Point mutations in the post-M2 region of human
-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 |
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
-ENaC
(
-hENaC) led to a decrease and increase in the macroscopic
conductance of
R586E,R587E
- and
R589E,R591E
-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
R586E,R587E
- and
R589E,R591E
-hENaC, and the maximum
amiloride-sensitive Na+ current was decreased for
R586E,R587E
-hENaC and increased for
R589E,R591E
-hENaC. The relative permeabilities of
Li+ and K+ vs. Na+ were increased
11.25- to 27.57-fold for
R586E,R587E
-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 |
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:
,
, and
(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
-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
-helix, as
predicted by
-helical wheel analysis (21). In
-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
-hENaC that is identical in
the five mammalian
-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
-ENaC homolog
(RRLRRAWFSWPR; Ref. 37) but is not present in any of the
- or
-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
-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 

-hENaC heterologously expressed in
Xenopus oocytes using double point mutants. Double point
mutations of arginine residues in the post-M2 region of
-hENaC
resulted in an increase in the
R589E,R591E
-hENaC- and a decrease in the
R586E,R587E
-hENaC-associated chord conductance but
left the single-channel Li+ conductance unaltered. However,
both the apparent equilibrium dissociation constant for Na+
(K
) and the maximal amiloride-sensitive Na+ current (I
) 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
R586E,R587E
-hENaC. Radii of channel pore
analyses revealed that the diameter of the
R586E,R587E
-hENaC channel is smaller, and the
diameter of the
R589E,R591E
-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 |
Construction of site-directed point mutations.
Full-length 

-hENaC cDNAs were a gift of Dr. Michael J. Welsh
(University of Iowa) (25, 26). Single point mutations in
the
-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
-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
-,
-, and
-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
-hENaC plus green fluorescent protein (GFP)-tagged
- and
-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 M
. 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 M
when filled with 100 mM
LiCl, 10 mM HEPES, and 2 mM CaCl2 (at pH 7.4). Currents
were collected using the Clampex 7.0 feature of pCLAMP at a sampling interval of 500 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
|
(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
|
(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)
|
(3)
|
where Km(Etest) is
an association constant at a given holding potential
(Etest),
Km(0) is the association constant
at 0 mV,
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)
|
(4)
|
where R, T, and F have their
usual meanings, z is the valence of the cation,
I
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
|
(5)
|
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)
|
(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)
|
(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 |
Generation of
-hENaC arginine mutants.
Figure 1 presents the linear amino acid
sequence alignments in the immediate post-M2 region of
-,
-, and
-hENaC. This concentration of arginine residues in the
-ENaC
subunit is highly conserved among the known members of the
degenerin/ENaC superfamily but not among the
- and
-ENaC
subunits. Because
-ENaCs by themselves form bona fide
amiloride-sensitive, highly Na+-selective cation channels,
we reasoned that the
-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.

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Fig. 1.
Amino acid alignment of the
post-M2 regions of -, -, and -subunits of human epithelial
Na+ channels (hENaC) and double point mutants of
-subunit. The 4 arginines in -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 

-hENaC,
R586E,R587E
-hENaC, and
R589E,R591E
-hENaC in oocytes are shown in Fig.
2. Currents induced by wild-type 

-hENaC were typically
1,300 nA at
100 mV (Fig.
2A). Macroscopic amiloride-sensitive Na+
currents produced by the double
-hENaC mutant
R586E,R587E
-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
R589E,R591E
-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,
R586E,R587E
-hENaC, and
R589E,R591E
-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.

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Fig. 2.
Macroscopic amiloride-sensitive Na+ and Li+
currents of wild-type   -hENaC and double point mutants
expressed in Xenopus oocytes. Representative whole cell,
amiloride-sensitive Na+ currents are presented in oocytes
expressing wild-type   - (A),
R586E,R587E - (B), and
R589E,R591E -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 

-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
R586E,R587E
-hENaC and
R589E,R591E
-hENaC were smaller and greater,
respectively, than those for the wild-type channel.

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Fig. 3.
Current-voltage (I-V) relationships of
wild-type   -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.
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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
  -, R586E,R587E -, and
R589E,R591E -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   -,
R586E,R587E -, and
R589E,R591E -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   - (solid), R586E,R587E - (dashed), and
R589E,R591E -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 

-hENaC,
R586E,R587E
-hENaC, and
R589E,R591E
-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
R586E,R587E
-hENaC and was virtually unchanged for
R589E,R591E
-hENaC. However, for
R586E,R587E
-hENaC and
R589E,R591E 
-hENaC,
PNa/PK decreased to 1.8 and 10.0, respectively. Likewise,
PLi/PK values for
R586E,R587E
-hENaC and
R589E,R591E
-hENaC were lower (41 and 14.9, respectively) than that for the wild-type channel (100.5) (Table 1).
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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,
R586E,R587E
-hENaC, and
R589E,R591E
-hENaC, respectively. The normalized
maximal current at saturating [Na+] (i.e.,
INa) was different for
R586E,R587E
-hENaC and
R589E,R591E
-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
-subunit.

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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   - (solid line)
R586E,R587E - (dotted line), and
R589E,R591E -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,
R586E,R587E
-hENaC, and
R589E,R591E
-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+.

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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
  -, R586E,R587E -, and
R589E,R591E -hENaC carried by external
Li+. D: maximal
INa-V curves for wild-type
  -, R586E,R587E -, and
R589E,R591E -hENaC carried by external
Na+.
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|
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
) in Fig.
6C associated with
R586E,R587E
-hENaC and
R589E,R591E
-hENaC were less than and greater
than wild type, respectively. Similar changes in the maximal amiloride-sensitive Na+ currents
(I
) were also found for these three
constructs (Fig. 6D). The retrieved parameters, including the fractional electrical distance from the outside
(
Na for Na+ and
Li
for Li+) and the values of Km at the
membrane potential of 0 mV
[K
(0) and
K
(0)] are summarized in
Table 2. Consistent with the steepness of
Km shown in Fig. 6, A and
B, the electrical distance (
) 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
for Li+ was significantly reduced for both double point
mutants. In other words, the double arginine mutations in the
-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
), 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.
Confocal imaging.
The most parsimonious explanation for the observation that
the Imax of the
R589E,R591E
-hENaC construct was higher, and the
Imax of the
R586E,R587E
-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
R589E,R591E
-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
R589E,R591E
-hENaC-injected oocytes than in
either the wild type or
R586E,R587E
-hENaC-injected
oocytes. In contrast, the fluorescence of the
R586E,R587E
-hENaC-injected oocytes was not
significantly lower than that of the wild type (102 ± 5, ±SE;
n = 10; P > 0.1).

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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
R589E,R591E
-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 

-hENaC was not significantly different for
either
R586E,R587E
-hENaC or
R589E,R591E
-hENaC (Table 3). However, calculated
values of single-channel open probability (Po)
revealed significant differences between the wild-type and mutant
channels (Table 3).

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Fig. 8.
Single-channel traces of hENaCs in inside-out patches
excised from oocytes. Oocytes expressing wild-type   -
(top) R586E,R587E - (middle),
and R589E,R591E -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.
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|

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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   - (A), R586E,R587E -
(B), and R589E,R591E -hENaC
(C) channels. Li+ (100 mM) was in the pipette
solution, and equimolar Li+ ( ),
Na+ ( ), and K+
( ) 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
R589E,R591E
-hENaC and to +60 mV for
R586E,R587E
-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


-,
R586E,R587E
-, and
R589E,R591E
-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).
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|
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
R586E,R587E
-hENaC than for
either the wild type or the
R589E,R591E
-hENaC.
From these data, the radius of the conduction pathway can be calculated
to be 1.53 Å for the wild-type 

-hENaC, 1.17 Å for the
R586E,R587E
-hENaC, and 1.83 Å for the
R589E,R591E
-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
-hENaC have differential effects on pore size.

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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   - (solid
line), R586E,R587E - (dashed line), and
R589E,R591E -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   -,
R586E,R587E -, and
R589E,R591E -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 |
In this paper, we present comparative data regarding the
functional effects of two double point mutations in the
-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 

-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 

-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
-ENaC, devoid of the
- and
-subunits,
displays a PNa much lower than that of


-ENaC. Interestingly, respiratory tract ENaC mRNA analysis
shows a pronounced decreased or even lack of the
-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 (
,
, and
) 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
- and
-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
-subunit containing at least four, one of which is
R589-S-R591. The increased macroscopic
amiloride-sensitive current and increased surface expression of
R589E,R591E
-hENaC in oocytes (but not
R586E,R587E
-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
-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.
 |
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