Flores Carlos A, Niemeyer María Isabel, Sepúlveda Francisco V, Cid L Pablo
Centro de Estudios Científicos (CECS), Valdivia, Chile.
Mol Membr Biol. 2006 Mar-Apr;23(2):149-56. doi: 10.1080/09687860500449978.
Members of the ClC family of membrane proteins have been found in a variety of species and they can function as Cl- channels or Cl-/H+ antiporters. Three potential ClC genes are present in the Drosophila melanogaster genome. Only one of them shows homology with a branch of the mammalian ClC genes that encode plasma membrane Cl- channels. The remaining two are close to mammalian homologues coding for intracellular ClC proteins. Using RT-PCR we have identified two splice variants showing highest homology (41% residue identity) to the mammalian ClC-2 chloride channel. One splice variant (DmClC-2S) is expressed in the fly head and body and an additional, larger variant (DmClC-2L) is only present in the head. Both putative Drosophila channels conserve key features of the ClC channels cloned so far, including residues conforming the selectivity filter and C-terminus CBS domains. The splice variants differ in a stretch of 127 aa at the intracellular C-terminal portion separating cystathionate beta synthase (CBS) domains. Expression of either Drosophila ClC-2 variant in HEK-293 cells generated inwardly rectifying Cl- currents with similar activation and deactivation characteristics. There was great similarity in functional characteristics between DmClC-2 variants and their mammalian counterpart, save for slower opening kinetics and faster closing rate. As CBS domains are believed to be sites of regulation of channel gating and trafficking, it is suggested that the extra amino acids present between CBS domains in DmClC-2L might endow the channel with a differential response to signals present in the fly cells where it is expressed.
膜蛋白ClC家族的成员已在多种物种中被发现，它们可作为Cl⁻通道或Cl⁻/H⁺反向转运体发挥作用。果蝇基因组中存在三个潜在的ClC基因。其中只有一个与编码质膜Cl⁻通道的哺乳动物ClC基因分支具有同源性。其余两个与编码细胞内ClC蛋白的哺乳动物同源物相近。我们利用逆转录聚合酶链反应（RT-PCR）鉴定出了两个剪接变体，它们与哺乳动物ClC-2氯通道具有最高的同源性（41%的残基同一性）。一个剪接变体（DmClC-2S）在果蝇的头部和身体中表达，另一个更大的变体（DmClC-2L）仅存在于头部。这两个推测的果蝇通道都保留了迄今为止克隆的ClC通道的关键特征，包括构成选择性过滤器的残基和C端CBS结构域。这两个剪接变体在细胞内C端部分分隔胱硫醚β合酶（CBS）结构域的127个氨基酸片段上有所不同。在HEK-293细胞中表达任何一种果蝇ClC-2变体都会产生具有相似激活和失活特征的内向整流Cl⁻电流。DmClC-2变体与其哺乳动物对应物在功能特征上有很大的相似性，只是开放动力学较慢且关闭速率较快。由于CBS结构域被认为是通道门控和运输的调节位点，因此有人提出，DmClC-2L中CBS结构域之间存在的额外氨基酸可能使该通道对其表达所在的果蝇细胞中存在的信号产生不同的反应。
