and gamma -subunits, displays a PNa much lower than that of alpha beta gamma -ENaC. Interestingly, respiratory tract ENaC mRNA analysis shows a pronounced decreased or even lack of the beta -ENaC subunit (36), consistent with the lower PNa value determined by Kroll et al. (20) in their studies.

Recent studies have employed site-directed mutagenesis to examine structure-function relationships of ENaC channels (16, 17, 21, 31, 33, 35). Most of these studies have examined the amino acid residues in the hydrophobic region preceding the M2 domain as well as the proximal M2 region of ENaC. In general, these studies have utilized methanethiosulfonate (MTS compounds) in combination with cysteine-scanning mutagenesis to examine the external accessibility of these MTS reagents to various sites within ENaC. Mutagenesis studies in the three subunits (alpha , beta , and gamma ) of ENaC revealed specific changes in cation selectivity, single-channel conductance, or amiloride sensitivity (16, 17, 21, 31, 33, 35), implicating all three subunits in pore formation. Despite these observations, both Snyder et al. (35) and Kellenberger et al. (16, 17) favor the idea that the pore structure of ENaC is distinct from that of KcsA, a bacterial K+ channel whose structure has been resolved by X-ray crystallography and whose pore is formed by the symmetrical arrangement of four identical subunits (2, 7). Conversely, Sheng et al. (33) argue for more similarity between the structures of ENaC and KcsA.

The new information that has been provided by the present work is that mutations made within an arginine-rich region, presumably located at the cytoplasmic face of the channel, affect ion selectivity but not single-channel conductance. These observations suggest that this region contributes to the selectivity pathway of the pore. However, the relative permeabilities of Na+ vs. K+ and Na+ vs. Li+ were not altered by R589E and R591E mutations. These results suggest that there may be a second selectivity region within the pore that an alkali metal cation, after passing the primary, more extracellularly located selectivity filter, must encounter before its exit from the membrane. This would be consistent with the conclusions previously made suggesting that ENaC is a multi-ion pore (12, 28, 33). It is also possible that lysines and arginines in the post-M2 region of the beta - and gamma -hENaC subunits contribute in a similar fashion to ionic selectivity. An additional possibility is that an extracellular cation binding site also influences selectivity.

Our data suggest that ion selectivity does not reside at a single site but that multiple sites contribute to ion discrimination. Thus the primary selectivity filter per se would consist of an extended number of residues encompassing both the pre-H2, H2, and proximal portion of the M2 domains, perhaps 28-30 amino acids in length, followed by the membrane-spanning M2 domain. If this membrane-spanning M2 region is inwardly tilted with respect to the normal plane of the membrane (as is the case for the KcsA channel), then a constriction in the more distal (i.e., cytoplasmic) part of the conduction pathway would occur, thereby accounting for the effects on ion selectivity produced by mutations in the arginine-rich region located at this point. This arrangement is plausible on the basis of calculations from molecular sieving theory showing that the double arginine mutations can influence the effective radii of the pore, presumably by conformational changes.

A series of elegant papers from the Jan laboratory has implicated a novel endoplasmic reticulum (ER) retention motif (R-X-R) that is hidden in properly assembled oligomeric ion channel complexes (32, 39). This motif is highly conserved in cystic fibrosis transmembrane regulator (6) and in the sulfonylurea receptor (24). This motif is also present in each subunit of ENaC, with the alpha -subunit containing at least four, one of which is R589-S-R591. The increased macroscopic amiloride-sensitive current and increased surface expression of alpha R589E,R591Ebeta gamma -hENaC in oocytes (but not alpha R586E,R587Ebeta gamma -hENaC) is consistent with the interpretation of the idea that mutations of arginine residues in such a motif increase ER export and surface delivery of ENaC.

In summary, our results suggest that the selectivity filter (at least that part contributed by the alpha -subunit of ENaC) is formed by an extended region encompassing amino acids in the pre-H2, H2, M2, and post-M2 domains. Our data further suggest that distal to a primary selectivity filter, another domain exists where discrimination between alkali metal cations can also occur. This is consistent with our previous hypothesis that the conduction pathway of ENaC has at least two barriers or interaction sites that a cation must encounter before its exit into the cytoplasmic compartment (12).


    ACKNOWLEDGEMENTS

We thank Jason Lockhart (Physiology, University of Alabama at Birmingham) and Eddie Walthall (Cystic Fibrosis Center, Univ. of Alabama at Birmingham) for technical support. We thank Cathy Guy and Isabel Quinones for providing excellent administrative support.


    FOOTNOTES

The present study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-37206 and DK-56095.

Present address of A. L. B. Langloh: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185.

Address for reprint requests and other correspondence: D. J. Benos, Dept. of Physiology and Biophysics, The Univ. of Alabama at Birmingham, 1918 Univ. Blvd., MCLM 704, Birmingham, AL 35294-0005 (E-mail: benos{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 2 November 2000; accepted in final form 5 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adams, DJ, Dwyer TM, and Hille B. The permeability of endplate channels to monovalent and divalent metal cations. J Gen Physiol 75: 493-510, 1985[Abstract].

2.   Aqvist, J, and Luzhkov V. Ion permeation mechanism of the potassium channel. Nature 404: 881-884, 2000[ISI][Medline].

3.   Bauer, RJ, Carl A, Kapicka CL, and Kenyon JL. Determination of channel open probabilities from multichannel data. J Neurosci Methods 68: 101-111, 1996[ISI][Medline].

4.   Bowman, CL. Application of the Goldman-Hodgkin-Katz current equation to membrane current-voltage data. J Theor Biol 108: 1-29, 1984[ISI][Medline].

5.   Canessa, CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, and Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 467-467, 1994[ISI][Medline].

6.   Chang, XB, Cui L, Hou YX, Jensen TJ, Aleksandrov AA, Mengos A, and Riordan JR. Removal of multiple arginine-framed trafficking signals overcomes misprocessing of Delta F508 CFTR present in most patients with cystic fibrosis. Mol Cell 4: 137-142, 1999[ISI][Medline].

7.   Doyle, DA, Morais CJ, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, and MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280: 69-77, 1998[Abstract/Free Full Text].

8.   Eskandari, S, Snyder PM, Kreman M, Zampighi GA, Welsh MJ, and Wright EM. Number of subunits comprising the epithelial sodium channel. J Biol Chem 274: 27281-27286, 1999[Abstract/Free Full Text].

9.   Fuchs, W, Larsen EH, and Lindemann B. Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin. J Physiol 267: 137-166, 1977[ISI][Medline].

10.   Hamill, OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 1: 85-100, 1981.

11.   Hille, B. Ionic Channels of Excitable Membranes (2nd ed.). Sunderland, MA: Sinauer, 1992, p. 337-361.

12.   Ismailov, II, Shlyonsky V, Gh., Alvarez O, and Benos DJ. Cation permeability of a cloned rat epithelial amiloride-sensitive Na+ channel. J Physiol (Lond) 504: 287-300, 1997[Abstract].

13.   Ji, HL, Chalfant ML, Jovov B, Lockhart JP, Parker SB, Fuller CM, Stanton BA, and Benos DJ. The cytosolic termini of the beta - and gamma -ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel. J Biol Chem 275: 27947-27956, 2000[Abstract/Free Full Text].

14.   Ji, HL, Fuller CM, and Benos DJ. Osmotic pressure regulates alpha beta gamma -rENaC expressed in Xenopus oocytes. Am J Physiol Cell Physiol 275: C1182-C1190, 1998[Abstract/Free Full Text].

15.   Ji, HL, Fuller CM, and Benos DJ. Peptide inhibition of constitutively activated epithelial Na+ channels expressed in Xenopus oocytes. J Biol Chem 274: 37693-37704, 1999[Abstract/Free Full Text].

16.   Kellenberger, S, Gautschi I, and Schild L. 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, 1999[Abstract/Free Full Text].

17.   Kellenberger, S, Hoffmann-Pochon N, Gautschi I, Schneeberger E, and Schild L. On the molecular basis of ion permeation in the epithelial Na+ channel. J Gen Physiol 114: 13-30, 1999[Abstract/Free Full Text].

18.   Kizer, N, Guo XL, and Hruska K. Reconstitution of stretch-activated cation channels by expression of the alpha -subunit of epithelial sodium channel cloned from osteoblasts. Proc Natl Acad Sci USA 94: 1013-1018, 1997[Abstract/Free Full Text].

19.   Kosari, F, Sheng S, Li J, Mak D-O, Foskett JK, and Kleyman TR. Subunit stoichiometry of the epithelial sodium channel. J Biol Chem 273: 3469-3474, 1998.

20.   Kroll, B, Bremer S, Tummler B, Kottra G, and Fromter E. Sodium dependence of the epithelial sodium conductance expressed in Xenopus laevis oocytes. Pflügers Arch 419: 101-117, 1991[ISI][Medline].

21.   Langloh, AL, Berdiev B, Ji HL, Keyser K, Stanton BA, and Benos DJ. Charged residues in the M2 region of alpha -hENaC play a role in channel conductance. Am J Physiol Cell Physiol 278: C277-C291, 2000[Abstract/Free Full Text].

22.   Lide, DR. Handbook of Chemistry and Physics (80th ed.). Cleveland, OH: CRC, 2000, p. 5/94-5/105.

23.   Lingueglia, E, Champigny G, Lazdunski M, and Barbry P. Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel. Nature 378: 730-733, 1995[ISI][Medline].

24.   Margeta-Mitrovic, M, Jan YN, and Jan LY. A trafficking checkpoint controls GABAB receptor heterodimerization. Neuron 27: 97-106, 2000[ISI][Medline].

25.   McDonald, FJ, Price MP, Snyder PM, and Welsh MJ. Cloning and expression of the beta - and gamma -subunits of the human epithelial sodium channel. Am J Physiol Cell Physiol 268: C1157-C1163, 1995[Abstract/Free Full Text].

26.   McDonald, FJ, Snyder PM, McCray PB, Jr, and Welsh MJ. Cloning, expression, and tissue distribution of a human amiloride-sensitive Na+ channel. Am J Physiol Lung Cell Mol Physiol 266: L728-L734, 1994[Abstract/Free Full Text].

27.   Methfessel, C, Witzemann V, Takahashi T, Mishina M, Numa S, and Sakmann B. Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels. Pflügers Arch 407: 577-588, 1986[ISI][Medline].

28.   Palmer, LG. Ion selectivity of epithelial Na+ channels. J Membr Biol 96: 97-106, 1987[ISI][Medline].

29.   Palmer, LG, Edelman IS, and Lindemann B. Current-voltage analysis of apical sodium transport in toad urinary bladder: effects of inhibitors transport and metabolism. J Membr Biol 57: 59-71, 1980[ISI][Medline].

30.   Palmer, LG, and Frindt G. Conductance and gating of epithelial Na+ channels from rat cortical collecting tubule. Effects of luminal Na+ and Li+. J Gen Physiol 92: 121-138, 1988[Abstract].

31.   Schild, L, Schneeberger E, Gautschi I, and Firsov D. Identification of amino acid residues in the alpha, beta, and gamma subunits of the sodium channel (ENaC) involved in amiloride block and ion permeation. J Gen Physiol 109: 15-26, 1997[Abstract/Free Full Text].

32.   Schwappach, B, Zerangue N, Jan YN, and Jan LY. Molecular basis for KATP assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter. Neuron 26: 155-167, 2000[ISI][Medline].

33.   Sheng, S, Li J, McNulty KA, Avery D, and Kleyman TR. Characterization of the selectivity filter of the epithelial sodium channel. J Biol Chem 275: 8572-8581, 2000[Abstract/Free Full Text].

34.   Snyder, PM, Cheng C, Prince LS, Rogers JC, and Welsh MJ. Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits. J Biol Chem 273: 681-684, 1998[Abstract/Free Full Text].

35.   Snyder, PM, Olson DR, and Bucher DB. A pore segment in DEG/ENaC Na+ channels. J Biol Chem 274: 28484-28490, 1999[Abstract/Free Full Text].

36.   Talbot, CR. Amiloride-sensitive sodium channels physiology and functional diversity. In: Current Topics in Membranes. Amiloride-Sensitive Sodium Channels: Physiology and Functional Diversity, edited by Benos DJ.. San Diego, CA: Harcourt, 1999, vol. 47, p. 197-217.

37.   Waldmann, R, Champigny G, and Lazdunski M. Functional degenerin-containing chimeras identify residues essential for amiloride-sensitive Na+ channel function. J Biol Chem 270: 11735-11737, 1995[Abstract/Free Full Text].

38.   Woodhull, AM. Ionic blockage of sodium channels in nerve. J Gen Physiol 61: 687-708, 1973[Abstract/Free Full Text].

39.   Zerangue, N, Schwappach B, Jan YN, and Jan LY. A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane KATP channels. Neuron 22: 537-548, 1999[ISI][Medline].


Am J Physiol Cell Physiol 281(1):C64-C74
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society