Abstract:
Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K+) solutions, we determined that CLIC6 is more permeable to chloride-(Cl-) as compared to bromide-(Br-), fluoride-(F-), and K+ ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 μM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl- channel.
摘要:
氯化物细胞内通道(CLIC)是以可溶性和跨膜形式存在的蛋白质家族。最新发现的CLIC6家族成员与乳房有关,卵巢,肺胃,和胰腺癌,并且还已知与多巴胺-(D(2)-样)受体相互作用。通道的可溶性结构已得到解决,但CLIC6的确切生理作用,生物物理表征,膜结构仍然未知。在这里,我们旨在使用膜片钳方法表征该通道的生物物理特性。为了确定CLIC6的生物物理特性,我们在HEK-293细胞中表达CLIC6。关于异位表达,CLIC6定位于HEK-293细胞的质膜。我们通过使用全细胞建立了CLIC6的生物物理特性,和细胞附着的配置。使用各种阴离子和钾(K+)溶液,我们确定CLIC6与溴化物-(Br-)相比,对氯化物-(Cl-)更具渗透性,Fluoride-(F-),K+离子在整个细胞配置中,加入10μMIAA-94(CLIC特异性阻断剂)后,CLIC6电流受到抑制.还发现CLIC6受pH和氧化还原电位调节。我们证明,组氨酸残基在648(H648)在C端,和N端半胱氨酸残基(C487)分别直接参与pH诱导的CLIC6的构象变化和氧化还原调节。使用qRT-PCR,我们发现CLIC6在肺和大脑中含量最高,我们记录了小鼠肺上皮细胞(MLE)中的CLIC6电流。总的来说,我们已经确定了CLIC6的生物物理特性,并将其确立为Cl-通道。