Carattino Marcelo D, Sheng Shaohu, Bruns James B, Pilewski Joseph M, Hughey Rebecca P, Kleyman Thomas R
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
J Biol Chem. 2006 Jul 7;281(27):18901-7. doi: 10.1074/jbc.M604109200. Epub 2006 May 11.
Epithelial sodium channels (ENaCs) mediate Na(+) entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed alpha, beta, and gamma, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the alpha subunit and at a single site within the gamma subunit. The alpha subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking alpha subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both alpha subunit cleavage and the tract alphaAsp-206-Arg-231 are active when expressed in oocytes, suggesting that alphaAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (alpha-26), corresponding to alphaAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na(+) channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC(50) for amiloride block of ENaC was not affected by the presence of alpha-26, indicating that alpha-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within alpha-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of alpha-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the alpha subunit activates ENaC by disassociating an inhibitory domain (alphaAsp-206-Arg-231) from its effector site within the channel complex.
上皮钠通道（ENaC）介导钠离子跨过高电阻上皮细胞顶端膜的转运，这些上皮细胞分布于远端肾单位、气道和肺泡以及远端结肠。这些通道由三个同源亚基组成，分别称为α、β和γ，它们具有细胞内的氨基和羧基末端以及由大的细胞外环连接的两个跨膜结构域。ENaC亚基的成熟涉及弗林蛋白酶依赖性在α亚基内的两个位点以及γ亚基内的一个位点对细胞外环的切割。α亚基必须在Arg - 205和Arg - 231之后立即进行两次切割，以便通道完全激活。缺乏α亚基切割的通道无活性，开放概率非常低。相比之下，缺乏α亚基切割和αAsp - 206 - Arg - 231片段的通道在卵母细胞中表达时是有活性的，这表明αAsp - 206 - Arg - 231作为一种抑制剂，使通道稳定在关闭构象。一种对应于αAsp - 206 - Arg - 231的合成26肽（α - 26）可逆地抑制非洲爪蟾卵母细胞中表达的野生型小鼠ENaC，以及小鼠集合管细胞系或人气道上皮细胞原代培养物中表达的内源性钠离子通道。α - 26的存在不影响氨氯地平对ENaC阻断的IC50，表明α - 26不与氨氯地平结合位点结合或相互作用。用Glu取代α - 26中的Arg残基，或用Ala取代Pro残基，显著降低了α - 26的效力。该肽通过降低通道开放概率来抑制ENaC。我们的结果表明，α亚基的蛋白水解通过将抑制结构域（αAsp - 206 - Arg - 231）与其在通道复合物中的效应位点解离来激活ENaC。
