Dulbecco Telethon Laboratory, Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, Italy; and.
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
J Am Soc Nephrol. 2019 Feb;30(2):293-302. doi: 10.1681/ASN.2018060593. Epub 2019 Jan 11.
The mechanism of anion selectivity in the human kidney chloride channels ClC-Ka and ClC-Kb is unknown. However, it has been thought to be very similar to that of other channels and antiporters of the CLC protein family, and to rely on anions interacting with a conserved Ser residue (Ser) at the center of three anion binding sites in the permeation pathway S. In both CLC channels and antiporters, mutations of Ser alter the anion selectivity. Structurally, the side chain of Ser of CLC channels and antiporters typically projects into the pore and coordinates the anion bound at S.
To investigate the role of several residues in anion selectivity of ClC-Ka, we created mutations that resulted in amino acid substitutions in these residues. We also used electrophysiologic techniques to assess the properties of the mutants.
Mutations in ClC-Ka that change Ser to Gly, Pro, or Thr have only minor effects on anion selectivity, whereas the mutations in residues Y425A, F519A, and Y520A increase the NO/Cl permeability ratio, with Y425A having a particularly strong effect.
s ClC-Ka's mechanism of anion selectivity is largely independent of Ser, and it is therefore unique in the CLC protein family. We identified the residue Y425 in ClC-Ka-and the corresponding residue (A417) in the chloride channel ClC-0-as residues that contribute to NO discrimination in these channels. This work provides important and timely insight into the relationship between structure and function for the kidney chloride channels ClC-Ka and ClC-Kb, and for CLC proteins in general.
人源肾氯通道 ClC-Ka 和 ClC-Kb 的阴离子选择性机制尚不清楚。然而，人们认为它与其他通道和 CLC 蛋白家族的转运体非常相似，并且依赖于阴离子与渗透途径 S 中三个阴离子结合位点中心保守的 Ser 残基（Ser）相互作用。在 CLC 通道和转运体中，Ser 的突变会改变阴离子的选择性。结构上，CLC 通道和转运体的 Ser 侧链通常会伸入孔道并与结合在 S 位的阴离子配位。
为了研究几个残基在 ClC-Ka 阴离子选择性中的作用，我们创建了导致这些残基发生氨基酸取代的突变。我们还使用电生理技术评估了突变体的特性。
突变 ClC-Ka 中的 Ser 突变为 Gly、Pro 或 Thr 仅对阴离子选择性产生较小影响，而突变 Y425A、F519A 和 Y520A 增加了 NO/Cl 通透性比，其中 Y425A 的影响特别强。
ClC-Ka 的阴离子选择性机制在很大程度上不依赖于 Ser，因此在 CLC 蛋白家族中是独特的。我们确定了 ClC-Ka 中的残基 Y425-以及氯离子通道 ClC-0 中的相应残基（A417）-是这些通道中对 NO 进行区分的残基。这项工作为肾氯通道 ClC-Ka 和 ClC-Kb 以及一般的 CLC 蛋白的结构与功能之间的关系提供了重要而及时的见解。
