Uncoupling and turnover in a Cl-/H+ exchange transporter.
Uncoupling and turnover in a Cl-/H+ exchange transporter.
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DOI：
10.1085/jgp.200709756
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发表时间：
2007-04
影响因子：
3.8
通讯作者：
Miller, Christopher
中科院分区：
文献类型：
--
作者：
Walden, Michael;Accardi, Alessio;Wu, Fang;Xu, Chen;Williams, Carole;Miller, Christopher
CLC 家族蛋白 CLC-ec1 是一种已知结构的细菌同源物，通过未知的膜转运机制以化学计量将两个 Cl− 交换为一个 H+。这项研究检查了保守酪氨酸残基 Y445 的突变，该残基直接协调位于膜中心附近的 Cl− 离子。该位置的突变会导致“解偶联”，使得 H+/Cl− 传输比率大致随取代侧链体积的减小而降低。当由 Cl− 梯度驱动时，未偶联的蛋白质仍然能够将质子向上泵送，但这种 H+ 泵送的程度和速率在更多未偶联的变体中较弱。解偶联伴随着传导性 Cl− 传输，与 H+ 的反向运动无关，即“泄漏”。通过传统的初始速度方法和新颖的泊松稀释方法在重构的脂质体中测量的单一 Cl− 转运速率对于野生型蛋白质为 ~4,000 s−1，并且未偶联的突变体以相似的速率转运 Cl−。
The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two Cl− for one H+ via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a Cl− ion located near the center of the membrane. Mutations at this position lead to “uncoupling,” such that the H+/Cl− transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a Cl− gradient, but the extent and rate of this H+ pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive Cl− transport that is not linked to counter-movement of H+, i.e., a “leak.” The unitary Cl− transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is ∼4,000 s−1 for wild-type protein, and the uncoupled mutants transport Cl− at similar rates.