Separate ion pathways in a Cl-/H+ exchanger.
Separate ion pathways in a Cl-/H+ exchanger.
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DOI：
10.1085/jgp.200509417
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发表时间：
2005-12
影响因子：
3.8
通讯作者：
Miller, Christopher
中科院分区：
文献类型：
--
作者：
Accardi, Alessio;Walden, Michael;Nguitragool, Wang;Jayaram, Hariharan;Williams, Carole;Miller, Christopher
CLC-ec1是一种原核细胞的clc型Cl−/H+交换转运体。关于H+与Cl−耦合的机理还知之甚少。一个关键的谷氨酸残基E148，先前被证明是通过调解蛋白质和细胞外溶液之间的质子转移来进行Cl−/H+交换所必需的。为了测试在蛋白质的胞内侧附近是否存在类似的H+受体，我们对向内的羧基残基进行了诱变扫描，并发现E203是唯一的残基，其中和消除了H+与Cl−运输的偶联。谷氨酸在转运蛋白亚类的所有已知CLC中都严格保守，而缬氨酸总是在CLC通道中发现。E203Q突变体的x射线晶体结构与野生型相似。在中性pH下，E203Q的Cl -转运速率受到抑制，双突变体E148A/E203Q与单突变体E148A一样，在不受pH影响的情况下表现出最大的Cl -转运速率。结果表明，CLC-ec1的底物交换涉及两个独立但部分重叠的渗透途径，一个是Cl -，一个是H+。这些途径从蛋白质的细胞外表面到E148是一致的，并且它们向细胞内侧发散。这种情况需要一种传输机制，它与我们熟悉的交替访问方案有着根本的不同。
CLC-ec1 is a prokaryotic CLC-type Cl−/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl−. A critical glutamate residue, E148, was previously shown to be required for Cl−/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl− transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl− transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl− transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl− and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.