Sheng Shaohu, Carattino Marcelo D, Bruns James B, Hughey Rebecca P, Kleyman Thomas R
Dept. of Medicine, Univ. of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Am J Physiol Renal Physiol. 2006 Jun;290(6):F1488-96. doi: 10.1152/ajprenal.00439.2005. Epub 2006 Jan 31.
Epithelial Na+ channels (ENaC) are inhibited by extracellular Na+, a process referred to as Na+ self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in alpha, or one site in gamma, blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant alpha-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the Na+ self-inhibition response, while channels with a mutant gamma-subunit showed a modestly enhanced Na+ self-inhibition response. Analysis of Na+ self-inhibition at varying [Na+] indicates that channels containing mutant alpha-subunits exhibit an increased Na+ affinity. At the single-channel level, channels with a mutant alpha-subunit had a low open probability (P(o)) in the presence of a high external [Na+] in the patch pipette. P(o) dramatically increased when trypsin was also present, or when a low external [Na+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving Na+ self-inhibition and that activation requires that the alpha-subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC P(o) and extracellular [Na+], supporting the notion that Na+ self-inhibition reflects a P(o) reduction due to high extracellular [Na+].
上皮钠通道（ENaC）受到细胞外钠离子的抑制，这一过程被称为钠离子自我抑制。我们之前证明，α亚基中两个弗林蛋白酶切割共有序列位点内的关键残基发生突变，或者γ亚基中的一个位点发生突变，当突变通道在非洲爪蟾卵母细胞中表达时，会阻断亚基蛋白水解并抑制通道活性（Hughey RP、Bruns JB、Kinlough CL、Harkleroad KL、Tong Q、Carattino MD、Johnson JP、Stockand JD和Kleyman TR。《生物化学杂志》279: 18111 - 18114，2004年）。当在Madin - Darby犬肾细胞中表达时，这些突变也会阻断亚基的切割，而当野生型亚基在弗林蛋白酶缺陷的中国仓鼠卵巢细胞中表达时，亚基切割和通道活性都会被阻断。我们现在报告，缺乏一个或两个弗林蛋白酶切割位点的突变α亚基的通道表现出钠离子自我抑制反应的显著增强，而具有突变γ亚基的通道则表现出适度增强的钠离子自我抑制反应。在不同钠离子浓度下对钠离子自我抑制的分析表明，含有突变α亚基的通道表现出钠离子亲和力增加。在单通道水平上，具有突变α亚基的通道在膜片吸管中存在高外部钠离子浓度时开放概率（P(o)）较低。当同时存在胰蛋白酶时，或者当膜片吸管中外部钠离子浓度较低时，P(o)会显著增加。我们的结果表明，ENaC亚基的弗林蛋白酶切割通过解除钠离子自我抑制来激活通道，并且激活需要α亚基被切割两次。此外，我们首次证明了ENaC的P(o)与细胞外钠离子浓度之间存在明确的关系，支持了钠离子自我抑制反映由于高细胞外钠离子浓度导致P(o)降低这一观点。
