Estévez Raúl, Pusch Michael, Ferrer-Costa Carles, Orozco Modesto, Jentsch Thomas J
Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Hamburg University, Falkenried 94, D-20246 Hamburg, Germany.
J Physiol. 2004 Jun 1;557(Pt 2):363-78. doi: 10.1113/jphysiol.2003.058453. Epub 2004 Jan 14.
All eukaryotic CLC Cl(-) channel subunits possess a long cytoplasmic carboxy-terminus that contains two so-called CBS (cystathionine beta-synthase) domains. These domains are found in various unrelated proteins from all phylae. The crystal structure of the CBS domains of inosine monophosphate dehydrogenase (IMPDH) is known, but it is not known whether this structure is conserved in CLC channels. Working primarily with ClC-1, we used deletion scanning mutagenesis, coimmunoprecipitation and electrophysiology to demonstrate that its CBS domains interact. The replacement of CBS domains of ClC-1 with the corresponding CBS domains from other CLC channels and even human IMPDH yielded functional channels, indicating a high degree of structural conservation. Based on a homology model of the pair of CBS domains of CLC channels, we identified some residues that, when mutated, affected the common gate which acts on both pores of the dimeric channel. Thus, we propose that the structure of CBS domains from CLC channels is highly conserved and that they play a functional role in the common gate.
所有真核生物的CLC氯离子通道亚基都拥有一个长长的胞质羧基末端，该末端包含两个所谓的CBS（胱硫醚β-合酶）结构域。这些结构域存在于所有生物门类的各种不相关蛋白质中。肌苷单磷酸脱氢酶（IMPDH）的CBS结构域的晶体结构是已知的，但尚不清楚该结构在CLC通道中是否保守。我们主要以ClC-1为研究对象，运用缺失扫描诱变、免疫共沉淀和电生理学方法来证明其CBS结构域相互作用。用其他CLC通道甚至人类IMPDH的相应CBS结构域替换ClC-1的CBS结构域，产生了功能性通道，这表明结构具有高度保守性。基于CLC通道CBS结构域对的同源模型，我们确定了一些残基，这些残基发生突变时会影响作用于二聚体通道两个孔的共同门控。因此，我们提出CLC通道的CBS结构域结构高度保守，并且它们在共同门控中发挥功能作用。
