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Extracellular Zn2+ activates the epithelial Na+ channel (ENaC) by relieving Na+ self-inhibition. However, a biphasic Zn2+ dose response was observed, suggesting that Zn2+ has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn2+, we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn2+ activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn2+ activating site, γH239A diminished Na+ self-inhibition and thus concealed the activating effects of Zn2+. Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn2+ inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp516 was predicted to be in close proximity to γHis88. Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn2+. Our results suggest that external Zn2+ regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His193, His200, and His202 and (ii) a low-affinity Zn2+ inhibitory site within the palm subdomain that includes His88 and Asp516.
plays an important role in the maintenance of extracellular fluid volume and regulation of blood pressure and in the regulation of airway surface liquid volume. Altered channel activity induced by mutations of ENaC genes results in several disorders, including Liddle syndrome and pseudohypoaldosteronism type 1, and contributes to the pathogenesis of cystic fibrosis (
). The response of ENaC to extracellular Zn2+ is biphasic. Low concentrations of Zn2+ activate the channel, with a maximal response at 100 μm Zn2+. Further increases in [Zn2+] reduce channel activity. This bell-shaped dose-response relationship suggests that Zn2+ enhances channel activity at low concentrations and is inhibitory at high concentrations (
). However, the Zn2+-binding sites within ENaC have not been identified, and the biphasic dose response to the heavy metal has not been explained.
ENaCs are typically composed of three homologous subunits, each of which contains short intracellular N and C termini, two transmembrane domains, and a large extracellular region. The crystal structure of the chicken acid-sensing ion channel 1 (cASIC1; homologous to ENaC) reveals a trimeric channel complex with a large extracellular domain (ECD) connected to the transmembrane domain via a wrist region (
). The extracellular region of a subunit is organized into five subdomains, referred to as palm, β-ball, knuckle, finger, and thumb. Recent studies suggest that the extracellular regions of ENaC subunits have a similar overall design to that of cASIC1 and that the ECD may sense various extracellular signals (
). We hypothesized that some of these residues mediate Zn2+ interaction with ENaC. Several lines of evidence suggest that the γ-subunit has a particularly important role in regulating channel gating in response to external factors (
). Mutations were introduced into His residues within the γ-subunit ECD to identify sites that have a role in the regulation of ENaC by Zn2+. Three His residues were identified within the finger subdomain that participate in a high-affinity Zn2+ activating site. Additionally, mutation of a palm subdomain His residue altered the low-affinity inhibitory response. These His residues are unique to the γ-subunit, as they are not found at the homologous sites in the α- and β-subunits.
Point or multiple mutations were generated in mouse γENaC (γmENaC) cDNA in the pBluescript SK− vector (Stratagene, La Jolla, CA) using the QuikChange II XL site-directed mutagenesis kit (Stratagene). Following mutagenesis, cDNAs were validated by sequencing. Wild-type ENaC α-, β-, and γ-subunit and mutant ENaC γ-subunit cRNAs were made using T3 RNA polymerase (Ambion), purified using an RNA purification kit (Qiagen), and quantified by spectrophotometry.
ENaC Expression and Two-electrode Voltage Clamp
ENaC expression in Xenopus oocytes and two-electrode voltage clamp were performed as reported previously (
). Stage V and VI oocytes free of follicle cell layers were injected with 1 ng of cRNA for each mENaC subunit per oocyte and incubated at 18 °C in modified Barth's saline (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 15 mm HEPES, 0.3 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, 10 μg/ml sodium penicillin, 10 μg/ml streptomycin sulfate, and 100 μg/ml gentamycin sulfate, pH 7.4).
All experiments were carried out at room temperature (20–24 °C). Two-electrode voltage clamp was performed using either an Axoclamp 900A amplifier (Molecular Devices, Sunnyvale, CA) or a TEV-200 voltage clamp amplifier (Dagan Corp., Minneapolis, MN) and a DigiData 1440A interface (Molecular Devices). Data acquisition and analyses were done with pCLAMP 8 or 9 (Molecular Devices). Oocytes were placed in a recording chamber from Warner Instruments (Hamden, CT) and perfused with bath solutions at a constant flow rate of 12–15 ml/min. Oocytes were continuously clamped at −60 or −100 mV as specified in the figure legends.
Na+ Self-inhibition Response
Na+ self-inhibition responses were examined as described previously (
). Briefly, Na+ self-inhibition was determined by measuring the decrease in current from the peak (Ipeak) to a steady state (Iss) elicited by a rapid increase in extracellular Na+ concentration from 1 to 110 mm. The [Na+] jump was done by rapidly replacing a 1 mm Na+ bath solution (NaCl-1 containing 1 mm NaCl, 109 mmN-methyl-d-glucamine, 2 mm KCl, 2 mm CaCl2, and 10 mm HEPES, pH 7.4) with a 110 mm Na+ bath solution (NaCl-110 containing 110 mm NaCl, 2 mm KCl, 2 mm CaCl2, and 10 mm HEPES, pH 7.4). Rapid solution exchange was performed with a 16-channel Teflon valve perfusion system (AutoMate Scientific, Inc., Berkeley, CA). The magnitude of Na+ self-inhibition was represented by the ratio of amiloride-sensitive Iss and Ipeak.
Zn2+ Dose Responses
A 1 m ZnCl2 stock solution (super pure, >99.999%, Sigma-Aldrich) was prepared in water and diluted to the desired concentrations in NaCl-110 bath solution. To examine Zn2+ dose responses, oocytes expressing mENaCs were continuously clamped at −60 mV while Zn2+ was applied at increasing concentrations in the range of 0.1 μm to 5 mm. Due to the limited solubility of ZnCl2 in NaCl-110 bath solution, Zn2+ concentrations >5 mm were not tested. At the end of an experiment, 10 μm amiloride was added to the bath to determine the amiloride-sensitive current. The changes in amiloride-sensitive currents in response to Zn2+ applications were used to analyze the dose-response relationship.
Because the amiloride-sensitive current demonstrated a biphasic dose-response relationship with [Zn2+], we assumed that ENaCs possess a high-affinity activating site and a low-affinity inhibitory site for Zn2+. Thus, the normalized currents in the presence of increasing concentrations of Zn2+ were fitted with Equation 1,
where IR is the relative current at certain [Zn2+] (i.e. current in the presence of Zn2+ normalized to current in the absence of Zn2+); Imax and Imin are the maximal and minimal normalized currents, respectively; C is the Zn2+ concentration; and EC50 and IC50 are the concentrations with 50% effects for the activating and inhibitory phases, respectively.
Data are presented as means ± S.E. To minimize bias from batch-to-batch variation in oocytes, the significance in the differences between mutant and WT channels was always analyzed from data obtained in the same batches of oocytes. Student's t tests were used for comparison, and the significance levels are specified below and in the figure legends. Curve fittings were performed with OriginPro 8.5 (OriginLab, Northampton, MA).
External Zn2+ Regulates mENaC by Interacting at Distinct Sites
Because extracellular Zn2+ activates ENaC by relieving Na+ self-inhibition (
), the Na+ self-inhibition response and the effect of Zn2+ were examined sequentially in each oocyte. The Na+ self-inhibition response reflects a decrease in current from a peak (Ipeak) to a steady state (Iss), which is elicited by a rapid increase in the extracellular Na+ concentration from 1 to 110 mm (Fig. 1). As currents approached a steady-state level, increasing concentrations of Zn2+ were applied, and changes in current were measured. Increasing extracellular [Zn2+] in the range of ∼0.1–100 μm led to a dose-dependent increase in current. The current fell when extracellular [Zn2+] was increased above 100 μm. The maximal current in the presence of Zn2+ (Iznm) was essentially identical to the Ipeak observed following the increase in extracellular [Na+] (Iznm/Ipeak = 1.00 ± 0.01, n = 32), consistent with the notion that Zn2+ relieves ENaC from inhibition by external Na+.
Fig. 1B illustrates the Zn2+ dose-response relationship. Mean amiloride-sensitive currents in the presence of increasing extracellular [Zn2+] were normalized to the base-line current. Similar to our previous report (
), we observed a bell-shaped dose response to Zn2+. This relationship fits a model of ENaC possessing a high-affinity Zn2+ activating site and a low-affinity Zn2+ inhibitory site (R2 = 0.997, Equation 1, see “Experimental Procedures”) with an estimated EC50 and IC50 of 2.1 ± 0.1 μm (n = 6) and 2.1 ± 0.1 mm (n = 6), respectively. The maximal relative current observed at 100 μm Zn2+ was 1.68 ± 0.07 (n = 6).
Ala Substitutions of Selected His Residues in the Extracellular Region of γmENaC Alter the Effect of Zn2+
There are a total of 10 His residues within the ECD of the γ-subunit. Four residues are located within the finger subdomain, three in the β-ball subdomain, two in the palm subdomain, and one in the thumb subdomain (Fig. 2). We individually mutated these His residues to Ala and expressed the mutant γ-subunits together with WT α- and β-subunits in oocytes. We sequentially examined Na+ self-inhibition and the response to Zn2+ (FIGURE 3, FIGURE 4) to determine whether a mutation selectively disrupted the effect of Zn2+ on channel activity or secondarily altered the Zn2+ effect by modifying the Na+ self-inhibition response.
Ala substitutions at 4 of the 10 extracellular γ-subunit His residues (γH193A, γH200A, γH202A, and γH239A) significantly reduced maximal ENaC activation by Zn2+ (FIGURE 3, FIGURE 4). Interestingly, all four of the extracellular His residues whose mutations reduced Zn2+ activation are within the predicted finger subdomain of the channel. In addition to suppressing Zn2+ activation, γH239A also diminished Na+ self-inhibition, similar to what was previously observed when γHis239 was substituted with other residues (
). As the γH239A mutant was relieved of Na+ self-inhibition prior to the addition of Zn2+, it is not possible to determine whether this residue is involved in Zn2+ binding to the channel. On the other hand, γH193A, γH200A, and γH202A did not significantly alter the Na+ self-inhibition response, suggesting that these three His residues in the finger subdomain have specific roles in Zn2+ activation of ENaC.
Although γH88A did not affect the maximal Zn2+ activation (p > 0.05 versus WT) or the Na+ self-inhibition response, it converted the Zn2+ dose response from bell-shaped to monophasic by selectively eliminating the inhibitory effect of high [Zn2+] (FIGURE 3, FIGURE 4). These results are consistent with a mutation-induced loss of a low-affinity Zn2+-binding site that mediates channel inhibition at high [Zn2+]. One mutation (γH283A) moderately increased the maximal Zn2+ activation (p < 0.01 versus WT) (Fig. 4). Collectively, these observations suggest that γHis88 within the palm subdomain and γHis193, γHis200, and γHis202 within the finger subdomain are involved in Zn2+-dependent regulation of ENaC.
Mutation of γHis193, γHis200, or γHis202 within the Finger Subdomain Reduces the Apparent Zn2+ Binding Affinity of the Activating Site
We analyzed the responses of WT and mutant channels to increasing [Zn2+] using a two-site model. Representative dose-response curves are shown in Fig. 5, and derived values for EC50 and IC50 are shown in Table 1. Our results show that γH88A did not affect the EC50 for the activating site, although it essentially eliminated the decrease in channel activity observed with higher [Zn2+]. In contrast, γH193A, γH200A, and γH202A reduced the sensitivity of mENaC to activation by extracellular Zn2+ and did not affect the inhibitory effect of high [Zn2+], leading to increased EC50 values and no change in the IC50 relative to WT. The results imply that γH193A, γH200A, and γH202A selectively reduce the apparent affinity of the potentiating site for Zn2+ while not interfering with the low-affinity Zn2+ inhibitory site.
We also examined whether substitutions at multiple His residues led to further increases in the EC50 for Zn2+. The Zn2+ dose-response relationships of γH200A/γH202A and γH193A/γH200A/γH202A were similar to those found in single mutants (Fig. 6). Thus, all three His residues within the finger subdomain are required for Zn2+-dependent channel activation. The lack of an additive effect of multiple His substitutions suggests that these His residues participate in a common Zn2+-binding site.
In addition to increasing the EC50 values of Zn2+ activation, γH193A, γH200A, γH202A, γH200A/γH202A, and γH193A/γH200A/γH202A also reduced the magnitude of the increase in current in response to Zn2+ (Imax) (Table 1). The reduced Imax observed in the mutants could be explained either by a reduction in the efficacy of channel activation by Zn2+ or by the presence of an inhibitory effect that limits the extent of channel activation. To distinguish these two possibilities, we examined channels with His substitutions at Zn2+ activating sites (γH200A/γH202A) and the Zn2+ inhibitory site (γH88A). As shown in Fig. 6E, γH88A/γH200A/γH202A reduced Zn2+ sensitivity by 8-fold compared with γH88A channels (EC50 = 20.4 ± 3.0 μm (n = 6) versus 2.6 ± 0.3 μm (n = 10); p < 0.001). However, both mutants had similar Imax values (1.81 ± 0.11 (n = 6) versus 1.79 ± 0.09 (n = 10); p > 0.05), demonstrating that mutations of the Zn2+ activating sites within the finger subdomain do not alter the magnitude of the maximal Zn2+ activation when the low-affinity Zn2+ inhibitory site is not present. Considered together, these results are consistent with the idea that γH200A/γH202A reduces the apparent Zn2+ binding affinity for the activating site without altering its efficacy. The inhibitory site containing γHis88 limits the efficacy of channel activation by Zn2+.
γHis239 Does Not participate in Zn2+ Binding
The γH239A mutation eliminated both Na+ self-inhibition and Zn2+ activation of ENaC (FIGURE 3, FIGURE 4, FIGURE 5). As Zn2+ activates ENaC through relief of Na+ self-inhibition, it is unclear whether γHis239 is a Zn2+-binding site similar to the other finger subdomain His residues or whether the γH239A mutation simply prevents channel inhibition by external Na+. To distinguish between these two possibilities, additional mutations were added to restore Na+ self-inhibition in the setting of the γH239A mutant. Previously, we found that the G481A mutation within the α-subunit greatly enhanced the Na+ self-inhibition response (
). Channels bearing both the αG481A and γH239A mutations exhibited a robust Na+ self-inhibition response and a biphasic response to Zn2+ (Fig. 7). Both αG481A and αG481A/γH239A channels had Imax values that were significantly greater than the WT values (3.4 ± 0.2 (n = 6) and 3.7 ± 0.2 (n = 6), respectively), presumably as a result of the enhanced Na+ self-inhibition. The EC50 for Zn2+ activation of αG481A/γH239A was similar to that of WT (4.2 ± 0.5 μm (n = 6) versus WT; p > 0.05), as was the IC50 of the Zn2+ inhibitory response (1.2 ± 0.0 mm (n = 6) versus WT; p > 0.05). Thus, γH239A did not weaken the apparent Zn2+ affinity of either the high- or low-affinity site.
Region Surrounding γHis88 Is a Determinant of the Low-affinity Inhibitory Site for Zn2+
Next, the importance of the palm subdomain γHis88 and surrounding region was examined to explore the nature of the low-affinity Zn2+ inhibitory site. As shown in FIGURE 3, FIGURE 4, FIGURE 5, mutation of γHis88 to Ala prevented ENaC inhibition by high concentrations of Zn2+. Mutation of γHis88 to either Arg or Asp also rendered the Zn2+ dose responses monophasic and did not significantly alter the Na+ self-inhibition response (Fig. 8, A and B). These results suggest that a His residue at this position is an essential determinant of the Zn2+ inhibitory site.
Zn2+ is often coordinated by multiple neighboring amino acid residues in metal-binding proteins (
). To define additional residues that contribute to the low-affinity Zn2+ inhibitory site, we searched for additional potential Zn2+-binding residues using sequence alignments of ENaC and ASIC and the resolved cASIC1 structure (
). γHis88 aligns with Ala82 in cASIC1, which is located immediately after the first β-strand of ECD (β1) within the palm subdomain (Fig. 8C). Multiple regions within the same subunit (strand β1 and loops β1-β2, β11-β12, and β5-β6) and an adjacent subunit (N-terminal end of helix α4 and loop α5-β10) are in close proximity to Ala82 in cASIC1. Eleven residues in these regions are within 6 Å of the Ala82 (Fig. 8C). Among these residues, only Ala413 and Met364 have homologous residues in mENaC that are often found within a Zn2+ coordination shell (γAsp516 and βGlu444, respectively). We examined whether these sites are involved in ENaC regulation by Zn2+. As shown in Fig. 8 (D and E), γD516A and γD516R largely prevented ENaC inhibition by high [Zn2+], similar to what we observed with the γH88A mutant (Fig. 5). Mutation of the other residue that was predicted to be in close proximity to γHis88 (βE444A) did not significantly alter the Zn2+ effects on ENaC (data not shown). The proximity of γAsp516 to γHis88 and their similar mutation-induced changes in the Zn2+ response suggest that the two residues share a functional role in constituting the low-affinity Zn2+ inhibitory site.
The residues corresponding to γHis88 and γAsp516 in cASIC1 (Ala82 and Ala413) are located at a subunit interface (Fig. 8C). According to the suggested counterclockwise arrangement (viewed from above) of the ENaC α-, β-, and γ-subunits (
), γHis88 (Ala82 in cASIC1) and γAsp516 (Ala413 in cASIC1) are expected to interact with residues within the thumb subdomain of βENaC. There are five negatively charged βENaC residues within loops β9-α4 and α5-β10 of the thumb subdomain: βGlu354, βAsp365, βGlu436, βGlu444, and βGlu448. Given their locations within flexible loops, they might be in close proximity to γHis88 and γAsp516 and contribute to the low-affinity inhibitory site. Mutations of these β-subunit residues to Ala did not alter the channel's response to Zn2+ (data not shown). Thus, it is unlikely that these residues within the thumb domain of βENaC are involved in Zn2+ regulation of the channel.
The major goal of this study was to probe the structural basis of the regulation of ENaC by Zn2+. The biphasic dose response of Zn2+ on the purinergic receptor P2X is believed to originate from Zn2+ binding at two distinct sites within the receptor: a high-affinity activating site and a low-affinity inhibitory site (
), we hypothesized that the bell-shaped Zn2+ dose-response relationship with ENaC activity is due to the presence of a high-affinity activating site and a low-affinity inhibitory site within the ECD. We found that a minimal two-site model sufficiently describes the bell-shaped dose response of mouse ENaC to external Zn2+. The presence of a high-affinity activating site and a low-affinity inhibitory site is supported by our observations that Zn2+-mediated activation or inhibition can be selectively eliminated by targeted mutations of specific residues in the activating site or the inhibitory site, respectively. On the basis of these observations, we propose that γHis193, γHis200, and γHis202 within the finger subdomain contribute to a high-affinity Zn2+-binding site and that γHis88 and γAsp516 in the palm subdomain are key determinants of a low-affinity Zn2+-binding site (Fig. 9).
The following observations suggest that γHis193, γHis200, and γHis202, presumably within the finger subdomain, contribute to a high-affinity Zn2+ activating site. (i) Mutation of these residues increased the EC50 of Zn2+ activation and had no effect on the IC50 (Fig. 5 and Table 1), suggesting that these residues are specifically involved in Zn2+ binding to an activating site. (ii) Although mutations of γHis193, γHis200, and γHis202 reduced the maximal ENaC current elicited by Zn2+, maximal Zn2+ activation was not affected when the Zn2+ inhibitory site was also mutated (γH88A/γH200A/γH202A). Thus, γH200A/γH202A appears to primarily affect the apparent affinity of the activating site and does not alter the efficacy of channel activation. (iii) γHis193, γHis200, and γHis202 appear to share a similar functional role in mediating Zn2+ effects on the channel. Individual mutation of any of the three His residues induced a similar phenotype, including an increase in EC50, a decrease in maximal Zn2+ activation, and a lack of effect on Na+ self-inhibition. Moreover, mutation-induced changes in EC50 were not additive in γH193A/γH200A/γH202A channels (Fig. 6D). (iv) Their proximity in the sequence makes it structurally possible for them to contribute to a common binding site. Interestingly, the His200-Val201-His202 sequence matches a common Zn2+-binding motif, HXH, where X can be any residue (
Channels with substitutions of all three His residues are still activated by Zn2+, suggesting that there are additional sites within the channel where Zn2+ binds and activates ENaC. It is notable that His residues are not present at sites in the α- and β-subunits that correspond to γHis193, γHis200, and γHis202 (Fig. 9). Further studies are needed to identify additional sites involved in Zn2+ activation of ENaC.
Our results demonstrate that γHis88 within the palm subdomain is a key determinant for the low-affinity inhibitory Zn2+-binding site. Interestingly, like the three finger subdomain His residues, γHis88 is also not conserved among ENaC subunits (Fig. 9). Taking advantage of the structural information of ASIC1, a negatively charged residue, γAsp516, was identified that lies in close proximity to γHis88 and is likely involved in the inhibitory effect of Zn2+ as well (Fig. 9). However, it is also possible that mutations of γHis88 and γAsp516 may diminish the inhibitory component in the biphasic Zn2+ dose response by disrupting conformational changes induced by Zn2+ binding, instead of abolishing Zn2+ binding. Nevertheless, our observations suggest that this region (loops β1-β2 and β11-β12), located at the middle of the palm subdomain, contributes to a general inhibitory site for different channel regulators (Fig. 9). Indeed, a putative Cl−-binding site and a Cu2+-binding site have been identified near this region in human ENaCs (
). External Zn2+ may disrupt either Na+ binding to its receptor site or Na+-induced conformational changes. Our observations that mutations at the implicated Zn2+-binding sites (γH193A, γH200A, γH202A, and γH88A) did not alter the Na+ self-inhibition response suggest that the binding sites for Zn2+ and Na+ are distinct. The lack of effect on the apparent affinity for Zn2+ activation of a mutation of a critical site for Na+ self-inhibition (γH239A) also supports the notion of distinct Zn2+- and Na+-binding sites. Therefore, it is likely that Zn2+ binding interferes with Na+-induced motions within the ECD. Similar to Zn2+, several extracellular factors such as proteases, Cl−, H+, and small molecules are thought to regulate ENaC gating in part by altering the Na+ self-inhibition response (
). Multiple external regulators of ENaC, including transition metals, Cl−, H+, and other small molecules, exert their effects on ENaC gating by binding to regions within the ECD that are structurally distant from the channel pore (
). These gating regulators are expected to remotely influence the channel gate through a series of conformational changes. The identification of these allosteric regulatory sites will advance our understanding regarding mechanisms by which the binding of allosteric regulators alters channel gating.
In summary, this study provides novel insights into the structural basis for Zn2+ regulation of ENaC. Our results suggest that external Zn2+ regulates ENaC activity by binding to multiple extracellular sites, including a high-affinity activating site in the finger subdomain and a low-affinity inhibitory site in the palm subdomain of the γ-subunit. These findings advance our understanding of the regulation of ENaC gating by extracellular factors.
We thank Dr. Thomas R. Kleyman for helpful discussions and Brandon M. Blobner for oocyte preparation.
Epithelial sodium channel and the control of sodium balance: interaction between genetic and environmental factors.