Surprises from an Unusual CLC Homolog
Surprises from an Unusual CLC Homolog
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DOI：
10.1016/j.bpj.2012.08.063
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发表时间：
2012-11-07
影响因子：
3.4
通讯作者：
Matulef, Kimberly
中科院分区：
文献类型：
--
作者：
Phillips, Sabrina;Brammer, Ashley E.;Matulef, Kimberly
氯离子通道（CLC）家族的独特之处在于，一些成员是Cl-离子通道，另一些是Cl-/H+反向转运蛋白。在反向转运蛋白中耦合H+和Cl-转运的分子机制仍然未知。我们的一种新的细菌同源物的表征从柠檬酸杆菌koseri，CLC-CK 2，产生了令人惊讶的发现CLC蛋白的Cl-和H+运输的要求。首先，即使CLC-ck 2缺乏保守的氨基酸附近的Cl-结合位点的CLC选择性签名序列的一部分，这种蛋白质催化Cl-运输，虽然缓慢。CLC-ck 2的离子选择性与CLC-ec 1相似，不同之处在于SO 42-通过CLC-ck 2与Cl-吸收强烈竞争，但对CLC-ec 1没有影响。第二，更令人惊讶的是，CLC-ck 2是Cl-/H+反向转运蛋白，尽管它在Glu（in）位置含有异亮氨酸，这在以前被认为是H+途径的关键部分。CLC-ck 2是第一个已知的在该位置含有非极性残基的反向转运蛋白。在CLC-ck 2中Glu（in）位点处引入谷氨酸不会增加H+通量。与其他CLC反向转运蛋白一样，CLC-ck 2中外部谷氨酸门（Glu（ex））的突变阻止H+通量。因此，Glu（ex）而不是Glu（in）对于CLC蛋白中的H+渗透是关键的。
The chloride channel (CLC) family is distinctive in that some members are Cl- ion channels and others are Cl-/H+ antiporters. The molecular mechanism that couples H+ and Cl- transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl- and H+ transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl--binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl- transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO42- strongly competes with Cl- uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl-/H+ antiporter, even though it contains an isoleucine at the Glu(in) position that was previously thought to be a critical part of the H+ pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Glu(in) site in CLC-ck2 does not increase H+ flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu(ex)) in CLC-ck2 prevents H+ flux. Hence, Glu(ex), but not Glu(in), is critical for H+ permeation in CLC proteins.