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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.
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.