Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
J Biol Chem. 2012 Oct 12;287(42):35589-35598. doi: 10.1074/jbc.M112.394734. Epub 2012 Aug 28.
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).
细胞外的 Zn(2+) 通过解除 Na(+) 的自我抑制来激活上皮钠离子通道（ENaC）。然而，观察到 Zn(2+) 存在双相剂量反应，表明 Zn(2+) 对通道具有双重作用（即激活和抑制）。为了研究 Zn(2+) 这种双相作用的结构基础，我们研究了突变小鼠 γENaC 的 10 个细胞外 His 残基对通道的影响。在指状亚域内的 4 个突变（γH193A、γH200A、γH202A 和 γH239A）显著降低了通道对 Zn(2+) 的最大激活作用。而 γH193A、γH200A 和 γH202A 降低了 Zn(2+) 激活位点的表观亲和力，γH239A 降低了 Na(+) 的自我抑制，从而掩盖了 Zn(2+) 的激活作用。手掌亚域内 His 残基的突变（γH88A）则消除了低亲和力 Zn(2+) 的抑制作用。基于与酸感应离子通道 1 的结构同源性，预测 γAsp(516)与 γHis(88)接近。残基（γD516A）的取代削弱了 Zn(2+) 的抑制作用。我们的结果表明，外部 Zn(2+) 通过与 γ 亚基内的多个细胞外位点结合来调节 ENaC 的活性，包括（i）指状亚域内的高亲和力刺激位点，涉及 His(193)、His(200)和 His(202)，以及（ii）手掌亚域内的低亲和力 Zn(2+) 抑制位点，包括 His(88)和 Asp(516)。
