Probing the Structural Basis of Zn2+ Regulation of the Epithelial Na+ Channel

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 gamma ENaC. Four mutations within the finger subdomain (gamma H193A, gamma H200A, gamma H202A, and gamma H239A) significantly reduced the maximal Zn2+ activation of the channel. Whereas gamma H193A, gamma H200A, and gamma H202A reduced the apparent affinity of the Zn2+ activating site, gamma H239A diminished Na+ self-inhibition and thus concealed the activating effects of Zn2+. Mutation of a His residue within the palm subdomain (gamma H88A) abolished the low-affinity Zn2+ inhibitory effect. Based on structural homology with acid-sensing ion channel 1, gamma Asp(516) was predicted to be in close proximity to gamma His(88). Ala substitution of the residue (gamma 516A) blunted the inhibitory effect of Zn2+. Our results suggest that external Zn2+ regulates ENaC activity by binding to multiple extracellular sites within the gamma-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 Zn2+ inhibitory site within the palm subdomain that includes His(88) and Asp(516).