Broadbent Steven D, Wang Wuyang, Linsdell Paul
Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS B3H 4R2, Canada.
Biochem Cell Biol. 2014 Oct;92(5):390-6. doi: 10.1139/bcb-2014-0066. Epub 2014 Aug 20.
Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is thought to be controlled by cytoplasmic factors. However, recent evidence has shown that overall channel activity is also influenced by extracellular anions that interact directly with the extracellular loops (ECLs) of the CFTR protein. Very little is known about the structure of the ECLs or how substances interacting with these ECLs might affect CFTR function. We used patch-clamp recording to investigate the accessibility of cysteine-reactive reagents to cysteines introduced throughout ECL1 and 2 key sites in ECL4. Furthermore, interactions between ECL1 and ECL4 were investigated by the formation of disulfide crosslinks between cysteines introduced into these 2 regions. Crosslinks could be formed between R899C (in ECL4) and a number of sites in ECL1 in a manner that was dependent on channel activity, suggesting that the relative orientation of these 2 loops changes on activation. Formation of these crosslinks inhibited channel function, suggesting that relative movement of these ECLs is important to normal channel function. Implications of these findings for the effects of mutations in the ECLs that are associated with cystic fibrosis and interactions with extracellular substances that influence channel activity are discussed.
囊性纤维化跨膜传导调节因子（CFTR）氯离子通道的活性被认为受细胞质因子控制。然而，最近的证据表明，通道的整体活性也受到细胞外阴离子的影响，这些阴离子直接与CFTR蛋白的细胞外环（ECL）相互作用。关于ECL的结构或与这些ECL相互作用的物质如何影响CFTR功能，人们了解甚少。我们使用膜片钳记录来研究半胱氨酸反应试剂对引入ECL1和ECL4关键位点的半胱氨酸的可及性。此外，通过在引入这两个区域的半胱氨酸之间形成二硫键交联来研究ECL1和ECL4之间的相互作用。R899C（在ECL4中）与ECL1中的多个位点之间可以以依赖于通道活性的方式形成交联，这表明这两个环的相对取向在激活时会发生变化。这些交联的形成抑制了通道功能，表明这些ECL的相对移动对正常通道功能很重要。本文讨论了这些发现对与囊性纤维化相关的ECL突变的影响以及与影响通道活性的细胞外物质相互作用的意义。
