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J. Biol. Chem., Vol. 277, Issue 51, 50098-50111, December 20, 2002

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External Nickel Inhibits Epithelial Sodium Channel by Binding to Histidine Residues within the Extracellular Domains of  and  Subunits and Reducing Channel Open Probability^*

Shaohu Sheng, Clint J. Perry, and Thomas R. Kleyman^§

From the Renal-Electrolyte Division, the Department of Medicine, and the ^§ Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Received for publication, September 30, 2002, and in revised form, October 18, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Epithelial sodium channels (ENaC) are regulated by various intracellular and extracellular factors including divalent cations. We studied the inhibitory effect and mechanism of external Ni²⁺ on cloned mouse -- ENaC expressed in Xenopus oocytes. Ni²⁺ reduced amiloride-sensitive Na⁺ currents of the wild type mouse ENaC in a dose-dependent manner. The Ni²⁺ block was fast and partially reversible at low concentrations and irreversible at high concentrations. ENaC inhibition by Ni²⁺ was accompanied by moderate inward rectification at concentrations higher than 0.1 mM. ENaC currents were also blocked by the histidine-reactive reagent diethyl pyrocarbonate. Pretreatment of the oocytes with the reagent reduced Ni²⁺ inhibition of the remaining current. Mutations at His²⁸² and His²³⁹ located within the extracellular loops significantly decreased Ni²⁺ inhibition of ENaC currents. The mutation H282D or double mutations H282R/H239R eliminated Ni²⁺ block. All mutations at His²³⁹ eliminated Ni²⁺-induced inward current rectification. Ni²⁺ block was significantly enhanced by introduction of a histidine at Arg²⁸⁰. Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni²⁺ block. Although H282C-- channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), -- H239C channels were insensitive to MTSET. From patch clamp studies, Ni²⁺ did not affect unitary current but decreased open probability when perfused into the recording pipette. Our results suggest that external Ni²⁺ reduces ENaC open probability by binding to a site consisting of His²⁸² and His²³⁹ and that these histidine residues may participate in ENaC gating.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Epithelial Na⁺ channels (ENaCs)¹ mediate Na⁺ transport across high resistance epithelia and participate in the regulation of extracellular fluid volume and blood pressure. Molecular cloning has revealed that ENaC subunits (, , and ) belong to a super gene family which is now often referred to as ENaC/Deg family and consist of ENaCs, degenerins (DEG and MEC), acid-sensing ion channels (ASIC), and FMRFamide-gated Na⁺ channels (1-5). These channel proteins are composed of subunits that share a common membrane topology with two transmembrane domains, intracellular N and C termini and large extracellular loops (ECL). Members of the ENaC/Deg family are Na⁺-selective and sensitive to the epithelial Na⁺ channel blocker amiloride, although differences in factors that activate, or gate, these channels have been reported (1). Combined application of site-directed mutagenesis and oocyte expression has yielded important findings regarding structure and function relationships of ENaC (6-9). Although the exact subunit stoichiometry of -- ENaC is still controversial (10-12), all three ENaC subunits (, , and ) contribute to formation of the core structure, the ion conduction pore (13). Two amiloride-binding sites have been identified within ENaC subunits; one within the pore regions and a second within the ECL of ENaC (13-15). The main selectivity filter resides at a 3-residue tract ((G/S)XS) within the putative pore region (or equivalently termed as pre-M2 region) (16-20). Mutations that alter ENaC gating have been found in the N termini (21, 22), pore regions (23, 24), and nearby regions (25), M2 domains (26), and the C termini (27).

Variability in the ionic selectivity, conductance, and gating of ENaCs in both native tissues and expression systems has been observed (1). In part, the functional diversity of the Na⁺ channels may reflect the influence of intracellular and extracellular factors that regulate channel activity. Divalent cations are important regulators of many ion channels, especially voltage-gated and ligand-gated channels (28). Several cations (Ca²⁺, Mg²⁺, and Ba²⁺) were reported to block Na⁺ currents from the external side with low affinity and complex features in native tissues (29). Recently, it was reported that external Ni²⁺ stimulated short circuit currents in A6 monolayers (30) and blocked whole-cell currents in oocytes expressing rat -- ENaC (31). We report the inhibitory effects of extracellular Ni²⁺ on the whole-cell and single channel currents in Xenopus oocytes expressing -- mouse ENaC (mENaC), and the identification of two histidine residues within the extracellular domain as putative Ni²⁺ binding sites. Preliminary description of this work was presented in abstract form (32, 33).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Site-directed Mutagenesis and Functional Expression of -- mENaC in Xenopus oocytes-- Point mutations were generated in , , or  mENaC cDNAs (34) cloned into pBluescript SK vector (Stratagene, La Jolla, CA) using two-step PCR methods as described previously (19). cRNAs were synthesized with T3 RNA polymerase (Ambion Inc., Austin, TX) and dissolved in nuclease-free water (Ambion Inc.). Stage V-VI Xenopus oocytes were treated with collagenase type IV (Sigma) to remove follicle cell layers and injected with 1-4 ng of cRNA for each mENaC subunit per cell in a total volume of 50 µl. Injected oocytes were maintained at 18 °C in modified Barth's saline.

Two-electrode Voltage Clamp-- Two-electrode voltage clamp was performed 24-72 h after cRNA injection at room temperature (20-24 °C) as described previously (19). The bath solution contained 110 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 10 mM HEPES, and the pH was adjusted to 7.40 with NaOH. Pipettes filled with 3 M KCl had a resistance of 0.5-2 megohms. Typically, oocytes were clamped from 140 to 60 mV in increments of 20 mV for 500 ms every 2 s. Amiloride at the concentration of 100 µM was added to the bath at the end of each experiment to determine the amiloride-insensitive current that was used to calculate amiloride-sensitive currents. Data acquisition and analyses were performed with pClamp 7.1 for Windows (Axon Instruments, Union City, CA).

Patch Clamp-- Vitelline membranes of oocytes were removed manually following incubation of the oocytes at room temperature in a hypertonic solution containing 200 mM sucrose. Oocytes were then transferred to a recording chamber with bath solution and allowed to recover for 30 min before clamping. The bath solution contained 110 mM NaCl, 2 mM CaCl₂, 10 mM HEPES, pH 7.40. For Na⁺ current recordings the pipette solution was identical to the bath solution, and for Li⁺ current recordings NaCl was replaced by LiCl. Glass pipettes with tip resistances of 5-20 megohms were used. Single channel currents were recorded in cell-attached configuration using PC ONE Patch Clamp amplifier (Dagan Corp., Minneapolis, MN), DigiData 1322A interface, and Clampex 8.1 software (Axon).

Examination of Effects of Inhibitors-- Inhibitors used in two-electrode voltage clamps (NiCl₂, MgCl₂, amiloride, diethyl pyrocarbonate, and sulfhydryl reagents) were prepared in the bath solution and delivered to the bath by gravity perfusion. To avoid hydrolysis, diethyl pyrocarbonate (DEPC) and sulfhydryl reagents were prepared immediately prior to use. Whole-cell Na⁺ currents were recorded before and after external application of an inhibitor. The effects of a certain inhibitor on ENaC currents were defined by comparison of the amiloride-sensitive currents before and after perfusion of the inhibitor in the same oocyte. Dose-response relationship for Ni²⁺ inhibition of mENaC currents was obtained by plotting the relative currents measured at 100 mV and in increasing concentrations of NiCl₂ (0.01, 0.1, 1, 10, 25, and 50 mM) against Ni²⁺ concentrations using a semi-logarithmic scale. The relative currents represent the ratios of whole-cell amiloride-sensitive Na⁺ currents in the presence of Ni²⁺ in bath solutions relative to the current measured immediately before application of Ni²⁺. The osmolarity of the bath solution containing NiCl₂ was not adjusted. Non-linear least squares curve fitting was used to obtain the dose-response parameters. In most cases dose-response data were fitted with both one-site and two-site equations using software OriginPro 7.0 for Windows (OriginLab Corp., Northampton, MA). The one-site Equation 1 is as follows:

(Eq. 1)

where I_R is the relative current in the presence of a specific concentration of inhibitor; K_i is the inhibition constant; C is the concentration of an inhibitor, and n represents Hill coefficient. The two-site Equation 2 is as follows:

(Eq. 2)

where I_R and C are as defined for Equation 1; K₁ and K₂ are the K_i values for two components in the dose-response curve; A and B are the fractions of each component, and their sum equals the total drop of I_R in the dose-response curve. The goodness of fitting of curves obtained from the one-site or two-site equation was compared by an F test designed to assess changes in both the sum-of-squares and degrees of freedom using Prism 3.0 (GraphPad Software Inc., San Diego). Correlation coefficients (R²) calculated from curve fittings were also used as a parameter of goodness of fitting.

Pipette perfusion of Ni²⁺ in patch clamps was performed by applying either positive pressure to the perfusion vial or negative pressure to the pipette using 2PK+ Whole-cell/Patch Perfusion Kit (ALA Scientific Instruments Inc., Westbury, NY). Success of pipette perfusion was judged by examining the increase in pipette solution and additional noise in the recordings.

Statistical Analyses-- Data are expressed as mean ± S.E. in most figures. Student's t test was used to assess significance of difference between WT and mutant channels or between mutant channels. Non-linear curve fitting was performed as above with Origin 7.0 Pro.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

External Ni²⁺ Inhibited Amiloride-sensitive Na⁺ Currents in Oocytes Expressing -- mENaC-- The effect of externally applied Ni²⁺ on whole-cell amiloride-sensitive Na⁺ currents was studied in Xenopus oocytes expressing -- mENaC. Ni²⁺ inhibited amiloride-sensitive Na⁺ currents in a dose-dependent manner in the concentration range of 0.01-50 mM (Fig. 1A). At 50 mM, Ni²⁺ inhibited about 80% of the amiloride-sensitive current. The dose-response data were fitted reasonably well with the one-site equation described under "Experimental Procedures" (Fig. 1C, dashed line). The estimated inhibitory constant (K_i) and Hill coefficient were 0.58 ± 0.09 (n = 6) and 0.37 ± 0.01 mM (n = 6), respectively (Table I). However, the dose-response curve appeared to contain more than one component. Data were fitted with the two-site equation as described under "Experimental Procedures," assuming that two classes of binding sites with different apparent affinities for Ni²⁺ account for the inhibitory effect of Ni²⁺ on ENaC currents (Fig. 1C, solid line). The non-linear least square fitting resulted in a higher correlation coefficient with the two-site equation (0.9958) than that obtained from fitting with the one-site equation (0.9545). According to the F test, the two-site equation was significantly better than the one-site equation to describe the data (p < 0.05). Two K_i values were 0.05 ± 0.00 and 11.00 ± 1.60 mM (n = 6).

View larger version (36K): [in this window] [in a new window]   Fig. 1.   External Ni2+ reduced whole-cell Na+ currents in oocytes expressing -- mENaC. A, representative recordings in an oocyte expressing WT -- mENaCs by two-electrode voltage clamp. Na+ currents were measured by clamping the oocyte from 140 to 60 mV in the presence of 0 (control), 0.01, 0.1, 1, 10, 25, and 50 mM NiCl2 in the bath solution. The oocyte was then washed with bath solution for 2 min and finally perfused with 0.1 mM amiloride prepared in the bath solution. Scales are identical and shown at the low right-hand corner. The dashed lines over current traces indicate zero current levels. B, current-voltage relationship (I-V) curves were generated by plotting amiloride-sensitive currents against clamping voltages from the above recordings. For clarity, only I-V curves obtained in 0 (), 0.01 (), 0.1 (), 1 (), 10 (), 25 (×), and 50 (+) mM NiCl2 are shown. C, dose-response relationship of Ni2+ inhibition of the Na+ currents. Relative currents represent currents in the presence of Ni2+ normalized to the control currents. Data points displayed as open circles with vertical bars represent mean ± S.E. from six oocytes. The dashed line is from curve fitting with a one-site equation (fitting parameters: Ki = 0.54, Hill coefficient = 0.37, R2 = 0.955), and the solid line is from fitting with a two-site equation (fitting parameters: K1 = 0.05 mM, K2 = 10.23 mM, A = 0.63, B = 0.2) as described under "Experimental Procedures." D, time course of Ni2+ inhibition. Oocytes were clamped to 60 mV and 60 mV for 450 ms every 5 s during experiments. Relative currents were obtained by normalizing the currents at 60 mV to the current level immediately before Ni2+ reached bath. Data are presented as mean ± S.E. (vertical lines) from five oocytes. Solid and open bars indicate the periods of application of 1 mM Ni2+ and 0.1 mM amiloride, respectively. E, lack of reversibility of Ni2+ inhibition at high concentration. Relative currents are shown in the absence and presence of 50 mM Ni2+, and after a 2-min washout. Vertical bars are S.E. (n = 8).

The current reduction by Ni²⁺ was accompanied by a moderate inward rectification as the concentration of Ni²⁺ exceeded 0.1 mM (Fig. 1, A and B). The current-voltage relationship curves (I-V) indicate that Ni²⁺ inhibition of ENaC currents was enhanced when oocyte membranes were depolarized. The Ni²⁺-induced current rectification is different from that induced by amiloride, which is generally considered as a pore blocker for ENaC and results in outward current rectification (35, 36).

The Ni²⁺ inhibition of mENaC currents was fast with a maximal effect observed around 1 min after initiation of Ni²⁺ perfusion (Fig. 1D). Removal of Ni²⁺ from the bath solution partially restored the Na⁺ currents when Ni²⁺ was applied at 1 mM. However, at a high concentration (50 mM) Ni²⁺ inhibition was not reversed by removal of Ni²⁺ from the bath for a period of 2 min (Fig. 1E).

It was reported that external Ca²⁺, Mg²⁺, Ba²⁺, and Sr²⁺ blocked Na⁺ channels in toad urinary bladder with complex voltage dependence. The blocking effects were rather weak with calculated concentrations for half-maximal inhibition of greater than tens of millimolar (29). To test whether the relatively high affinity Ni²⁺ inhibition of -- mENaC was unique among divalent cations, we examined several other divalent cations for their effects on -- mENaC. As shown in Fig. 2, external Mg²⁺ produced a weak inhibition of amiloride-sensitive Na⁺ currents with an estimated K_i of 73.5 ± 10.9 mM (n = 5) and a Hill coefficient of 0.47 ± 0.05 (n = 5). Furthermore, current rectification was not observed in the presence of external Mg²⁺. The inhibition potency of external Mg²⁺ on mENaC is similar to that observed in toad bladder (29). We previously reported that external Cd²⁺ moderately increased amiloride-sensitive Na⁺ currents at a concentration of 5 mM (24). Schild et al. (13) reported that inward Li⁺ currents in oocytes expressing -- rat ENaC were insensitive to external Zn²⁺ even in the millimolar range, and a small current reduction (~20%) was shown with 10 mM Zn²⁺. We observed no inhibition of amiloride-sensitive Na⁺ currents with 5 mM Zn²⁺ in the bath solution. However, at low concentrations (0.01-1 mM), Zn²⁺ produced a moderate potentiation of the Na⁺ currents, similar in amplitude to the increase in Na⁺ currents observed with Cd²⁺.² Therefore, among divalent cations (Ca²⁺, Mg²⁺, Ba²⁺, Sr²⁺, Zn²⁺, Cd²⁺, and Ni²⁺), Ni²⁺ appears to be the most potent external inhibitor of ENaC.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effect of external Mg2+ on ENaC. A, dose-response of external Mg2+ on -- mENaC Na+ currents. Dashed line is from fitting the data with one-site equation. The fitting parameters are as follows: Ki, 72 mM; Hill coefficient, 0.46. B, current-voltage relationships in the absence () and presence of 0.01 (), 0.1 (), 1 (), 10 (), 25 (×), and 50 (+) mM MgCl2 were generated the same way as Fig. 1B.

DEPC Inhibited -- mENaC Currents and DEPC Pretreatment Rendered the Channels Less Sensitive to Ni²⁺-- Nickel is a transition ion with polarizability between those of hard ions and soft ions. Nitrogen is considered as its most preferred ligand, although sulfur and oxygen can also coordinate Ni²⁺. Nickel is often coordinated by nitrogen atoms from histidine residues in nickel enzymes (37-39), and it is conceivable that Ni²⁺ binds to histidine residue(s) within ENaC subunits to exert its inhibitory effect on the channel. We therefore examined if the histidine-modifying reagent DEPC had any effect on -- mENaC expressed in oocytes and whether DEPC-modified channels exhibited an altered response to Ni²⁺. Perfusion of oocytes with bath solutions containing DEPC at concentrations of 0.1, 1, and 10 mM blocked amiloride-sensitive Na⁺ currents in a dose-dependent manner. The dose-response relationship was satisfactorily fitted with the one-site equation (R² = 0.998) (Fig. 3A). The estimated K_i was 0.45 ± 0.07 mM and the Hill coefficient was 0.99 ± 0.05 (n = 4). The Na⁺ currents were almost (~94%) completely blocked by 10 mM DEPC. DEPC block was essentially irreversible. In contrast to Ni²⁺-induced inward rectification, the amiloride-sensitive Na⁺ currents in the presence of DEPC were linear in the range of 140 to 60 mV (Fig. 3B). After perfusion of 1 mM DEPC, the remaining currents were essentially insensitive to Ni²⁺ (Fig. 3C). The currents from oocytes pretreated with DEPC and perfused with Ni²⁺ did not exhibit rectification (Fig. 3D). These results suggest that histidine residues within ENaC, accessible from extracellular space, participate in Ni²⁺ inhibition of ENaC.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of DEPC on ENaC currents and Ni2+ inhibition. A, dose-response of DEPC inhibition of ENaC currents. Amiloride-sensitive Na+ currents were measured at 100 mV before and after perfusion of 0.1, 1, and 10 mM DEPC prepared in the bath solution for 3 min. Relative currents were calculated by normalizing the currents in the presence of DEPC to the current before DEPC application. The mean relative currents are displayed as open square and vertical bars are S.E. (n = 4). Dashed line is from fitting the data with one-site equation. The fitting parameters are as follows: Ki, 0.44 mM; Hill coefficient, 0.98; and R2, 0.999. B, current-voltage relationship curves in the absence () and presence of 0.1 (), 1 mM (), and 10 mM (×) DEPC in the bath solution and 2 min after washout of DEPC from the bath solution () were generated from amiloride-sensitive currents (mean ± S.E., n = 4). C, Ni2+ dose response following treatment of 1 mM DEPC was examined the same way as above after obtaining control currents measured after washout of DEPC for 2 min. The amiloride-sensitive currents in the presence of Ni2+ were normalized to the control currents, which gave the relative currents expressed in the dose-response curve as mean ± S.E. (n = 4). D, I-V curves in the absence of DEPC and Ni2+ () and presence of 0.01 (×), 0.1 (), 1 (+), 10 (), 25 (), and 50 mM () Ni2+ following 3 min of perfusion of 1 mM DEPC () and washout of DEPC for 3 min () were generated from amiloride-sensitive Na+ currents and are representative of four oocytes.

Ni²⁺ Inhibition of mENaC Currents Was Attenuated by Mutations at His²⁸² and His²³⁹ within the Extracellular Domains of  and  mENaC-- Our results suggest that Ni²⁺ is not a pore blocker of ENaC as it produced inward current rectification rather than outward rectification, and the inhibition of currents was only partially reversible following removal of Ni²⁺ from the bath. The experiments with DEPC suggested that histidine residues might be involved in Ni²⁺ block of ENaC currents. There are numerous histidine residues within the ECLs of each mENaC subunit, 6 in  mENaC, 11 in  mENaC, and 10 in  mENaC. However, only one histidine is conserved within the ECLs of the three subunits (His³⁸¹, His³¹⁹, and His³³⁸). These conserved histidine residues were individually mutated to an arginine, and Ni²⁺ sensitivity of -- mENaCs containing a mutation within one subunit was examined and compared with wild type (WT) mENaC. Significant changes in Ni²⁺ sensitivity were not observed with channels containing a mutation of these conserved histidine residues, suggesting that these conserved histidine residues are not essential for Ni²⁺ inhibition of ENaC (Table I).

We previously identified an amiloride-binding domain within the ECL of  rat ENaC based on homology with an amiloride-binding site within an anti-amiloride antibody (14, 40). Selected mutations within a six-residue tract (residues 278-283, WYRFHY), including His²⁸², altered the amiloride sensitivity of channels composed solely of -subunits reconstituted in planar lipid bilayers, although the amiloride sensitivity of -- ENaC containing an His²⁸² mutation was unchanged (11, 17). We examined whether His²⁸² served as one of the Ni²⁺-binding ligands by generating point mutations at this site in  mENaC. As shown in Fig. 4A, all four mutations (H282C, H282R, H282W, and H282D) dramatically decreased Ni²⁺ inhibition as evidenced by the shift in the Ni²⁺ dose-response curves of the mutant channels to the right with respect to that of WT. The changes in Ni²⁺ inhibition observed with these mutant channels were H282D > H282W > H282R > H282C. Only the dose-response data for H282C-- mENaCs were fitted reasonably well with the one-site equation that yielded parameters as follows: K_i, 7.46 mM; Hill coefficient, 0.45; and R², 0.968. No inhibition of amiloride-sensitive Na⁺ currents by Ni²⁺ was observed in oocytes expressing H282D-- mENaCs. On the contrary, external Ni²⁺ induced a significant increase in H282D-- currents at concentrations of 0.01, 0.1, and 1 mM, with peak stimulation at 0.1 mM. At higher Ni²⁺ concentrations, no change in Na⁺ currents was observed when compared with control currents. The data suggest that His²⁸² participates in the binding of Ni²⁺ to ENaC. Interestingly, inward current rectification was observed in the presence of Ni²⁺ with all four mutant channels despite significantly reduced Ni²⁺ inhibition of the Na⁺ currents (Fig. 4, B-E). For H282D-- mENaC, current rectification was evident in the absence of Ni²⁺ (Fig. 4C).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Mutations at His282 reduced Ni2+ inhibition. A, dose-response curves of external Ni2+ on H282R-- (), H282D-- (), H282C-- (), and H282W-- () mENaCs. WT dose-response () is shown for comparison. Data are shown as mean ± S.E. Numbers of clamped oocytes are 4 for H282C-- and H282W--, 5 for H282R--, and 14 for H282D--. B-E, I-V curves in the absence and presence of external Ni2+ were obtained as above from amiloride-sensitive Na+ currents. Symbols used in the I-V curves are identical and shown at the lower right-hand corner. The curves represent the results from 5 oocytes for B, D, and E and 14 oocytes for C.

The above results encouraged us to examine whether an equivalent histidine residue in mENaC (His²³⁹) was also involved in Ni²⁺ inhibition. The mutations H239C, H239R, and H239D significantly attenuated Ni²⁺ inhibition (Fig. 5). Surprisingly, Na⁺ currents in oocytes expressing the mutant channels did not exhibit rectification in the presence of Ni²⁺ (Fig. 5, B-D), suggesting that His²³⁹ is required for Ni²⁺-induced current rectification. The double mutations (H282R and H239R) eliminated Ni²⁺ inhibition of Na⁺ currents (Fig. 6A); these channels did not display current rectification (Fig. 6B). These results provided strong evidence that His²⁸² and His²³⁹ provide Ni²⁺-binding sites and are primarily responsible for Ni²⁺ inhibition of ENaC currents. Introduction of a histidine residue at the corresponding site in  mENaC (Q220H) resulted in a modest increase in Ni²⁺ K_i, suggesting that this residue does not have an important role in Ni²⁺ inhibition of ENaC (Table I).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Mutations at His239 reduced Ni2+ inhibition. A, dose-response curves of Ni2+ on --H239R (), --H239D (), and --H239C () mENaCs were generated by fitting the data with the one-site equation (dashed lines) and two-site equation (solid lines) as described under "Experimental Procedures." WT dose-response curve () is shown for comparison. Data are shown as mean ± S.E. Numbers of clamped oocytes are 6, 5, and 7 for --H239R, --H239D, and --H239C, respectively. B-D, I-V curves in the absence and presence of varying concentrations of external Ni2+ were obtained by plotting the amiloride-sensitive Na+ currents against clamping voltages. Mutant channels are identified as point mutation names. Symbols used in the I-V curves are the same as in Fig. 4. The curves represent the results from 5 to 7 oocytes.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Double mutations (H282R/H239R) eliminated Ni2+ inhibition. A, dose response of Ni2+ on amiloride-sensitive Na+ currents was examined in oocytes expressing H282R--H239R mENaCs. Relative currents in the presence of varying concentrations of external Ni2+ are displayed as filled squares (mean ± S.E., n = 5). Dashed line was from fitting the data with the one-site equation. The data could not be fitted with the two-site equation. B, I-V curves in the absence and presence of Ni2+ were generated same way as above, and symbols are identical to those in Fig. 4. The curves are representative of five oocytes.

Introduction of Additional Histidine Residues at Sites Neighboring His²⁸² Altered Ni²⁺ Inhibition of ENaC Currents-- In nickel-binding proteins, a Ni²⁺ can be coordinated by one (41), two (42-44), three (45, 46), or four (47) histidine residues, and two histidine residues are often separated by one or three residues (HXH or HXXXH) (37, 48-50). A sequence "HEXXH" located in an -helix has been considered as a signature metal-binding motif for metalloproteins (45, 46, 51, 52). To gain more knowledge about the putative Ni²⁺-binding site, we examined whether the introduction of additional histidine residue(s) in the vicinity of His²⁸² enhanced Ni²⁺ inhibition of ENaC currents, presumably by altering Ni²⁺ binding affinity. Histidine residues were individually introduced at positions Trp²⁷⁸, Tyr²⁷⁹, Arg²⁸⁰, Phe²⁸¹, Tyr²⁸³, Ile²⁸⁴, and Asn²⁸⁵. In addition, two -subunit mutants with consecutive 4-histidine tracts, Y279H/R280H/F281H/H282 (referred to as His^279-282) and H282/Y283H/I284H/N285H (referred to as His^282-285), were also generated. The effects of Ni²⁺ on these mutant channels are shown in Fig. 7. The mutant channel R280H-- showed a significant increase in Ni²⁺ sensitivity, greatly shifting the high affinity component in the dose-response curve to the left with less effect on the low affinity component compared with WT dose response (Table II). Interestingly, double mutations (R280H and H239R) shifted the high affinity component in the dose-response curve to the right compared with R280H-- but to the left compared with WT mENaCs. The low affinity component was shifted to the right compared with both R280H-- and WT mENaCs (Fig. 7E and Table II). Like other mutations at His²⁸², R280H did not affect the Ni²⁺-induced current rectification (Fig. 7F). Three other mutations (W278H, Y283H, and N285H) slightly decreased Ni²⁺ block of the Na⁺ currents, whereas three other mutations (Y279H, F281H, and I284H) did not significantly change Ni²⁺ block (Fig. 7A). As observed with R280H-- channels, the mutant channels with a consecutive 4-histidine tract including R280H exhibited a large decrease in the Ni²⁺ K_i (His^279-282, K_i = 0.12 ± 0.03, n = 8). In contrast, the -subunit mutant with a consecutive 4-histidine tract C-terminal to (and including) His²⁸² exhibited a large increase in the Ni²⁺ K_i (His^282-285, K_i = 10.37 ± 0.92 mM, n = 5). These data suggest that R280H, in addition to His²⁸² and His²³⁹, participates in Ni²⁺ binding. Fig. 7G provides three models illustrating the coordination of Ni²⁺ by these residues, generated based on the changes in Ni²⁺ inhibition of ENaC currents observed with mutant channels and the preferred coordination geometries of Ni²⁺ complexes and Ni²⁺-binding peptides and proteins (see "Discussion").

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Effect of histidine substitution at His282 neighboring sites altered Ni2+ sensitivity. Histidine residues were introduced at varying sites within the tract WYRFHYIN (278-285). Sequences of the tract with the introduced histidine (boldface letter) are shown in C. A naturally occurring histidine (His282) is boxed throughout the sequences. WT sequence is underlined, and mutant channels are identified by the point mutations. His279-282 and His282-285 represent two multiple mutations: Y279H/R280H/F281H and Y283H/I284H/N285H. The dose responses of the mutant channels were examined as above. Data were fitted with both the one-site and two-site equations. For ease of comparison, Ki values and Hill coefficients from one-site fittings are shown in A and B. Data are displayed as mean ± S.E. from 5 to 12 oocytes. Open bars indicate no significant difference between mutant and WT channels (p > 0.05), and filled bars indicate the values of mutant channels that are statistically different from those of WT channels (p < 0.01). D, inward amiloride-sensitive Na+ currents measured at 100 mV in oocytes expressing WT or mutant mENaCs are shown as mean ± S.E. (number of oocytes). The negative sign indicating inward currents was removed for ease of display. The currents were not measured in the same batch of oocytes. E, dose responses of Ni2+ on WT (), R280H-- (), and R280H--H239R () mENaCs were examined with nine concentrations of Ni2+. Relative currents represent mean ± S.E. from 6 to 8 oocytes. Solid lines are from fitting the data with two-site equation. F, I-V curves of amiloride-sensitive Na+ currents in the absence () and presence of 0.001 (), 0.003 (), 0.01 (), 0.03 (), 0.1 (×), 1 (+), 10 (), 25 (), 50 (), and 100 (no symbol) mM Ni2+ in the bath solution from an oocyte expressing R280H-- mENaCs were made as above. They represent the results from six oocytes. G, proposed Ni2+ coordination models for WT (left), R280H (middle), and R280H/H239R mENaCs (right). For WT channels, Ni2+ is coordinated by three nitrogen atoms from two His282 and one His239 and the fourth ligand (water oxygen). For R280H-- mENaCs, the introduced histidines provide two additional ligands besides the four ligands in WT. A solvent (water) replaces the eliminated His239 in R280H--H239R channels.

MTSET Inhibited Amiloride-sensitive Na⁺ Currents in Oocytes Expressing H282C-- mENaCs-- The above results suggested that a Ni²⁺-binding site consisting of His²⁸² and His²³⁹ has a key role in Ni²⁺ inhibition of ENaC currents. These histidine residues may also have an important functional role. To explore further the role of His²⁸² in channel activity, we examined the response of the mutant channels (H282C--) to external sulfhydryl reagents. The positively charged reagent MTSET irreversibly inhibited amiloride-sensitive Na⁺ currents by 45% when applied externally at the concentration of 1 mM (Fig. 8, A and B). As observed with Ni²⁺, inward rectification was observed following MTSET modification of the channel (Fig. 8B). The remaining currents following MTSET modification were less sensitive to Ni²⁺ (Fig. 8C). MTSET at 1 mM did not alter amiloride-sensitive Na⁺ currents in oocytes expressing WT -- mENaCs (24) or --H239C mENaCs (Fig. 8A), and pretreatment of the oocytes with the reagent did not alter Ni²⁺ inhibition of these channels. The negatively charged reagent MTSES [sodium (2-sulfonatoethyl) methanethiosulfonate] at 5 mM also inhibited about 40% of the Na⁺ currents of H282C-- channels, whereas it did not change the currents of WT channels (24). A possible explanation of why MTSET failed to inhibit --H239C mENaC is illustrated in Fig. 8D. We propose that His²³⁹ is located deeper within a putative Ni²⁺-binding pocket than His²⁸² and is not accessible to MTSET that requires a cylindrical space of 6 × 10 Å (53).

View larger version (34K): [in this window] [in a new window]   Fig. 8.   MTSET reduced Na+ currents in oocytes expressing H282C-- mENaC and further reduced Ni2+ inhibition of the remaining currents. A, effects of MTSET on H282C-- and --H239C mENaCs. Amiloride-sensitive Na+ currents were measured at 100 mV before, 2 min after perfusion of 1 mM MTSET prepared in the bath solution, and 2 min after washout of MTSET from the bath. Relative currents were obtained by normalizing the currents to the levels before application of MTSET. Open, filled, and shaded bars are the relative currents (mean ± S.E., n = 5 for H282C-- and n = 13 for --H239C) obtained before and after MTSET and after washout of MTSET, respectively. B, I-V curves of amiloride-sensitive Na+ currents before () and after () MTSET and after washout of MTSET () were generated as before. Data are mean ± S.E. from five oocytes. C, Ni2+ dose responses on H282C-- mENaC currents without () and with () treatment of the oocytes with 1 mM MTSET. The dose response of Ni2+ on H282C-- mENaCs without MTSET is the same as in Fig. 5A. Relative currents in solid circles (mean ± S.E., n = 5) were obtained by normalizing the currents in the presence of Ni2+ to the current levels following 1 mM MTSET perfusion for 3 min and washout of the reagent for 2 min, and immediately prior to Ni2+ application. Dashed lines are from fitting the data with the one-site equation. D shows one of the possible reasons why MTSET inhibited H282C-- mENaC without effect on --H239C mENaC. The shaded area shows part of the ENaC ECLs with a semicircle pocket designated as a Ni2+-binding site. The three open rectangles represent three Ni2+-binding ligands out of a possible total number of four or six ligands. Side chains of His282 and His239 provide three of the ligands. A circle inside the pocket represents a bound Ni2+ ion to WT channels. One molecule of MTSET is drawn with space-filled model, and its thiol head is placed at the entrance of the pocket. Attachment of MTSET to sulfhydryl group of H282C would prevent Ni2+ from entering the binding site to cause inhibition of the channel currents. The SH group of H239C may be too deep for interaction with MTSET.

In summary, we observed that amiloride-sensitive Na⁺ currents were blocked by three different reagents. Ni²⁺ and DEPC blocked WT ENaC. MTSET and MTSES blocked H282C. Moreover, pretreatment of channels with either DEPC or MTSET resulted in a reduced response to Ni²⁺, suggesting that these three reagents interact with a common site within the channel. Furthermore, these results suggest that His²⁸² and His²³⁹ have an important role in channel function.

Ni²⁺ Block Was Reduced by Extracellular Acidification but Not by Amiloride-- The imidazole rings from histidine residues have pK_a values of 6.0 and 14 and may exist in different protonation states including a protonated imidizolium, a neutral imidazole, and a negatively charged imidazolate. Protonated imidizolium cannot coordinate a metal ion (54). Therefore, we examined whether Ni²⁺ inhibition of ENaC currents was affected by acidification of the bath solution. The Ni²⁺ inhibitory effect on Na⁺ currents was examined with bath solutions buffered to pH 7.4 with HEPES (buffer range 6.8-8.2), 5.5 with MES (buffer range 5.5-6.5), or 4.4 with citric acid (buffer range 2.2-6.5). Fig. 9 shows that lowering the bath pH reduced (at pH 5.5) or eliminated (at pH 4.4) the Na⁺ current inhibition by Ni²⁺. We observed a biphasic effect of extracellular acidification on amiloride-sensitive Na⁺ currents, a brief stimulation (10% for pH 5.5 and 70% for 4.4) with peak effect at 1-2 min followed by a return to basal current or below basal current.² These results are consistent with a recent report (55) and inconsistent with others (56, 57). Because of the biphasic pH effects, a Ni²⁺ dose response could not be accurately obtained. Instead, we compared the effects of 1 mM Ni²⁺ on the Na⁺ currents at different pH values. As different effects of extracellular acidification were observed in native tissues and oocytes expressing cloned ENaC, the physiological significance of these pH effects is not clear (55).

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Ni2+ inhibition of ENaC was reduced by extracellular acidification. The effects of 1 mM external Ni2+ on amiloride-sensitive Na+ currents at three external pH values (7.4, 5.5, and 4.4) were examined in oocytes expressing -- mENaCs. Bars represent relative currents calculated by normalizing the amiloride-sensitive Na+ currents in the absence (open bars) and presence (solid bars) of 1 mM Ni2+ to the current levels in the absence of Ni2+ in each bath solution with the corresponding pH value. Data are mean ± S.E., and the numbers of observations are indicated in the parentheses. The * indicates the absolute currents in the presence of 1 mM Ni2+ are significantly lower than the absolute control currents with pH 7.4 and 5.5 (p < 0.001). The difference between the control currents and currents in the presence of 1 mM Ni2+ at pH 4.4 is not statistically significant (p > 0.05). Triangles indicate that the relative currents with 1 mM Ni2+ at pH 5.5 and 4.4 are significantly higher than the relative current with 1 mM Ni2+ at pH 7.4.

Residue His²⁸² is located within a previously identified amiloride-binding domain in the ECL of the  subunit (14, 40). We have demonstrated that His²⁸², together with His²³⁹, is involved in Ni²⁺ inhibition of ENaC. We performed two experiments to examine whether Ni²⁺ and amiloride compete for the same binding site when both inhibitors were present in the bath solution. First, we determined the response of WT -- mENaC to increasing concentrations of amiloride in the presence or absence of an intermediate concentration of Ni²⁺ (1 mM). Ni²⁺ did not significantly alter the sensitivity of the channel to amiloride.² Second, the response of WT channels to increasing concentrations of Ni²⁺ was examined in the presence or absence of an intermediate concentration (100 nM) of amiloride. Amiloride did not significantly alter the sensitivity of the channel to Ni²⁺.² These results suggest that Ni²⁺ and amiloride do not compete for the same site in -- ENaC.

Ni²⁺ Reduced ENaC Open Probability without Altering Single Channel Current Levels-- Reduction of whole-cell Na⁺ currents by external Ni²⁺ could be due to a decrease in single channel conductance, open probability (P_o) of individual channels, number of functional channels on the cell surface, or electrochemical driving force, or by a combination of these factors. We performed single channel recordings to distinguish these possibilities. The unitary Na⁺ currents were not altered by including 10 mM Ni²⁺ in the pipette solution (Fig. 10, A and B). As ENaC open probability is highly variable from patch to patch, it is difficult to determine whether Ni²⁺ reduces ENaC P_o by comparing the P_o values from patches performed in the absence and presence of Ni²⁺ in the pipette solution. We therefore examined channel transitions while Ni²⁺ was being added to the patch pipette by pipette perfusion. The results from pipette perfusion experiments indicated that Ni²⁺ reduced NP_o of mENaC without effect on unitary Li⁺ current (Fig. 10, C-F). Single channel conductances before and after pipette perfusion of Ni²⁺ were 8.3 ± 0.4 pS (n = 4) and 8.1 ± 0.3 pS (n = 4), respectively. Since Ni²⁺ inhibition was not accompanied by a decrease in the reversal potential in oocytes, it was unlikely that external Ni²⁺ in the millimolar range significantly altered the driving force for Na⁺ influx. Moreover, the density of channel proteins was not likely reduced by external Ni²⁺ within 1 min. Therefore, our results suggest that Ni²⁺ inhibition of ENaC currents is due to a reduction of channel P_o.

View larger version (34K): [in this window] [in a new window]   Fig. 10.   Ni2+ reduced Po without changing single channel current. Patch clamp recordings were performed in cell-attached configuration in oocytes expressing -- mENaCs. Oocytes were bathed in the same bath solution as used in the two-electrode voltage clamp studies. Na+ currents were recorded at 100 mV (membrane potential) in a pipette solution either same as the bath solution (A) or with the bath solution containing 1 mM NiCl2 (B). Li+ current was recorded at 60 mV before pipette perfusion (C) with a pipette solution containing 110 mM LiCl, 2 mM CaCl2, 10 mM HEPES with pH of 7.4. Traces D-F were from the same patch as in C but recorded at 2, 5, or 8 min following pipette perfusion of 10 mM NiCl2. All recordings are shown on the same scale (right to each trace) with total length of 120 s. Dashed lines indicate the closed state, and solid lines indicate open levels. Current traces were filtered at 100 Hz with Clampfit 8.1 (Axon). All-points amplitude histograms (G) were generated using Fetchan 6 (Axon) from the current recording before (0-400 s, left panel) or after pipette perfusion (600-1500 s, right panel). Letters C, O1, O2, and O3 indicate closed, or the first, second, or the third open state, respectively. The dashed lines were from least square fitting with the Levenberg-Marquardt Method using Pstat 6 (Axon).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ni²⁺ Is an External Inhibitor of ENaC-- In this study, we found that external Ni²⁺ inhibited amiloride-sensitive whole-cell Na⁺ currents from oocytes expressing -- mENaC in a dose-dependent manner. The blocking effect of Ni²⁺ is in good agreement with that observed by Segal and colleagues (31) who reported that 2.5 mM Ni²⁺ inhibited 60% of -- rENaC currents. Another group recently reported (58) that Ni²⁺ and other Ca²⁺ channel blockers partially blocked amiloride-sensitive short circuit currents in fetal rat alveolar type II epithelia. Apical membranes of this type of epithelia are believed to have two types of Ca²⁺-activated and amiloride-sensitive channels (28 pS nonselective cation channel and 12 pS Na⁺ channel) whose properties are clearly different from those of -- ENaC (59, 60). Therefore, the identity of the Ni²⁺-blocked channels is not clear, although under certain cultural conditions the predominant channels expressed in these cells show single channel properties similar to those of ENaCs (61). It appears that Ni²⁺ is the only divalent cation that externally blocks ENaC at submillimolar concentrations. Why is Ni²⁺ a high affinity blocker of ENaC? A simple explanation is that metal ion-binding site(s) are located in functional domains within the ENaC complex that exhibit higher affinity for Ni²⁺ than other divalent cations. In metalloproteins, formation of a metal-ligand complex is dependent on many factors including the size, relative charge, polarizability (softness and hardness), preferential coordination geometry of the ion, the liganding donor atoms, and solvent effects (62, 63). The ionic radius of the divalent cation does not appear to be the most important determinant in blocking ENaC. A smaller cation Mg²⁺ (0.65 Å, Pauling (64)) is a much weaker inhibitor of ENaC than Ni²⁺ (0.72 Å), and a larger cation Cd²⁺ (0.97 Å) does not inhibit ENaC at a concentration of 5 mM (24). Moreover, Zn²⁺ (0.74 Å) with very close radius to Ni²⁺ has no inhibitory effect on ENaC at millimolar concentrations (13). As transition metal ions, Ni²⁺ and Zn²⁺ have a number of common properties including size, charge, intermediate polarizability (not as "hard" as Ca²⁺ and Mg²⁺ and not as "soft" as Cd²⁺ and Hg²⁺), and favor nitrogen and sulfur atoms as their binding ligands (62). It is not clear why Zn²⁺ and Ni²⁺ do not have similar inhibitory effects on ENaC. It was recently reported that Zn²⁺ might be a physiological co-activator of the acid-sensing ion channels (111). Therefore, the observed ENaC current stimulation by low concentrations of external Zn²⁺ suggests that Zn²⁺ might be a high affinity stimulator of ENaC. It has been known for a long time that sulfhydryl group-reactive reagents, including Cd²⁺, stimulate epithelial Na⁺ transport, possibly by relieving Na⁺ self-inhibition; however, its physiological significance and mechanism are still unknown (1).

The inhibition of ENaC currents by external Ni²⁺ could be due to a direct blocking mechanism (pore plugging) or an indirect one, often referred to as an allosteric mechanism. The inward current rectification observed in the presence of Ni²⁺ does not fit with a typical voltage-dependent block of a cation channel from the external side. For example, current-voltage relationship curves in the presence of submaximal concentrations of amiloride show outward rectification, indicating a reduced block of ENaC when the membrane is depolarized and consistent with the idea that amiloride is an external pore blocker of ENaC (1). The poor reversibility of Ni²⁺ inhibition of the ENaC currents suggests a complex feature of the blocking mechanism and is inconsistent with the notion of a direct pore plugging. We favor the idea that Ni²⁺ is an external inhibitor of ENaC but does not directly interact with the pore.

It has been known that external Ni²⁺ affects a variety of ion channels including voltage-gated Ca²⁺ channels (65-68), voltage-gated K⁺ channels (69-71), voltage-gated Na⁺ channels (72), voltage-gated H⁺ channels (73-75), CNG channels (76), P2X receptors (77, 78), -aminobutyric acid type A receptors (79), and glutamate receptors (80, 81). These channels display different sensitivities to Ni²⁺ with inhibitory constants in the range of micromolar to millimolar. The mechanism of Ni²⁺ effects on these channels is not clear, although both direct and allosteric blocking mechanisms have been suggested (67, 69, 70, 77, 82). Xenopus oocytes express several kinds of endogenous channels whose activity is normally small compared with that of overexpressed foreign channels (83). However, expression of selected exogenous proteins has been reported to activate certain endogenous channels that are normally rather "silent" (84, 85). Therefore, it is possible that the expressed whole-cell currents in oocytes may contain a significant level of endogenous channel activity that is sensitive to both external Ni²⁺ and amiloride if ENaC expression activates these channels. This is unlikely the case under our experimental conditions. First, there is no clear evidence that ENaC expression in Xenopus oocytes activates endogenous channels. When extracellular Na⁺ is replaced by K⁺ or Ca²⁺, no inward currents were detected in oocytes expressing ENaC that are clamped at 100 mV (16, 18, 19). These data indicated that endogenous channels do not provide a significant contribution to the whole-cell currents measured in ENaC-expressed oocytes under our typical clamping protocols (i.e. protocols that are not designed to elicit voltage-dependent currents). Second, we evaluated the Ni²⁺ effect on ENaC currents at a clamp potential of 100 mV, a potential at which many voltage-gated channels should not be activated. The linear I-V relationship in the range of 140 to 60 mV of the currents measured in ENaC-expressed oocytes indicated a lack of voltage-gated current. Moreover, the observed Ni²⁺ inhibition of ENaC currents was specifically eliminated by H282D mutation and by the double (H282R/H239R) mutation, which strongly suggests that the whole-cell current reduction is due to Ni²⁺ interaction with ENaC rather than with other channels. It was recently reported that external Ni²⁺ (less potent than Cd²⁺ and Zn²⁺) blocked a slowly activating Na⁺ current elicited by sustained depolarization of the Xenopus oocyte membrane, a current that is clearly different from ENaC currents (86).

Whether Ni²⁺ has any physiological role in channel regulation in humans is unclear, although it is known that Ni²⁺ is essential for bacteria and plant growth (87-89). As nickel is considered a hazardous ion to human, the inhibitory effect of Ni²⁺ on ENaC activity may contribute to its toxic effects on kidneys, lungs, and digestive system where ENaC is expressed. We anticipate that Ni²⁺ will be a useful tool to study the structure and function of epithelial Na⁺ channels. Nickel has been used successfully as a pharmacological tool to distinguish different types of voltage-gated Ca²⁺ channels and to gain useful information regarding gating mechanisms of CNG channels (90, 91).

Histidine Residues His²⁸² and His²³⁹ within the Extracellular Loops of ENaCs Form the Ni²⁺-binding Site-- The histidine-reactive reagent DEPC inhibited amiloride-sensitive Na⁺ currents with an estimated K_i of 0.45 mM, suggesting that histidine residue(s) in ENaC complex are located in a functional domain. The potential role of histidyl groups in amiloride-sensitive Na⁺ channels was proposed nearly 20 years ago based on the identification of a titratable group with a pK_a of 6.7 and DEPC inhibition of short circuit current in toad urinary bladder (92). Moderate DEPC treatment (1 mM) of oocytes expressing WT mENaCs significantly reduced Ni²⁺ inhibition on the remaining currents, suggesting that Ni²⁺ and DEPC act at a common site.

Our mutagenesis studies suggest that His²⁸² and His²³⁹ are the primary structural determinants for Ni²⁺ inhibition of mENaC currents. However, there were distinct changes associated with mutations at these two sites. The -mutation-induced changes in Ni²⁺ inhibition followed the order: H239R > H239D > H239C, consistent with the preferential coordination of Ni²⁺. Cysteine residues coordinate Ni²⁺ in hydrogenase, dehydrogenase, and deformylase enzymes (38, 39, 42, 44). Aspartic acid participates in Ni²⁺ coordination in urease (37). The relationship between substituted residues at His²⁸² and the degrees of alteration in Ni²⁺ inhibition did not follow the order observed with His²³⁹. The largest change in Ni²⁺ dose response on mENaC currents was observed with H282D (Fig. 4A). The most striking difference observed with the mutations His²⁸² and His²³⁹ was that all channels with mutations at His²³⁹ showed no Ni²⁺-induced current rectification (Fig. 5, B-D), whereas all channels with -subunit mutations (His²⁸² or Arg²⁸⁰) showed inward rectification in the presence of Ni²⁺ (Fig. 4, B-E, and Fig. 7F). These results suggest that the interaction of Ni²⁺ with His²³⁹ is required for Ni²⁺-induced current rectification.

Our results suggest that His²⁸² and His²³⁹ are involved in Ni²⁺ binding to the channel complex. However, other residues may be needed to complete the Ni²⁺ coordination geometry as Ni²⁺ is coordinated by 4, 5, or 6 ligands in most Ni²⁺-binding proteins (89). In order to identify other residues involved in Ni²⁺ binding and to probe the secondary structure of residues near His²⁸², one or three additional histidines were engineered in the tract WYRFHYIN (residues 278-285) of mENaC. Among 7 single histidine substitutions in this tract, R280H was the only mutation associated with enhanced Ni²⁺ inhibition of channel currents, suggesting that the introduced histidine at 280 contributed to Ni²⁺ coordination (Fig. 7G). The enhanced Ni²⁺ inhibition by R280H was attenuated by mutation of His²³⁹ (R280H--H239R). Although these data suggest that Arg²⁸⁰ and His²⁸² might be located in a -sheet structure, other secondary structures can also place these two residues in close proximity favoring metal coordination by both residues, such as  helix, 3₁₀ helix, and random coil. An -helical structure containing both residues would place Arg²⁸⁰ and His²⁸² on opposing faces of the helix (Fig. 11D) and is not consistent with the observed enhancement of Ni²⁺ inhibition by R280H. Another possibility is that Arg²⁸⁰ and His²⁸² are not located in the same secondary structure, in agreement with structural predictions that place Arg²⁸⁰ within an -helix and His²⁸² immediately adjacent to the -helix (Fig 11C). We generated a structural model of a Ni²⁺-binding site (Fig. 11G) based on consensus structural predictions of the regions flanking His²⁸² and His²³⁹ (Fig. 11, C and E) and our results from mutagenesis studies. The Ni²⁺-binding domain in the -subunit was modeled as an N-terminal -helix followed by a -sheet with His²⁸² at the transition point. Our model places R280H and His²⁸² at positions that allow both residues to participate in Ni²⁺ coordination. This proposed arrangement of R280H and His²⁸² is strikingly similar to the arrangement of a pair of metal-coordinating histidine residues, His¹¹² within an -helix and His¹¹⁰ adjacent to the helix in the Ni²⁺-binding metallochaperone UreE (48). The corresponding region in  mENaC was modeled as an -helix based on secondary structure predictions.

View larger version (48K): [in this window] [in a new window]   Fig. 11.   Sequence alignments, structure prediction, and working models for the Ni2+-binding domain and mechanism of Ni2+ inhibition. A, sequence alignments were performed with Vector NTI 7.0 (InforMax Inc., Bethesda) from sequences as follows: , , and  mouse ENaCs (GenBankTM accession numbers AF112185, AF112186, and AF112187); , , and  rat ENaCs (GenBankTM accession numbers X70497, X77932, and X77933); , , and  human ENaCs (GenBankTM accession numbers L29007, L36593, and L36592);  bovine ENaC (GenBankTM accession number U14944); and  human ENaC (GenBankTM accession number U38254). The first amino acid residue number for each sequence is listed in parentheses. Homology is presented in the following colors codes: identical, red background; similar, yellow background; and block of similar residues, green background. The histidine residues studied in this report are in boldface type. B, a linear model is shown to indicate the location of the histidine residues (His282 and His239, as a red sphere) in one ENaC subunit. Secondary structural predictions were performed on the entire amino acid sequence of mENaC (C) or mENaC (E) with five different methods and only predictions of Cys263-Pro291 and Cys216-247 are shown. GOR4 (115), PHD (116, 117), and Predator (118) predictions were done at the web site "Network Protein Sequence Analysis" (npsa-pbil.ibcp.fr). The predictions are presented as the following single letters: h for -helix; e for extended strand; and c for random coil. NNPredict was done at the web site (www.cmpharm.ucsf.edu/~nomi/nnpredict.html) (119, 120). A dash means no prediction by NNPredict. The four lines above the "Consensus" are predictions by DNASis software 2.6 for Windows (Hitashi Software Engineering Co., Ltd., South San Francisco, CA) using Chou-Fasman algorithm (121). The letters H (h), S (s), and T represent -helix, -strand, and turn, respectively. Uppercase letters indicate probability, and lowercase letters indicate possibility. Helical wheel analyses of  and  of mENaC were performed on Val272-His282 (D) and Asn230-His239 (E). Residue His282 is predicted beyond the helical region by most methods. It is included in the analysis to show that His282 and Arg280 would be on the opposite faces of the helix if His282 were considered within the helix. G, a structural model for Ni2+-binding site was generated based on the secondary structure predictions and mutagenesis results from this study (see text under "Discussion"). The model was built with HyperChem 6.03 (Hypercube, Inc., Gainesville, FL). Backbones are displayed as 9 thin green lines, and residues are shown in sticks. Colors for carbon, oxygen, nitrogen, and sulfur atoms are gray, red, blue, and yellow. For clarity, only selected residues are labeled with the one-letter symbol and sequence number. Two ENaC domains on the top were modeled as -helix (Val272-Phe281) and -sheet (His282-Asn285), and the  ENaC domain was built as a long helix on the bottom (Asn230-Gln246). A Ni2+ is shown as green sphere and a solvent (water) is shown as a red sphere. H, working models for Ni2+ inhibition of ENaC. The "gate" model (left) places the Ni2+-binding site (Ni2+ as green sphere) within a putative gate that swings into the outer vestibule of the pore during channel closing. The transduction model (right) proposes that Ni2+ binding (green sphere) causes a local conformational change near the binding site that is remotely transmitted to the outer vestibule of the channel pore through regions connecting the binding site and the pore. Yellow arrows indicate movement directions, and the red sphere represents one Na+ entering the ENaC pore from outside. The pore is shown in blue, and the portions of ECLs are drawn in green or orange.

The dose-response relationship of Ni²⁺ inhibition of Na⁺ currents of WT mENaC was best described with an equation providing two classes of binding sites for Ni²⁺ (Fig. 1). For several mutations, such as R280H, H282C, and H239C, the high affinity component of the Ni²⁺ dose-response curve appeared to be shifted more than the low affinity component (Figs. 4A, 5A, and 7). Ni²⁺ dose-response curves for other mutant channels (except H282D) showed similar shifts in both the high and low affinity binding sites. Although these data do not allow us to distinguish whether His²⁸² and His²³⁹ form independent Ni²⁺-binding sites or form a single binding site, we favor the view that these histidine residues contribute to a common Ni²⁺-binding site. Substitution of His²⁸² or His²³⁹ with cysteine or arginine produced similar shifts in the Ni²⁺ dose-response curves, and channels with mutations at both sites (H282R--H239R) were Ni²⁺-insensitive. These data suggest collaborative coordination of Ni²⁺ binding by these histidine residues. The elimination of Ni²⁺ inhibition by point mutation H282D, the enhanced Ni²⁺ inhibition by R280H, and partial reversal of the enhancement by a mutation in ENaC (H239R) also support this view. Intersubunit coordination of Ni²⁺ has been demonstrated in CNG channels and the Ni²⁺-binding metallochaperone UreE (47, 48, 76, 91). Alternatively, the high and low affinity sites for Ni²⁺ may be formed by His²⁸² (or more than one His²⁸²) and His²³⁹, respectively. The relative shift of the high affinity component of the dose-response relationship by mutations at His²³⁹ appeared to be smaller than shift of the low affinity component (Fig. 5A), whereas a greater shift of the high affinity component of the dose-response relationship was observed with His²⁸² mutations (Fig. 4A). As the osmolarity of Ni²⁺-containing bath solutions was not adjusted in this study and hypertonicity may affect ENaC currents (93, 94), we cannot rule out the possibility of osmotic effects on ENaC or endogenous channels at high concentrations of Ni²⁺ (25 and 50 mM). The observations that H282D or H282R/H239R eliminated both high and low affinity inhibition are not consistent with the possibility.

Based on above results, preferences of Ni²⁺ coordination, and assuming that the channel complex contains more than one -subunit (10-12), we propose a Ni²⁺-binding site within WT ENaC where Ni²⁺ is coordinated by four ligands, three nitrogen atoms from two His²⁸² residues and one from His²³⁹. A fourth unknown ligand could be an oxygen atom from solvent (water) or an oxygen-bearing residue (Figs. 7G and 11G). Although we do not have direct evidence for the involvement of two His²⁸² residues in Ni²⁺ coordination, the loss of Ni²⁺ block observed with H282D-- channels, compared with the modest change in Ni²⁺ sensitivity observed with --H239D channels, is consistent with the notion that more than one -subunit participates in the coordinated binding of Ni²⁺. Our proposed Ni²⁺ coordination pattern is similar to that of the endonuclease domain of the bacterial toxin colicin E9 and the zinc endopeptidase astacin. Both proteins utilize three nitrogen atoms from histidine residues and one oxygen atom from either a solvent or a tyrosine residue as Ni²⁺ ligands (45, 49).

External Ni²⁺ Inhibits ENaC by Reducing Channel Open Probability-- Our single channel results indicate that external Ni²⁺ decreased the open probability of -- mENaC without affecting single channel currents. This notion is consistent with the recent observation that Ni²⁺ decreased apparent open channel density without changing the single channel current as determined by noise analysis (31). A decrease in channel number is less likely a mechanism for Ni²⁺ inhibition of ENaC currents, given the rapidity of the current response to Ni²⁺ (Fig. 1D) and the lack of change in oocyte capacitance in response to Ni²⁺ (31).

The open probability of ENaC has been shown to be highly variable in different systems (1, 95). ENaCs display slow gating with long open and close times, and in some cases two or more distinct gating modes with high and low P_o values were observed. ENaC may naturally transition among different open states that can be regulated by various factors (96, 97). We observed that Ni²⁺ inhibition was saturated at the concentrations near 50 mM (~100-fold greater than the estimated K_i for Ni²⁺) and did not eliminate amiloride-sensitive Na⁺ currents. These data are consistent with the view that Ni²⁺ binding to ENaC results in a conformational change that shifts channels to a gating mode with a lower P_o or, alternatively, that Ni²⁺ stabilizes a closed conformation and thus reduces ENaC P_o. Ni²⁺-induced conformational changes in proteins have been observed in Ni²⁺-dependent enzymes and other native proteins or synthetic peptides (41, 98, 99).

Fig. 11H illustrates two potential mechanisms by which Ni²⁺ binding leads to a decrease in ENaC P_o, based on our presumption that the Ni²⁺-binding site is located within the extracellular region outside of the conduction pore. One model (the "gate" model) places the Ni²⁺-binding site within a putative gate that swings into the outer vestibule of the pore during channel closure. Ni²⁺ binding stabilizes the gate in the closed channel state. An extracellular gate has been proposed to explain state-dependent accessibility of an introduced cysteine within the pore region to sulfhydryl reagents (23). The second model (the "transduction" model) proposes that Ni²⁺ binding induces a local conformational change near the binding site that is transmitted to the outer vestibule of the channel pore through a "linker" region connecting the Ni²⁺-binding site and the pore. Ni²⁺ binding to the ECLs ultimately results in conformational changes in the outer pore that favor the closed state, which is associated with a decreased P_o. The second model is similar to the gating mechanism proposed for ionotropic glutamate receptors (100, 101).

His²⁸² and His²³⁹ May Be Located within a Gating Domain in the ECLs of ENaC Subunits-- A number of observations suggest that His²⁸² and His²³⁹ are located within domains that participate in the control of ENaC gating. Ni²⁺ decreased ENaC open probability (see above and Ref. 31) through interactions with His²⁸² and His²³⁹. MTSET inhibition of H282C-- mENaC may be due to a decrease in P_o, although we have not determined channel P_o following MTSET treatment. Furthermore, we observed that H282R--H239R mENaC has a high P_o,² and Applebury et al. (102, 103) have reported that mutations within the WYRFHY tract (residues 278-283 in rat ENaC) altered gating kinetics of -subunit channels expressed in Chinese hamster ovary cells. Primary structure analyses also support the notion that His²⁸² and His²³⁹ are located in important functional domains. These tracts are among the most conserved domains within ENaC subunits (Fig. 11A) but are not found in the ECLs of other members of the ENaC/Deg family. Secondary structural predictions suggest that these domains exhibit a high degree of organization, including regions that are predicted to form -helices or -sheets (Fig. 11, C and E). Helical wheel analyses of the predicted -helical regions revealed that charged or highly polar residues are arranged on one face of the helix, whereas hydrophobic residues are located on the opposing face (Fig. 11, D and F), a structure capable of interacting with different environments (i.e. aqueous or lipophilic) or domains.

The potential role of histidine residues in ENaC gating is consistent with the role of histidine residues in mediating conformational changes in enzymes (104-107) or ion channels. Okada et al. (108) proposed protonation of a histidine (His³⁷) in the M2 ion channel protein from influenza A virus, and its interaction with Trp⁴¹ is a key step in channel activation. The role of histidine residues in CNG gating has been well characterized (91).

The mechanism by which domains containing His²⁸² and His²³⁹ affect ENaC gating are unclear and deserve further investigation with methods involving structural approaches. Information regarding ENaC gating mechanisms is emerging through mutagenesis studies. Previous studies have shown that ENaC gating is affected by mutations in several different domains in epithelial Na⁺ channel subunits including the N-terminal domain (21, 22, 109), the pore region (23, 24), the C-terminal part of the ECL (25), the M2 domain (26), and the intracellular C-terminal domain (27, 110). However, it is unclear whether these different regions exert effects on channel gating in an independent manner or whether interactions between these domains influence channel gating through a common pathway.

The proposed role of ECLs in ENaC gating is consistent with the notion that ECL domains within ENaC-related channels are involved in channel gating. ASIC are activated by extracellular acidification, and a histidine (His⁷²) in the ECL of ASIC-2a has been identified as a putative H⁺ sensor (111). FaNaCh is a peptide-gated Na⁺ channel, and the peptide (FMRFamide) binding domain was proposed to reside within the ECL (112). Residues or domains within the ECLs of subunits of mechanotransducing channels in Caenorhabditis elegans (i.e. Deg-1 and Mec-4) were proposed to modulate channel gating (113, 114). ENaC may share a similar design in gating machinery with other members in the ENaC/DEG family, although it is clear that they are gated by different factors.

In summary, we have characterized Ni²⁺ inhibition of ENaC activity at whole-cell and single channel levels, identified His²⁸² and His²³⁹ as primary Ni²⁺-binding residues, and demonstrated that Ni²⁺ reduces channel P_o. Our results also suggest that these two residues are present in domains within the extracellular loops that are associated with channel gating.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK51391 and DK54354.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.

To whom correspondence should be addressed: 929 Scaife Hall, Renal-Electrolyte Division, University of Pittsburgh, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9295; Fax: 412-648-9166; E-mail: shaohu@pitt.edu.

Published, JBC Papers in Press, October 22, 2002, DOI 10.1074/jbc.M209975200

² S. Sheng, C. J. Perry, and T. R. Kleyman, unpublished data.

	ABBREVIATIONS

The abbreviations used are: ENaC, epithelial Na⁺ channel; mENaC, mouse epithelial Na⁺ channel; I-V, current-voltage relationship; K_i, inhibitory constant; DEPC, diethyl pyrocarbonate; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate bromide; WT, wild type; MES, 2-(N-morpholino)ethanesulfonic acid; ASIC, acid-sensing ion channels; CNG, cyclic nucleotide-gated channel; ECL, extracellular loops.

	REFERENCES

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