title: Probing the structural basis of Zn2+ regulation of the epithelial Na+ channel.
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).
authors.affiliation: Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
authors.forename: Jingxin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
authors.forename: Katie L
authors.initials: KL
authors.lastname: Winarski
authors.affiliation: Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
authors.forename: Mike M
authors.initials: MM
authors.lastname: Myerburg
authors.affiliation: Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Pitt
authors.affiliation: Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. Electronic address: shaohu@pitt.edu.
authors.forename: Shaohu
authors.initials: S
authors.lastname: Sheng
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002413:Cations, Divalent / Q000493:pharmacokinetics / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D026941:Sodium Channel Blockers / Q000493:pharmacokinetics / Q000494:pharmacology*; D040681:Structural Homology, Protein; D014982:Xenopus laevis; D015032:Zinc / Q000493:pharmacokinetics / Q000494:pharmacology*
chemical_list: D002413:Cations, Divalent; D053503:Epithelial Sodium Channels; C507053:Scnn1g protein, mouse; D026941:Sodium Channel Blockers; D015032:Zinc