Jayaram Hariharan, Accardi Alessio, Wu Fang, Williams Carole, Miller Christopher
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454, USA.
Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11194-9. doi: 10.1073/pnas.0804503105. Epub 2008 Aug 4.
The CLC family of Cl(-)-transporting proteins includes both Cl(-) channels and Cl(-)/H(+) exchange transporters. CLC-ec1, a structurally known bacterial homolog of the transporter subclass, exchanges two Cl(-) ions per proton with strict, obligatory stoichiometry. Point mutations at two residues, Glu(148) and Tyr(445), are known to impair H(+) movement while preserving Cl(-) transport. In the x-ray crystal structure of CLC-ec1, these residues form putative "gates" flanking an ion-binding region. In mutants with both of the gate-forming side chains reduced in size, H(+) transport is abolished, and unitary Cl(-) transport rates are greatly increased, well above values expected for transporter mechanisms. Cl(-) transport rates increase as side-chain volume at these positions is decreased. The crystal structure of a doubly ungated mutant shows a narrow conduit traversing the entire protein transmembrane width. These characteristics suggest that Cl(-) flux through uncoupled, ungated CLC-ec1 occurs via a channel-like electrodiffusion mechanism rather than an alternating-exposure conformational cycle that has been rendered proton-independent by the gate mutations.
氯离子转运蛋白的CLC家族既包括氯离子通道，也包括氯离子/氢离子交换转运体。CLC-ec1是转运体亚类中结构已知的细菌同源物，以严格、强制的化学计量比每质子交换两个氯离子。已知两个残基Glu(148)和Tyr(445)处的点突变会损害氢离子移动，同时保留氯离子转运。在CLC-ec1的X射线晶体结构中，这些残基形成了位于离子结合区域两侧的假定“门”。在形成门的两个侧链尺寸都减小的突变体中，氢离子转运被消除，单位氯离子转运速率大大增加，远高于转运体机制预期的值。随着这些位置侧链体积的减小，氯离子转运速率增加。一个双无门突变体的晶体结构显示出一条狭窄的管道贯穿整个蛋白质的跨膜宽度。这些特征表明，通过未偶联、无门控的CLC-ec1的氯离子通量是通过类似通道的电扩散机制发生的，而不是通过门控突变使其与质子无关的交替暴露构象循环。
