From the School of Natural and Environmental Sciences, Newcastle University, Ridley Building 2, Newcastle upon Tyne NE1 7RU, United Kingdom and.
the Institute of Animal Physiology, Department of Animal Physiology and Molecular Biomedicine, Justus-Liebig University, 35392 Giessen, Germany.
J Biol Chem. 2018 May 4;293(18):6647-6658. doi: 10.1074/jbc.RA118.002543. Epub 2018 Mar 25.
The epithelial sodium channel (ENaC) is a critical regulator of vertebrate electrolyte homeostasis. ENaC is the only constitutively open ion channel in the degenerin/ENaC protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. The canonical ENaC is composed of three subunits (α, β, and γ), but a fourth δ-subunit may replace α and form atypical δβγ-ENaCs. Using as a model, here we found that mRNAs of the α- and δ-subunits are differentially expressed in different tissues and that δ-ENaC predominantly is present in the urogenital tract. Using whole-cell and single-channel electrophysiology of oocytes expressing αβγ- or δβγ-ENaC, we demonstrate that the presence of the δ-subunit enhances the amount of current generated by ENaC due to an increased open probability, but also changes current into a transient form. Activity of canonical ENaCs is critically dependent on proteolytic processing of the α- and γ-subunits, and immunoblotting with epitope-tagged ENaC subunits indicated that, unlike α-ENaC, the δ-subunit does not undergo proteolytic maturation by the endogenous protease furin. Furthermore, currents generated by δβγ-ENaC were insensitive to activation by extracellular chymotrypsin, and presence of the δ-subunit prevented cleavage of γ-ENaC at the cell surface. Our findings suggest that subunit composition constitutes an additional level of ENaC regulation, and we propose that the δ-ENaC subunit represents a functional example that demonstrates the importance of proteolytic maturation during ENaC evolution.
上皮钠离子通道（ENaC）是脊椎动物电解质稳态的关键调节剂。ENaC 是退行性/ENaC 蛋白家族中唯一的组成型开放离子通道，因此其表达、膜丰度和开放概率受到严格控制。典型的 ENaC 由三个亚基（α、β 和 γ）组成，但第四个 δ-亚基可能取代 α 并形成非典型的 δβγ-ENaC。以 作为模型，我们在这里发现，α-和 δ-亚基的 mRNA 在不同组织中差异表达，并且 δ-ENaC 主要存在于泌尿生殖道中。通过表达 αβγ-或 δβγ-ENaC 的卵母细胞的全细胞和单通道电生理学，我们证明由于开放概率增加，δ-亚基的存在会增加 ENaC 产生的电流量，但也会使电流变为瞬态形式。典型 ENaC 的活性严重依赖于 α-和 γ-亚基的蛋白水解加工，并且用表位标记的 ENaC 亚基进行免疫印迹表明，与 α-ENaC 不同，δ-亚基不会被内源性蛋白酶 furin 进行蛋白水解成熟。此外，δβγ-ENaC 产生的电流对细胞外糜蛋白酶的激活不敏感，并且 δ-亚基的存在阻止 γ-ENaC 在细胞表面的切割。我们的发现表明亚基组成构成了 ENaC 调节的另一个层次，我们提出 δ-ENaC 亚基代表了一个功能实例，证明了蛋白水解成熟在 ENaC 进化过程中的重要性。
