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ABSTRACT: Epithelial Na(+) channel (ENaC) mutations are associated with several human disorders, underscoring the importance of these channels in human health. Recent human genome sequencing projects have revealed a large number of ENaC gene variations, several of which have been found in individuals with salt-sensitive hypertension, cystic fibrosis and other disorders. However, the functional consequences of most variants are unknown. In this study, we used the Xenopus oocyte expression system to examine the functional properties of a human ENaC variant. Oocytes expressing αβγL511Q human ENaCs showed 4.6-fold greater amiloride-sensitive currents than cells expressing wild type channels. The γL511Q variant did not significantly alter channel surface expression. Single channel recordings revealed that the variant had 4.0-fold higher open probability than wild type. In addition, γL511Q largely eliminated the Na(+) self-inhibition response, which reflects a downregulation of ENaC open probability by extracellular Na(+). Moreover, γL511Q diminished chymotrypsin-induced activation of the mutant channel. We conclude that γL511Q is a gain-of-function human ENaC variant. Our results suggest that γL511Q enhances ENaC activity by increasing channel open probability, and dampens channel regulation by extracellular Na(+) and proteases.
AJP Renal Physiology 11/2012; · 4.42 Impact Factor
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ABSTRACT: Extracellular Zn(2+) activates the epithelial Na(+) channel (ENaC) by relieving Na(+) self-inhibition. However, a biphasic Zn(2+) dose response was observed, suggesting that Zn(2+) has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn(2+), 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 Zn(2+) activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn(2+) activating site, γH239A diminished Na(+) self-inhibition and thus concealed the activating effects of Zn(2+). Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn(2+) inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp(516) was predicted to be in close proximity to γHis(88). Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn(2+). Our results suggest that external Zn(2+) 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 His(193), His(200), and His(202) and (ii) a low-affinity Zn(2+) inhibitory site within the palm subdomain that includes His(88) and Asp(516).
Journal of Biological Chemistry 08/2012; 287(42):35589-98. · 4.77 Impact Factor
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ABSTRACT: Epithelial Na(+) channels (ENaCs) play an essential role in the regulation of body fluid homeostasis. Certain transition metals activate or inhibit the activity of ENaCs. In this study, we examined the effect of extracellular Cu(2+) on human ENaC expressed in Xenopus oocytes and investigated the structural basis for its effects. External Cu(2+) inhibited human αβγ ENaC with an estimated IC(50) of 0.3 μM. The slow time course and a lack of change in the current-voltage relationship were consistent with an allosteric (non pore-plugging) inhibition of human ENaC by Cu(2+). Experiments with mixed human and mouse ENaC subunits suggested that both the α and β subunits were primarily responsible for the inhibitory effect of Cu(2+) on human ENaC. Lowering bath solution pH diminished the inhibition by Cu(2+). Mutations of two α, two β, and two γ His residues within extracellular domains significantly reduced the inhibition of human ENaC by Cu(2+). We identified a pair of residues as potential Cu(2+)-binding sites at the subunit interface between thumb subdomain of αhENaC and palm subdomain of βhENaC, suggesting a counterclockwise arrangement of α, β, and γ ENaC subunits in a trimeric channel complex when viewed from above. We conclude that extracellular Cu(2+) is a potent inhibitor of human ENaC and binds to multiple sites within the extracellular domains including a subunit interface.
Journal of Biological Chemistry 06/2011; 286(31):27436-46. · 4.77 Impact Factor
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ABSTRACT: The activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na(+). A His residue within the extracellular domain of gammaENaC (gammaHis(239)) was found to have a critical role in Na(+) self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na(+) self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na(+) self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na(+) self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the gammaH239F mutant showed that, in the absence of Na(+) self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that gammaHis(239) functions within an extracellular allosteric regulatory subdomain of the gamma subunit that has an important role in conferring the response of the channel to external Na(+).
Journal of Biological Chemistry 08/2010; 285(34):26088-96. · 4.77 Impact Factor
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ABSTRACT: Activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na(+). We searched for determinants of Na(+) self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at gammaMet(438) altered the Na(+) self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na(+) self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na(+) self-inhibition. Mutations at the homologous site in the alpha subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na(+) self-inhibition. Mutations at the homologous betaAla(422) resulted in minimal or no change in Na(+) self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing alphaG481Mbetagamma, alphabetagammaM438V, and alphaG481M/betagammaM438V, respectively. This pair of residues map to thealpha5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both alphaG481Cbetagamma and alphabetagammaM438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that alphaGly(481) and gammaMet(438) are functional determinants of Na(+) self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.
Journal of Biological Chemistry 02/2009; 284(12):7756-65. · 4.77 Impact Factor
