Zifarelli Giovanni, Pusch Michael
Institute of Biophysics, National Research Council, Genova, Italy.
J Gen Physiol. 2008 Feb;131(2):109-16. doi: 10.1085/jgp.200709899.
ClC-1 belongs to the gene family of CLC Cl(-) channels and Cl(-)/H(+) antiporters. It is the major skeletal muscle chloride channel and is mutated in dominant and recessive myotonia. In addition to the membrane-embedded part, all mammalian CLC proteins possess a large cytoplasmic C-terminal domain that bears two so-called CBS (from cystathionine-beta-synthase) domains. Several studies indicate that these domains might be involved in nucleotide binding and regulation. In particular, Bennetts et al. (J. Biol. Chem. 2005. 280:32452-32458) reported that the voltage dependence of hClC-1 expressed in HEK cells is regulated by intracellular ATP and other nucleotides. Moreover, very recently, Bennetts et al. (J. Biol. Chem. 2007. 282:32780-32791) and Tseng et al. (J. Gen. Physiol. 2007. 130:217-221) reported that the ATP effect was enhanced by intracellular acidification. Here, we show that in striking contrast with these findings, human ClC-1, expressed in Xenopus oocytes and studied with the inside-out configuration of the patch-clamp technique, is completely insensitive to intracellular ATP at concentrations up to 10 mM, at neutral pH (pH 7.3) as well as at slightly acidic pH (pH 6.2). These results have implications for a general understanding of nucleotide regulation of CLC proteins and for the physiological role of ClC-1 in muscle excitation.
ClC-1属于CLC Cl(-)通道和Cl(-)/H(+)反向转运蛋白的基因家族。它是主要的骨骼肌氯离子通道，在显性和隐性肌强直中发生突变。除了膜嵌入部分外，所有哺乳动物的CLC蛋白都具有一个大的细胞质C末端结构域，该结构域带有两个所谓的CBS（来自胱硫醚-β-合酶）结构域。多项研究表明，这些结构域可能参与核苷酸结合和调节。特别是，贝内茨等人（《生物化学杂志》，2005年。280:32452 - 32458）报道，在HEK细胞中表达的hClC-1的电压依赖性受细胞内ATP和其他核苷酸的调节。此外，最近，贝内茨等人（《生物化学杂志》，2007年。282:32780 - 32791）和曾等人（《普通生理学杂志》，2007年。130:217 - 221）报道，细胞内酸化增强了ATP效应。在这里，我们表明，与这些发现形成鲜明对比的是，在非洲爪蟾卵母细胞中表达并采用膜片钳技术的内面向外模式进行研究的人ClC-1，在中性pH（pH 7.3）以及微酸性pH（pH 6.2）条件下，对浓度高达10 mM的细胞内ATP完全不敏感。这些结果对于全面理解CLC蛋白的核苷酸调节以及ClC-1在肌肉兴奋中的生理作用具有重要意义。
