The Role of Pre-H2 Domains of �- and �-Epithelial Na� Channels inIon Permeation, Conductance, and Amiloride Sensitivity*

Received for publication, November 3, 2003, and in revised form, December 1, 2003Published, JBC Papers in Press, December 2, 2003, DOI 10.1074/jbc.M312012200

Hong-Long Ji‡, LaToya R. Bishop§, Susan J. Anderson, Catherine M. Fuller, and Dale J. Benos

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

Epithelial Na� channels (ENaC) regulate salt and wa-ter re-absorption across the apical membrane of absorp-tive epithelia such as the kidney, colon, and lung. Struc-ture-function studies have suggested that the secondtransmembrane domain (M2) and the adjacent pre- andpost-M2 regions are involved in channel pore formation,cation selectivity, and amiloride sensitivity. BecauseNa� selectivity, unitary Na� conductance (�Na), andamiloride sensitivity of �-ENaC are strikingly differentfrom those of �-ENaC, the hypothesis that the pre-H2domain may contribute to these characterizations hasbeen examined by swapping the pre-H2, H2, and both(pre-H2�H2) domains of �- and �-ENaCs. Whole-cell andsingle channel results showed that the permeation ratioof Li� and Na� (PLi/PNa) for the swap � chimeras co-expressed with ��-ENaC in Xenopus oocytes decreasedsignificantly. In contrast, the ratio of PLi/PNa for theswap � constructs was not significantly altered. Singlechannel studies confirmed that swapping of the H2 andthe pre-H2�H2 domains increased the �Na of �-ENaC butdecreased the �Na of �-ENaC. A significant increment inthe apparent inhibitory dissociation constant for amilo-ride (Ki

amil) was observed in the � chimeras by swappingthe pre-H2, H2, and pre-H2�H2 domains. In contrast, astriking decline of Ki

amil was obtained in the chimeric �constructs with substitution of the H2 and pre-H2�H2domains. Our results demonstrate that the pre-H2 do-main, combined with the H2 domain, contributes to thePLi/PNa ratio, single channel Na� conductance, andamiloride sensitivity of �- and �-ENaCs.

The epithelial Na� channel (ENaC)1 was the first subgroupof the ENaC/DEG superfamily cloned from mammals. The to-pology of ENaC/DEG comprises two short N- and C-terminalintracellular tails, two hydrophobic membrane-spanning do-mains (M1 and M2), and a large, extracellular loop with two (orthree) cysteine-rich domains (1, 2). There is an overall �37%amino acid identity between �-, �-, �-, and �-subunits. Both the

�- and �-subunits can form independent conducting channelswith similar amiloride sensitivities and ion selectivities tothose co-expressed with ��-subunits. The �- and �-subunits aremodifying subunits that regulate the trafficking and conduct-ance of �- and �-ENaCs (1, 2). The ���-ENaC has a higherpermeability to Na� and Li� compared with the other alkalimetals. For example, ���-ENaC has a permeability ratio ofPLi/PNa up to 2, but the channel is virtually impermeant to K�

ions (1, 2). Similar to other ENaC/DEG superfamily members,ENaC is very sensitive to amiloride, displaying an apparentinhibitory dissociation constant (Ki

amil) in the nanomolar range(1, 2).

Previous publications (1, 2) have demonstrated that thepre-M2 region of �-ENaC, more precisely, the second hydropho-bic domain (H2) preceding the M2 region, serves as the outermouth of the ENaC pore and is involved in channel gating. Anamiloride-binding site has also been identified functionally inthe H2 domain in addition to the one more proximal to thisdomain (3, 4). The H2 domain also forms part of the ion selec-tivity filter, as deduced by mutation analysis of the pre-M2region (5–12).

The 20 amino acids comprising the M2 domain mainlyfunction as part of the conduction pathway. Studies on mice,rat, and human �-ENaC support this hypothesis (8, 9, 13, 14).We have shown previously (15) that positively charged resi-dues downstream of the M2 region of �-hENaC (post-M2domain) also contributed to ion permeation and gating be-havior and that the functional diameter of the channel pore,in constructs in which these post-M2 positively chargedamino acids were mutated to negatively charged glutamicacids, was altered.

There is 63% diversity in the predicted amino acid se-quences between �- and �-ENaC subunits. �- and �-ENaCshave three essential biophysical and pharmacological differ-ences (16). First, the whole-cell amiloride-sensitive Na� cur-rent of ���-ENaC is greater than the amiloride-sensitive Li�

current, yielding a Li�/Na� permeation ratio (PLi/PNa) of 0.6rather than 2.0 (for ���-ENaC). There is no difference inPNa/PK. Second, the value of Ki

amil for ���-ENaC (up to 2.6�M) is 30-fold greater than that for ���-ENaC (16). Third, theunitary Na� conductance (�Na) measured for ���-ENaC incell-attached patches is 2.4-fold greater than that of wt ���-ENaC, but the unitary Li� conductance (�Li) is unchanged(16). Nothing is known about the molecular basis for thebiophysical and pharmacological differences between �- and�-ENaC.

The ectodomains of Na/K-ATPase, gastric H/K-ATPase (18),inward-rectified and hERG K� channel (19, 20), and C5a re-ceptor (21) have been shown recently to mediate ion selectivity,channel conductance, and agonist/ion affinity. The contribu-

* This work was supported by National Institutes of Health GrantsDK37206 and DK56095. The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed. Tel.: 205-934-3758;Fax: 205-934-1445; E-mail: ji@physiology.uab.edu.

§ Present address: Dept. of Environmental Health, University of Al-abama at Birmingham, Birmingham, Alabama 35294.

1 The abbreviations used are: ENaC, epithelial sodium channel;rENaC, rat ENaC; hENaC, human ENaC; H2, the second hydrophobicdomains preceding M2 region; M2, the second membrane-spanningregion; DEG, cloned degenerin family; �Na, unitary Na� conductance;�Li, single channel Li� conductance; wt, wild type.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 9, Issue of February 27, pp. 8428–8440, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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tions of the extracellular pre-H2 domains of ENaC to the ionselectivity filter and amiloride inhibition are unknown. Theaim of the present study was to test the hypothesis that thepre-H2 domains of �- and �-ENaC subunits may be integrallyassociated with amiloride affinity, ion selectivity, and conduct-ance, therefore contributing to the different pharmacologicaland biophysical properties between �- and �-ENaC. Our resultsshow that swapping the pre-H2 and/or H2 regions of �- and�-ENaC carried over the features associated with their paren-tal wild type constructs partly in cation selectivity, single chan-nel conductance, and almost entirely amiloride sensitivity tothe chimeric mutations.

EXPERIMENTAL PROCEDURES

Site-directed and Swap Mutagenesis and Functional Expression—Swap and triple mutants were generated in rat �- and human �-ENaCcRNAs cloned into pSP70 vector (Promega, WI) using the ChameleonDouble-stranded Mutagenesis kit (Stratagene) (15). cRNAs weresynthesized using the T7 promoter (Ambion, TX) and dissolved innuclease-free water. Defolliculated oocytes were injected with 50 nl ofcRNAs containing �- (or �-), �-, and �-ENaC (ratio 1:1:1) for eachsubunit. Injected oocytes were incubated in 1⁄2 strength L-15 medium at18 °C (22, 23). The triple mutants and swap chimeras were engineeredas follows (Fig. 1): the pre-H2 chimeras (�pH2 and �pH2), swapping�545–568 with �459–518; the H2 chimeras (�H2 and �H2), exchanging�569 –587 with �519 –537; the pre-H2�H2 chimeras (�pH2H2

and �pH2H2), switching �545–587 with �495–537; the � triple mutant(�trip), �S564E,S566A,S568V; the � triple mutant (�trip),�E514S,A516S,V518S.

Dual Electrode Voltage Clamp—Whole-cell cation currents weremeasured in oocytes expressing ENaC using dual electrode voltageclamp 48 or 72 h post-injection (23). Oocytes were impaled with two3 M KCl-filled electrodes, having resistances of 0.5–2 megohms. ADagan TEV-200 voltage clamp amplifier was used to clamp oocyteswith concomitant recording of currents. Two reference electrodeswere connected to the bath by 3 M KCl, 3% agar bridges. The contin-uously perfused bathing solution was ND96 (in mM): 96 NaCl, 1MgCl2, 1.8 CaCl2, 2 KCl, and 5 HEPES (pH 7.4). Equimolar concen-trations of LiCl were used to replace NaCl to examine ion selectivity.The chamber volume was less than 100 nl, and the switching ofsolutions was controlled by SF-77B Perfusion Fast-Step system(Warner, Hamden, CT). Sampling protocols were given by pCLAMP8.0 software (Axon Instruments, Union City, CA), and currents at�100 and �60 mV were continuously monitored at an interval of 2 susing a strip chart recorder. Oocytes were clamped at a holdingpotential of 0 mV. The current-voltage (I-V) relationships were ac-quired by stepping the holding potential in 20-mV increments from�120 to � 80 mV. I-V data were recorded after the monitoringcurrents were stable before and after the application of 10 �M amilo-ride to the bath except where stated. Data were sampled at the rateof 1 kHz and filtered at 500 kHz.

Single Channel Patch Clamp—Oocytes were shrunk in a hyperos-motic medium, and the vitelline membrane was removed before patchclamping (15). The on-cell and inside-out configurations were used torecord single channel currents, using an Axopatch 1B amplifier (AxonInstruments, Union City, CA) (15). The patch pipettes were pulled fromfire-polished borosilicate glass (WPI Instruments) by using a multi-stepped micropipette puller (model M97, Flaming/Brown). The elec-trode tips were fire-polished. The resistance of the electrode was 5–10megohms when filled with pipette solution (NaCl 100 mM, HEPES-Na10 mM (pH 7.5)). Currents were collected using the software CLAMPEX7.0 at a sampling interval of 500 �s. The current traces were filteredwith the 0.1 kHz built-in low pass filter of CLAMPEX 7.0 and digitizedby DigiData 1200 (Axon Instrument, Union City, CA). After collectingdata for the on-cell configuration, inside-out patches were excised inbath solution (100 mM LiCl, 10 mM HEPES free acid (pH 7.5), LiOH),simply by pulling the electrode back. Depolarizing potentials (0 to �120mV) were applied to the on-cell patch, and both depolarizing and hy-perpolarizing potentials (�100 mV to �100 mV) were applied to theinside-out patches.

Data Analysis—All macroscopic currents presented in this paperwere amiloride-sensitive currents (except the traces in Fig. 6), whichwere derived by subtracting, by CLAMPFIT, the amiloride-resistantcurrent measured in the presence of 10 �M amiloride from the totalcurrent amplitude measured in the absence of the drug. Amiloride-

sensitive currents, measured between 200 and 800 ms after applicationof the test potentials, were averaged.

Analysis of single channel data was performed using the FETCHANand pSTAT programs of pCLAMP version 8.0 or Clampfit 9.0 (AxonInstruments, Union City, CA) as described previously (15). The unitarycurrent level was computed by making all-point histograms or meas-uring the amplitude of the current transient. Single channel conduct-ance was calculated by linear regression of I-V curves. Values of �Na and�Li under bi-ionic conditions were estimated by fitting the inward andoutward current sections of I-V curves, respectively.

The absolute permeabilities for Na� (PNa) and Li� (PLi) and intraoo-cyte ion concentrations were retrieved from the fitting of the macro-scopic current-voltage curves (I-V) with the Goldman-Hodgkin-KatzEquation 1 (15),

Iamilx

Acell� Px � z � Etest� F2

�X��out � �X�]in� eF�Etest/R�T

R � T� (1 � eF�Etest/R�T) (Eq. 1)

where R, T, and F have their usual meanings; z is the valence of thecation; Iamil

x represents the amiloride-sensitive current carried by thecation X�; Px is the absolute permeability value for the cation X�, and[X�]out and [X�]in indicate extraoocyte and intraoocyte concentrations,respectively. Acell is the calculated surface of an oocyte, assuming thatthe oocyte is a spheroid with a diameter of 1.0 mm.

The absolute cation permeability coefficients from single channelrecordings were computed by fitting the bi-ionic I-V curves with themodified Goldman-Hodgkin-Katz current Equation 2,

iLi � iNa � (PNa�[Na�]out � PLi�[Li�]in) �z � Etest � F2

RT�

eEtest� F/R�T

1 � eEtest� F/R�T

(Eq. 2)

where iLi and iNa indicate the unitary current carried by Na� in thepipette solution ([Na�]out) and Li� in the bath solution ([Li�]in), respec-tively. The corresponding permeabilities were expressed as PNa and PLi,respectively.

To analyze the affinity of amiloride to the channel, perfusates con-taining different concentrations of amiloride (ranging from 0 to 1 mM)were switched into the chamber. Both the Ki

amil and Hill coefficientwere retrieved by fitting the dose-response curves of amiloride with theHill Equation 3,

I �1

�1 � �AmilKi

amil�n� (Eq. 3)

where Kiamil is the concentration required for inhibiting half of the

maximal current; I is the measured amiloride-sensitive current at theconcentration of Amil; and n represents the Hill coefficient.

The voltage dependence of amiloride inhibition was also character-ized by fitting the plot of Ki

amil as function of membrane voltage withthe Woodhull Equation 4 (24),

Kiamil �Etest� � Kd�0� � exp�� �

z � F � Etest

R � T � (Eq. 4)

where Kiamil(Etest) is the equilibrium inhibitory dissociation constant at

a test potential (Etest); Kiamil(0) is the Ki

amil at 0 mV; � is the fractionaldistance across the electric field that can be sensed by amiloride; z is �1for amiloride; and R, T, and F have their usual meanings.

Both whole-cell and single channel current traces carried by cat-ions moving from the extra-oocyte to the intra-oocyte side were de-picted as inward (negative) currents and vice versa. Average datawere reported as mean S.E. if not stated otherwise. Statisticalanalysis was performed using one-way analysis of variance combinedwith the Bonferroni test for variance and mean of unpaired data. A pvalue of 0.05 between experimental groups was considered statis-tically significant.

RESULTS

The purpose of the study was to test the role of the pre-H2regions of �- and �-ENaC subunits in ion permeation, conduct-ance, and amiloride inhibition. Because these properties of�-rENaC and �-hENaC are significantly different from eachother at the macroscopic and microscopic current levels, theseparticular subunits were chosen for analysis. The chimeras

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were engineered by swapping the pre-H2 region as well as theH2 domain. Fig. 1 shows that there is 45.8 and 52.6% identity(37% identity for full-length�-rENaC and �-hENaC) betweenthe pre-H2 and H2 regions, respectively.

Ion Selectivity, Whole-cell and Single Channel Studies

Whole-cell Studies—To test the role of the pre-H2 region inion selectivity, whole-cell amiloride-sensitive currents carriedby extracellular Na� or Li� (101 mM) were recorded in oocyteswith the conventional two-electrode voltage clamp technique.Wild type (wt) rat ��-ENaC subunits were co-expressed withwt �-rENaC and its swap chimeras, whereas �-hENaC and theswapped constructs were expressed with wt human ��-ENaCsubunits. As shown in Fig. 2, the activation of the amiloride-sensitive currents evoked at membrane potentials was nottime-dependent between �120 mV to �80 mV. Oocytes wereheld at 0 mV, which was close to the resting membrane poten-tial as ENaC expression caused an overloading of the cytosolicNa� and Li� concentration, leading to a depolarization of theresting membrane potential. Both wt and chimeric constructshad a slightly inward rectified I-V curve with a reversal poten-tial (Erev) of more than �10 mV (Fig. 3). The macroscopicamiloride-sensitive Na� and Li� currents were �1520.37 361 and �3524.33 606 nA, respectively, at a membranepotential of �120 mV for wt ���-rENaC (n � 33). Currentcarried by Li� was markedly greater than that of Na� (p 0.001). On the other hand, the whole-cell amiloride-sensitiveNa� and Li� currents of �pH2��-rENaC were �2970.99 832(p 0.01 compared with that of wt ���-ENaC) and �4011.51 1178 nA, respectively (n � 25). Conversely, the amiloride-sensitive Na� current in oocytes expressing ���-hENaC wasgreater than the Li� current (�2602.61 530 versus�1660.94 386 nA for Na� and Li�, respectively, n � 34). Thecurrent amplitudes of both amiloride-sensitive Na� and Li�

currents associated with �pH2��-hENaC were greater than wtconstruct (�3302.67 670 nA for Na� and �2490.11 528 nAfor Li�, respectively, n � 26, p 0.05). These results for wt���-rENaC and ���-hENaC were consistent with those re-ported previously (16, 25, 26).

The whole-cell Na� and Li� current levels associated withthe pre-H2 swapped chimeras (Figs. 2 and 3) were not iden-tical to those of wt constructs, indicating that their perme-

abilities to Na� (PNa) and Li� (PLi) have been altered. Toaddress whether the pre-H2 domain regulates Na� or Li�

permeation, we calculated the absolute permeation constant.The retrieved values were listed in Table I. Only �pH2 and�pH2H2 chimeras shifted the Erev significantly to the hyper-polarizing direction. The value of PNa for wt rat ENaC wasabout half of PLi, yielding a PLi/PNa ratio of 1.8. However, thePNa of wt human ENaC was greater and the PLi/PNa ratio wasless compared with its rENaC counterpart. In contrast, thePLi for wt �-hENaC was smaller (approximately up to 50%that of rENaC), whereas PNa was greater than these of wtrENaC, yielding a PLi/PNa ratio of 0.62. The selectivity ratiosof wt rat and human ENaC constructs calculated from thepermeation constant were completely consistent with previ-ous studies (16, 25).

An essential G(S)XS tract has been identified in the H2region of ENaC, which controls Na� permeability (10, 12). Asshown in Fig. 1, there is a serine-rich domain (564–568,SESPS) within the pre-H2 region of �-ENaC, which is notpresent in the equivalent stretch of �-hENaC subunit. To de-termine the role of this SXS tract in ion selectivity, Na� andLi� permeabilities of triple mutants �564E,S566A,S568V��-rENaC (�trip) and �E514S,A516S,V518S��-hENaC (�trip) wereexamined. For comparison, chimeras swapping the H2 andpre-H2�H2 domains were constructed. A significant incrementin PNa was observed for the �trip, �pH2, and �pH2H2 chimeras,but there was a significant decrease in PLi for the �H2 chimera(Table I). The PLi/PNa ratio reduced markedly for all four chi-meric �-rENaC constructs. In contrast, only the �trip mutationexhibited an increment in PLi, whereas the PLi/PNa ratio wasnot statistically different from that of wt �-ENaC.

Single Channel Studies—In order to re-confirm the macro-scopic results under strictly controlled ionic conditions, weperformed single channel studies under bi-ionic conditions. Theon-cell and inside-out patches were obtained from injected oo-cytes, and single channel traces were digitized (Fig. 4). Theaverage unitary currents were plotted as I-V curves shown inFigs. 5 and 6. Wild type ���-ENaC and �pH2�� chimera func-tion as slight outward rectifiers compared with the macroscopicI-V relationships (Figs. 3 and 5A). Replacement of the pre-H2region with the corresponding regions of �-ENaC shifted theErev to �5 from �15 mV. By comparison, the I-V relationship of

FIG. 1. Sequence alignment and schematic model of �- and �-ENaC. As shown in the top panel, both �- and �-ENaCs have twotransmembrane domains (M1 and M2), a large extracellular loop (70% of the whole molecule), and two intracellular termini (N- and C-terminaltails). The swap pre-H2 and H2 regions are displayed in colors and amino acids are numbered at each end. The bottom panel shows alignment ofthe relevant segments including the pre-H2, H2, and part of M2 domains with ClustalW. The amino acids are in colors matching those in the toppanel. The ‘‘SXSXS’’ motif, degenerin (deg) site, and amiloride (amil)-binding site are indicated

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���-hENaC revealed an inwardly rectified curve with an Erev of15 mV (Fig. 5B).

We also applied the Goldman-Hodgkin-Katz equation to theI-V relationships generated under bi-ionic conditions to deter-mine the permeability constant and ratio. The permeabilityratio was also recalculated using the Erev (Table II). Consistentwith the whole-cell observations, the PLi was greater than PNa

for wt ���-rENaC, yielding a PLi/PNa ratio of 1.82 (Table II).Substitution of the pre-H2 and pre-H2�H2 domains of �-ENaCwith the corresponding regions of �-ENaC resulted in an incre-ment in PNa, whereas swapping of the H2 domain with that of�-ENaC led to a decrease in PLi. Both the pre-H2 and/or H2domain substitution of �-ENaC resulted in reduced discrimi-nation between Na� and Li�. With respect to wt �-hENaC, thePLi was less than its PNa with a calculated PLi/PNa ratio of 0.55.The chimeric �-ENaC constructs with replacement of thepre-H2 and/or H2 domains of �-ENaC showed no markedchange in Na� and Li� permeabilities.

Single Channel Conductance

The results of on-cell single channel experiments furtherverified the whole-cell observations (Fig. 4A). The unitary cur-rent level recorded at �100 mV in on-cell patches displayeddifferences when the pipette was filled with Na� or Li� (100mM). For the wt ���-rENaC and swap chimeric constructs, theNa� current was much smaller than Li� current (Fig. 4A, toppanel and Table III). In contrast, the single channel Na� cur-rent of wt ���-hENaC was much greater (12 versus 4 pS for wt���-ENaCs), whereas the Li� current associated with ���-hENaC was almost identical to that of ���-ENaCs (7 pS).Swapping the H2 and pre-H2�H2 domains increased �Na of �chimeras, but the �Na was significantly reduced for the swap�-ENaC constructs in on-cell patches.

For wt ���-rENaC, there was no significant difference in�Na and �Li between the on-cell and inside-out configurations.In contrast, both the �Na and �Li of �-ENaC in the inside-outpatches were strikingly smaller than those from the cell-

attached patches (57 and 66% of the on-cell conductance,respectively, see Table III). With regard to wt ���-hENaC, the�Li was reduced by 44%. Consistent with the results in the on-cellpatches, an increment for the �H2 and a decrease for the �H2 and�pH2H2 constructs in �Na were observed in the inside-out patches(Table III). Inconsistent with data obtained from the on-cell con-figuration, both the �Na and �Li for the other constructs declinedsignificantly in the inside-out patches (Table III).

Amiloride Sensitivity

To test the hypothesis that the pre-H2 region preceding theamiloride-binding site located in the H2 domain modified thekinetics of amiloride inhibition of the ENaC channel, we usedthe whole-cell mode to record amiloride-sensitive Na� currentsin the presence of various amiloride concentrations (range, 0–1mM). Fig. 7A shows representative traces of the time course andconcentration dependence of the amiloride inhibitory effect forwt and pre-H2 swap constructs. Generally speaking, stablecurrents could be achieved within a few seconds following theapplication of amiloride. Fig. 7, B and C shows the dose-re-sponse curves at �100 mV fitted with the Hill equation. Thevalue of Ki

amil for wt ��� rENaC was 54.7 19 nM (n � 7). Forthe �pH2�� chimeric mutation, the Ki

amil increased approxi-mately by 3-fold to 150.0 16.5 nM (n � 5, p 0.01). Consist-ent with a previous report (16), the Ki

amil for ���-hENaC was52-fold greater than that of ���-rENaC (2860 501 nM, n �12). Exchanging the pre-H2 region of �-hENaC with that of�-rENaC significantly decreased the Ki

amil to 745 247 nM forthe �pH2��-hENaC (n � 6).

We constructed swap chimeras by switching the H2 domainand the pre-H2�H2 domains, in which an amiloride-bindingsite is located as positive controls (1, 2). Fig. 8 presents thecomputed Ki

amil and Hill coefficient as a function of membranepotential. The voltage dependence of Ki

amil was fitted with theWoodhull equation (24). The retrieved parameters includingthe fractional distance across the electrical field sensed byamiloride (�), the experimental Ki

amil at 0 mV, and the esti-

FIG. 2. Representative current traces of wt and the swap ENaC. A, amiloride-sensitive Na� currents in oocytes expressing wt ���-rENaC,�pH2��-rENaC, ���-hENaC, and �pH2��-hENaC. The test potential ranges from �120 to �80 mV in an increment of 20 mV. The holding potentialwas 0 mV. Extracellular Na� concentration, 100 mM; amiloride concentration, 10 �M. B, amiloride-sensitive Li� currents. The middle iconrepresents the mode used for these experiments is the whole-cell two-electrode voltage clamp. The dotted lines indicate the zero current levels.

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mated Kiamil by the Woodhull equation fitting are compiled in

Table IV.The experimental Ki

amil at 0 mV for the �pH2 construct de-creased significantly as compared with that for wt �-ENaC,whereas an increment was observed for the �pH2 chimera (Ta-ble IV). Swapping of the H2 domains between �- and �-ENaCsalmost completely exchanged the values of Ki

amil for each other

(Fig. 8, A and B). However, the pre-H2�H2 domain swapchannels did not show an additional shift in amiloride affinityto the others, instead the change in Ki

amil was smaller com-pared with those of the H2 swap chimeras. These results sug-gest that there are intramolecular domain-domain interactionsbetween the pre-H2 and H2 domains and that their interac-tions with amiloride are not synergistic.

FIG. 3. Macroscopic current-voltage (I-V) relationships. A, I-V curves of ���-rENaC (left) and �pH2 chimera (right). Amiloride-sensitive (AS) Na�

and Li� currents are as indicated by the inset legend. Vm, membrane potential. The reversal potential is shown by arrow and number. The dotted linesbetween the symbols are created by fitting average data with the Goldman-Hodgkin-Katz equation (see ‘‘Experimental Procedures’’ for details). B, I-V curvesof ���-hENaC (left) and swap � pH2 mutant (right). C, I-V curves of � triple (�trip, left) and � triple mutants (�trip, right). D, I-V curves of swap �H2 (left) and�pH2H2 mutants (right). E, I-V curves of swap �H2 (left) and �pH2H2 mutants (right). F, I-V curve of wild type ���-hENaC.

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In comparison to hyperpolarizing membrane potentials, theKi

amil was more voltage-dependent at depolarizing membranepotentials from �20 to �80 mV (Fig. 8, A and B). All estimatedvalues of the Ki

amil at 0 mV (Kiamil(0)) were pretty close to those

computed from the experimental data. For example, the calcu-lated Ki

amil(0) for wt ���-rENaC was 354 and 330 nM for theexperimental data. The value of � was 0.35 for wt rENaC, closeto those yielded by noise analysis of the macroscopic current

FIG. 3—continued

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(28, 33). In contrast, the value of � for ���-hENaC was greaterthan that for ���-rENaC (Table IV).

As shown in the right panel in Fig. 8A, all swap chimeric�-ENaC constructs had an increased Hill coefficient. On theother hand, the Hill coefficient reduced markedly for all chi-meric � constructs at depolarizing membrane potentials (Fig.8B, right panel).

DISCUSSION

The goal of the present study was to examine whether thepre-H2 domain regulates ion selectivity, unitary conductance,and amiloride sensitivity of ENaC. Our experimental strategywas to swap 24 amino acid residues preceding the H2 regions of�- and �-ENaCs. The main findings of our studies are as fol-lows. 1) The pre-H2 and H2 domains and an SXSXS motif of �-and �-ENaCs are involved in Na� and/or Li� permeation. 2)Single channel studies confirmed that the �Na of ���-hENaCincreased for the �H2 and �pH2H2 chimeras, supportive of thewhole-cell results; the �Na of chimeric �-ENaC constructs wasreduced in on-cell patches. 3) The �Li of all chimeric � and �constructs decreased in the inside-out mode with the exceptionof �Na for the �H2 and �pH2 chimeras. 4) The pre-H2 clusterscombined with the H2 domains of �- and �-subunits contributeto amiloride inhibition by modifying the Ki

amil, the Hill coeffi-cient, and voltage dependence.

Ion Permeation—Our results suggest that the swap chimeric�pH2-, �H2-, and �pH2H2-ENaC channels discriminate less be-tween Na� and Li� due to the elevated Na� permeation, whichwas inherited from the wild type ���-hENaC (Tables I and II).These results for wild type ���-hENaC are consistent with theobservations Waldmann et al. (17). Replacement of hydropho-bic region 2 (H2) of �-hENaC with the corresponding sequenceof MEC-4 significantly increased �Na to 14 pS and the �Na/�Li

ratio increased to 1.6 (17). Serines 589 and 593 located in thepore region of �-ENaC were further identified as modifying �Na

to the same extent (17). Additionally, mutations of the pre-M2region revealed that the H2 domain regulated ion permeation;in particular, �Na was altered (4, 8, 9, 11, 14). However, thesetwo key residues are highly conserved in both �- and �-ENaCisoforms. The present studies with an �-triple mutant sug-gested that an SXSXS motif partially contributes to the in-creased Na� permeability of the �pH2-ENaC chimera. In com-parison to the �pH2-chimera, the swap � constructs did notsignificantly affect Na� permeability.

Our results suggest that multiple domains integrally con-trol Li� and Na� permeation. They support the idea that theselectivity filter consists of a chain of amino acid residues (i.e.

FIG. 4. Single channel current traces recorded from on-celland inside-out patches. A, the abscissa is 2 s and the ordinate is 1pA. These traces were digitized at membrane potential of �100 mV.The upper trace was recorded with 100 mM Na�, and the bottom tracewas made with 100 mM Li�. The icon in the middle represents thepatch clamping configuration: the on-cell mode. B, single channeltraces obtained from the inside-out patches under bi-ionic conditions.On the right side are the patch potentials (Vm). Left panel is from thewt constructs, and the right panel is from the swap chimeras. Inside-out patches were clamped at command potentials (Vp) from �100 mVto � 100 mV. The scale bar for the x axis is 10 s and for the y axisis 1 pA.

TABLE IIon permeation ratio estimated by fitted the macroscopic currents with the Goldman-Hodgkin-Katz Equation 1

The pre-H2 (�pH2 and �pH2), H2 (�H2 and �H2), and pre-H2H2 (�pH2H2 and �pH2H2) swap chimeras as well as triple mutants (�trip and �trip) arecompared with wild type constructs. Erev, reversal potential of Na� current; PNa and PLi, absolute permeation constants for Na� and Li�; andPLi/PNa, permeation ratio of Na� and Li�. Numbers in parentheses in the 1st column are oocytes tested.

Constructs Erev PNa PLi PLi/PNa

mV cm/s � 10�6

���-rENaC (33) 30 2.3 4.57 0.2 8.31 0.4 1.82�trip��-rENaC (6) 27 2.5 6.12 0.1a 9.23 0.2 1.51a

�pH2��-rENaC (25) 10 1.2a 6.86 0.5a 8.30 0.6 1.21a

�H2��-hENaC (6) 23 3.1 4.83 0.3 5.52 0.3a 1.14a

�pH2H2��-hENaC (6) 14 5.2a 7.52 0.3a 7.76 0.3 1.04a

���-hENaC (11) 14 1.8 4.73 0.7 6.24 0.5 1.32���-hENaC (34) 15 1.4 6.19 0.1 3.84 0.2 0.62�trip��-hENaC (6) 12 1.5 6.67 0.4 5.01 0.4a 0.75�pH2��-hENaC (26) 13 1.1 6.21 0.4 4.75 0.2 0.76�H2��-hENaC (9) 13 1.1 6.10 0.2 4.36 0.2 0.72�pH2H2��-hENaC (6) 14 1.5 6.55 0.2 4.55 0.2 0.73

a p 0.05 compared with the corresponding wild type channels.

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FIG. 5. Unitary current-voltage (I-V) relationships for wild type and swap ENaC constructs from on-cell and inside-out patches.A, I-V curves of wt and the swap rENaC from the on-cell patches at pipette voltage (Vp) from �120 to �20 mV. The dotted line was drawn by fittingthe data with a linear fitter to calculate single channel conductance of Na� (square) and Li� (circle). Bottom panel shows I-V curves obtained fromthe inside-out patches for wt (n � 11) and the swap rENaC (n � 10). Reversal potential is labeled by arrow and number. The dotted lines werecreated by fitting the current data with the Goldman-Hodgkin-Katz equation strictly for bi-ionic conditions (Equation 2). The unitary Na� (�Na)and Li� (�Li) conductances under bi-ionic conditions were computed by linear regression of inward and outward currents, respectively. B, I-V curvesfor wt and the swap �-hENaC constructs from the on-cell and inside-out patches.

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the pre-H2, H2, M2, and post-M2 domains) and that theselectivity to Li�, Na�, and K� is determined by differentresidues. Moreover, more than one residue can influence thepermeation for each individual alkali cation species (8–10, 12).This idea explains why the Na� permeation of the swap chimeric�pH2H2-ENaC channels was not changed and why the PNa of the�pH2H2 is not identical to that of wt ���-hENaC. Our observa-

tions suggested that the pre-H2 and H2 domains of �-ENaC arenot the only key regions for its greater Na� permeability over�-ENaC.

Why are the �Na and �Li of wt and chimeric constructs in theinside-out patches lower than these of the on-cell patches (Ta-ble III)? Our previous studies have demonstrated that cytoskel-etal elements physically interact with and functionally regu-

FIG. 6. Unitary current-voltage (I-V) relationships for the H2 and the pre-H2�H2 swap chimeras from inside-out patches underbi-ionic conditions. Reversal potential and unitary conductance are shown by values. See the legend for Fig. 5 for details.

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late ���-ENaC activity (34, 35). In the excised patches, theprecise architectural arrangement of the cytoskeleton is un-doubtedly disrupted, and the channel conformation and/or in-teractions with these cytoskeletal proteins may be changed.Also, tension on �-ENaC during the formation of the inside-outpatches may contribute to an alteration in cation conductance.We cannot rule out the involvement of soluble intracellularelements and salt components. In the on-cell patches, swappingthe pre-H2 domain with the corresponding sequence of �-ENaCresulted in a decrease in �Na of �-ENaC, suggesting that thepre-H2 region of �-ENaC may be an interactive motif, whichdirectly cross-talks with the extracellular loop of other mem-brane proteins or serves as a regulator of other protein-proteininteractive motifs. Finally, the cytosolic Li� concentration (100mM) used for the inside-out configuration is much greater thanthe concentrations for the cell-attached mode. As predicted bythe reversal potential of the macroscopic I-V curves, intra-oocyte Na� content is approximately 60 mM for �-ENaC andless for �-ENaC. The decreased �Na and �Li in the inside-outpatches excised from �-ENaC-expressing cells may thus be dueto the feedback regulation and self- regulation, respectively (2).If so, wt ���-hENaC is more sensitive to the inhibitory regu-lation than wt ���-rENaC.

As indicated by the Goldman-Hodgkin-Katz current equa-tion (Equation 1), the permeability to cations positively corre-lates to whole-cell current. Whole-cell current is determined bythe number of electrically detected active channels (N), channelopen probability (Po), as well as single channel conductance.Our results, namely that the PNa of the �pH2��-rENaC is dou-bled whereas the single channel conductance is not changed,imply that the �pH2��-rENaC may increase either or bothactive channel number or channel open probability.

As shown in Figs. 1F and 6E, as well as Table III, wt ���-hENaC differs from the rat counterpart in their biophysicalfeatures including the PNa/PLi ratio, Erev of the macroscopicNa� current, and �Li in the inside-out patches. If these differ-ences are mainly determined by wt �- and �-hENaC subunits,then the properties of the swap �-ENaC constructs may beaffected by ��-hENaC subunits.

Amiloride Inhibition—Multiple amiloride-binding sites atthe extracellular loops of ��� subunits have been biochemi-cally and functionally identified (3, 4). However, no amiloridebinding or regulatory sites have yet been localized to thepre-H2 region. Our observations on amiloride inhibition ofthe wt and swap constructs suggested that the pre-H2 do-main is also important for amiloride interaction with thechannel, particularly for �-ENaC. The values of Ki

amil for thechimeras that swap the H2 and pre-H2�H2 regions are notidentical to that of the other parental constructs, suggestingthat domains beside these two regions influence amilorideaffinity. Because �- and �-subunits also contribute to amilo-ride affinity, co-expression of wt ��-ENaC subunits may com-pensate for the effects of swapping pre-H2 and H2 regions onamiloride inhibition.

Our observations also confirm previous studies (16) thatthe Ki

amil of ���-ENaC is in the micromolar rather than thenanomolar range. Amiloride inhibition of both �- and�-ENaCs is voltage-dependent, especially at depolarizingmembrane potentials above 0 mV (Fig. 7). Amiloride is apositively charged molecule when protonated and is thussensitive to membrane potential (36). Cations compete withamiloride for at least one of these two binding sites. Althoughamiloride affinity is regulated by extracellular cation concen-tration, external protons, membrane potential, and intracel-

TABLE IISingle channel permeation of the wt and swap ENaC constructs, inside-out patches

The absolute permeation constant for Li� (PLi) and Na� (PNa) ions was computed by fitting the I-V curves with the Goldman-Hodgkin-KatzEquation 2. Erev indicates reversal potential; PLi/PNa denotes the permeation ratio of Li� and Na�. Numbers in parentheses in the 1st column areoocytes tested.

Constructs PNa PLi PLi/PNa Erev PLi/PNaa

cm/s � 10�6 mV

���-rENaC (12) 4.99 0.3 10.0 1.2 2.00 -16 1.4 1.82�pH2��-rENaC (11) 8.50 0.4a 9.85 1.1 1.18a -5 1.6 1.23a

�H2��-rENaC (7) 5.28 0.4 5.07 0.3a 0.96a 0.51 0.1 0.97a

�pH2H2��-rENaC (4) 8.84 0.2a 9.28 0.2 1.05a 0.5 0.1 1.02a

���-hENaC (10) 10.0 0.6 7.32 0.6 0.73 15 1.2 0.55�pH2��-hENaC (10) 10.0 0.7 8.29 0.4 0.83 14 1.1 0.57�H2��-rENaC (3) 8.87 0.3 6.02 0.3 0.68 13.5 0.7 0.61�pH2H2��-rENaC (5) 9.21 0.2 6.81 0.4 0.74 12.9 0.5 0.73

a PLi/PNa, permeability ratio that is computed with the Erev.b p 0.05 compared with wild type ���-rENaC.

TABLE IIISingle channel conductance of the wt and swap ENaC constructs, on-cell and inside-out patches

The unitary Na� (� Na) and Li� (� Li) conductance was estimated by linear fitting of unitary I-V curves. Numbers in parentheses indicate thenumber of patches measured.

ConstructsOn-cell patch (4–15) Inside-out patch (5–9)

�Na �Li �Na �Li

pS pS

���-rENaC 4.03 0.11 6.86 0.20 4.31 0.22 6.55 0.18�pH2��-rENaC 4.22 0.16 6.92 0.26 3.42 0.12a 5.16 0.24a

�H2��-hENaC 7.92 1.22b 7.31 0.48 5.32 0.31a 3.27 0.24b

�pH2H2��-hENaC 5.68 0.49a 6.33 0.65 3.35 0.36a 2.58 0.12b

���-hENaC 4.00 0.18 7.30 0.16 4.30 0.37 3.21 0.29���-hENaC 12.09 0.53 7.14 0.34 6.92 0.33 4.74 0.19�pH2��-hENaC 8.16 0.44a 7.38 0.32 7.01 0.41 3.88 0.24a

�H2��-hENaC 6.98 0.15b 7.11 0.37 5.08 0.61b 3.69 0.36a

�pH2H2��-hENaC 5.00 0.71b 6.92 0.44 4.55 0.56b 2.93 0.22a

a p 0.05.b p 0.01 compared with wt constructs.

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lular amiloride accumulation, the fact that the identical ex-perimental conditions were used regardless of the ENaCconstructs under investigation excluded contributions fromthese other factors (36).

Comparing data from ���-rENaC and ��-rENaC revealedthat ��-ENaC channel was more permeable to Na� than Li�

(PNa � PLi), had an identical fractional distance across theelectrical field sensed by amiloride, and an increased Ki

amil (4�M, Refs. 6 and 28). These results raised the question that thechimeric �pH2-ENaC may assemble a channel with only a�-subunit (and not a �-subunit). However, the �pH2��-ENaChas a greater permeability to Li (�Na 4.2 versus �Li 6.9 pS)and a smaller Ki

amil (0.13 �M), which were similar to those ofwt ���-ENaC. Our studies also showed that the whole-cellNa� current for the �pH2�-ENaC at a holding potential of�120 mV was less than 200 nA (not shown), �10% of theamplitude of �pH2��-ENaC (Fig. 2). Thus, the possibility thatchimeric �-construct may assemble with only a �-subunit isunlikely.

The voltage dependence of amiloride inhibition in toad uri-nary bladder (31, 32) and cloned ENaC (27, 33) has beenconfirmed. Furthermore, depolarization led to an increment inamiloride koff but a decrease in kon for the native epithelial Na�

conductance (31, 32), whereas only a weak voltage dependenceof kon and/or koff was observed for ���- and ��-rENaC (28, 33).A simple plug-type blocking model was proposed whereby thepositively charged protonated amiloride is attracted by nega-tively charged amino acids located in the vestibule of the chan-nel (31). Because the negative charges in the M2 domain didnot contribute to amiloride affinity as evidenced by pore-regiontruncation mutants (37), and the two amiloride-binding sitesidentified by Ismailov et al. (3) and Snyder et al. (12) are notnegatively charged, it was speculated that the negative chargesof the pre-H2 region preceding the H2 cluster contributed toamiloride affinity. However, because the pre-H2 domain of�-hENaC contains more negative charges than the correspond-ing region within �-rENaC and yet has a lower Ki

amil, it isunlikely that these negative charges are important in amilo-ride-ENaC interactions.

We also found that the fractional distance across the electri-cal field sensed by amiloride for �-ENaC is greater (0.48) thanthat of �-ENaC (0.35). As summarized in Table IV, the valuefor ���-ENaC is close to the result by Segal et al. (33) andsimilar to other studies (26–32) on endogenous and clonedENaC/DEG channels. Like the observation by Woodhull (24) onthe proton block of voltage-dependent Na� channels, the steep-

FIG. 7. Amiloride sensitivity of wt and the pre-H2 swap ENaC channels. A, current traces of time course for amiloride inhibition from 0nM to 1 mM of macroscopic Na� current at �100 mV are shown in the top panel. B, dose-response curves for wild type and swap �-rENaC atmembrane potential of �100 mV. Circle, wt ENaC; square, the swap mutant. Both solid and dotted lines are created by fitting the data with theHill equation to calculate the Ki

amil and Hill coefficients. C, dose-response curves for wild type and swap �-hENaC.

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ness of the voltage dependence of block increases, whereas the� value elevates for wt ENaC. Their results suggested thatamiloride indeed blocks the channel pore. An alternative inter-pretation is that the amiloride-binding site is not in the chan-nel pore (3). A depolarizing membrane potential may act onintracellular cations as well as on amiloride. The shorter elec-

trical distance from the inside for �-ENaC (0.52 versus 0.65 for�-ENaC) may facilitate intracellular cation binding for out-ward transport, competitively decreasing amiloride affinityand showing more voltage dependence. However, our observa-tions for the swap chimeric constructs, where a different Ki

amil

can be obtained for the pre-H2 swap chimeras with an almost

FIG. 8. Voltage dependence of amiloride inhibition for wild type and swap constructs. A, the Kiamil (left) and Hill coefficient (right) of

wt and swap �-rENaC plotted against membrane potential (Vm). Kiamil was calculated by fitting the dose-response curves with the Hill equation.

Lines on the left panel are created by fitting the data with the Woodhull equation. B, the Kiamil (left) and Hill coefficients (right) of wt and swap

�-hENaC are plotted against membrane potential (Vm).

TABLE IVThe parameters retrieved by fitting of the voltage-dependent plots of Ki

amil with the Woodhull equation� is the fractional distance across the electrical field sensed by amiloride. Ki

amil(0) indicates the amiloride inhibitory dissociation constant atholding potential of 0 mV. Numbers in parentheses in the 1st column indicate the number of patches measured.

Constructs �(/1)a EstimatedKi

amil(0)Experimental

Kiamil(0)

nm

���-rENaC (7) 0.35 0.03 354 27 330 54�pH2��-rENaC (5) 0.30 0.02 550 29b 563 84b

�H2��-rENaC (5) 0.45 0.02b 10981 356c 11618 2909c

�pH2H2��-rENaC (7) 0.46 0.03b 3098 250c 3400 192c

���-hENaC (6) 0.48 0.02 13515 354 13089 3110�pH2��-hENaC (5) 0.43 0.03 3738 279c 3416 2296c

�H2��-hENaC (3) 0.32 0.03b 1087 79c 699 186c

�pH2H2��-hENaC (8) 0.31 0.02b 1724 76c 1685 342c

a � is 0.15–0.48 for ���-rENaC (4, 27), 0.3 for ��-rENaC (28), 0.54–0.62 for MEC4/MEC10 (29), 0.65–0.68 for UNC-105d (30), and 0.12–0.23 fortoad urinary bladder (31, 32).

b p 0.05.c p 0.01.

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unchanged electrical distance (Table IV), do not support eitherof these two interpretations. Swapping of the H2 domains withor without combination of the pre-H2 segment suggested thatsome key residues within the H2 domain contribute to 90% ofthe difference in amiloride affinity, because the identifiedamiloride-binding site is identical between �- and �-ENaCs.Changes in the fractional distance sensed by amiloride of theH2 swap chimeras also suggested that the H2 domain deter-mines the diversity in amiloride sensitivity between �- and�-ENaCs. The pre-H2 domain secondarily mediates amilorideinhibition.

It is well known that ENaC has a different closing confor-mation from DEG members (1, 2). Swapping of the M2 domainof �-ENaC with that of MEC-4 decreased amiloride affinityaccompanied by an alteration in closing conformation (17).Regulation of channel conformation and ligand binding hasbeen found in other channels as well (18–21). We recentlyfound that the conformational change of �-ENaC induced byH�, Zn2�, and other external cations was not akin to �-ENaC.2

Other indirect effects subsequent to swapping of the pre-H2and H2 regions, i.e. interactions with associated proteins, chan-nel structure, and external cation affinity, cannot be excludedby our results. Therefore, whether the changes in ion selectiv-ity, single channel conductance, and amiloride affinity associ-ated with chimeric �- and �-ENaC constructs are due to directeffects or conformation alterations is not clear.

Physiological Relevance—The possible physiological rele-vance for the pre-H2 region involvement of amiloride sensitiv-ity, unitary conductance, and ion selectivity is not clear. Theextraordinarily large M1-M2 connecting loop, comprising over70% of the mass of the entire channel sequence, implies thatthe extracellular loop may function as a receptor or sensor ofdifferent stimuli. The ENaC/DEG channels are regulated bymany external ligands, for example, channel-activating prote-ase 1, protons, amiloride, bivalent cations, and mechanicalstretch (10, 22, 36, 38). The regulatory sites for most of theseexternal inputs are likely located in the extracellular loop. Theectodomains of ENaC may also play a role in the interactionswith other cytoplasmic membrane proteins (i.e. cystic fibrosistransmembrane regulator, the cytoskeleton) (35, 39). Our re-sults indicate that the pre-H2 domain of ENaC may mediatethe regulation of chemical and physical stimuli on amilorideaffinity and cation selectivity.

In conclusion, our studies demonstrate that the pre-H2regions of both �-ENaC and �-ENaCs contribute to ion selec-tivity, single channel conductance, and amiloride inhibition.The pre-H2 domain functionally interacts with the H2 do-main as evidenced by our pre-H2�H2 domain swap studies.

Acknowledgments—We thank Hannah Mebane for preparing oo-cytes. We are grateful for the kind gifts of the �-ENaCs clone providedby Drs. R. Waldman and M. Lazdunski (Institut de PharmacologieMoleculaire et Cellulaire, CNRS, France) and rENaC clones from Drs.

C. Canessa (Molecular and Cellular Physiology, Yale University) and B.Rossier (Institute of Pharmacology and Toxicology, Lausanne, Switzer-land). We acknowledge Isabel Quinones for superb secretarialassistance.

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Pre-H2 and H2 Domains Swapping of �- and �-ENaC8440

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BenosHong-Long Ji, LaToya R. Bishop, Susan J. Anderson, Catherine M. Fuller and Dale J.

Permeation, Conductance, and Amiloride Sensitivity Channels in Ion+-Epithelial Naδ- and αThe Role of Pre-H2 Domains of

doi: 10.1074/jbc.M312012200 originally published online December 2, 20032004, 279:8428-8440.J. Biol. Chem. 

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