Determinants of anion-proton coupling in mammalian endosomal CLC proteins
Determinants of anion-proton coupling in mammalian endosomal CLC proteins
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DOI：
10.1074/jbc.m708368200
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发表时间：
2008-02-15
影响因子：
4.8
通讯作者：
Pusch, Michael
中科院分区：
文献类型：
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作者：
Zdebik, Anselm A.;Zifarelli, Giovanni;Pusch, Michael
CLC基因家族的许多蛋白质是Cl-通道，而其他蛋白质，如细菌ecClC-1或哺乳动物ClC-4和-5，介导Cl-/H-交换。突变“门控谷氨酸”（ClC-4中的Glu-224和ClC-5中的Glu-211）将这些交换剂转化为阴离子电导，就像ecClC-1中另一种细胞内“质子谷氨酸”的中和一样。我们在这里表明，中和质子谷氨酸的ClC-4（Glu-281）和ClC-5（Glu-268），但不取代它与天冬氨酸，组氨酸，或酪氨酸，而取消Cl-和H+的运输。表面表达未被这些突变改变。在ClC-4（E281 A）和ClC-5（E268 A）质子谷氨酸突变中，通过额外中和门控谷氨酸，可以恢复非偶联的Cl-转运，这表明野生型蛋白质只有在质子通过细胞质H（+）供体供应时才转运阴离子。发现二聚体蛋白质的每个单体单元能够独立于相邻亚基的运输活性进行Cl-/H-交换。NO3-或SCN-转运与H+反向转运部分解耦，但仍依赖于质子谷氨酸。将质子谷氨酸盐插入CLC通道改变了它们的门控，但未能将它们转化为Cl-/H+交换剂。噪声分析表明，ClC-5之间的沉默和运输状态切换的表观单位电导为0.5皮西门子。我们的结果是一致的想法，内体CLC-4和-5蛋白的Cl-/H+交换依赖于质子传递从细胞内可滴定的残基在位置268（编号CLC-5）和电流的强整流来自电压依赖性质子转移从Glu-268到Glu-211。
Many proteins of the CLC gene family are Cl- channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl-/H- exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl- and H+ transport. Surface expression was unchanged by these mutations. Uncoupled Cl- transport could be restored in the ClC-4(E281A) and ClC-5(E268A) proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H (+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl-/H- exchange independently from the transport activity of the neighboring subunit. NO3- or SCN- transport was partially uncoupled from H+ countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl-/H+ exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl-/H+ exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211.