Xie J, Drumm M L, Ma J, Davis P B
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
J Biol Chem. 1995 Nov 24;270(47):28084-91. doi: 10.1074/jbc.270.47.28084.
The cystic fibrosis transmembrane conductance regulator (CFTR) contains two membrane-spanning domains; each consists of six transmembrane segments joined by three extracellular and two intracellular loops of different length. To examine the role of intracellular loops in CFTR channel function, we studied a deletion mutant of CFTR (delta 19 CFTR) in which 19 amino acids were removed from the intracellular loop joining transmembrane segments IV and V. This mutant protein was expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild-type CFTR cDNA, while cells transfected with the mutant gene expressed only a core-glycosylated form (approximately 140 kDa). The chloride efflux rate (measured by 6-methoxyl-N-(3-sulfopropyl) quinolinium SPQ fluorescence) from cells expressing wild-type CFTR increased 600% in response to forskolin. In contrast, delta 19 CFTR-expressing cells had no significant response to forskolin. Western blotting performed on subcellular membrane fractions showed that delta 19 CFTR was located in the same fractions as delta F508 CFTR, a processing mutant of CFTR. These results suggest that delta 19 CFTR is located in the intracellular membranes, without reaching the cell surface. Upon reconstitution into lipid bilayer membranes, delta 19 CFTR formed a functional Cl- channel with gating properties nearly identical to those of the wild-type CFTR channel. However, delta 19 CFTR channels exhibited frequent transitions to a 6-picosiemens subconductance state, whereas wild-type CFTR channels rarely exist in this subconductance state. These data suggest that the intracellular loop is involved in stabilizing the full conductance state of the CFTR Cl- channel.
囊性纤维化跨膜传导调节因子（CFTR）包含两个跨膜结构域；每个结构域由六个跨膜片段组成，这些片段由三个不同长度的细胞外环和两个细胞内环连接。为了研究细胞内环在CFTR通道功能中的作用，我们研究了CFTR的一个缺失突变体（δ19 CFTR），其中从连接跨膜片段IV和V的细胞内环中去除了19个氨基酸。这种突变蛋白在人胚肾细胞系（293 HEK）中表达。从用野生型CFTR cDNA转染的细胞中免疫沉淀出完全成熟的糖基化CFTR（约170 kDa），而用突变基因转染的细胞仅表达核心糖基化形式（约140 kDa）。表达野生型CFTR的细胞的氯离子外流率（通过6-甲氧基-N-（3-磺丙基）喹啉鎓SPQ荧光测量）对福斯可林的反应增加了600%。相比之下，表达δ19 CFTR的细胞对福斯可林没有明显反应。对亚细胞膜组分进行的蛋白质印迹分析表明，δ19 CFTR与CFTR的加工突变体δF508 CFTR位于相同的组分中。这些结果表明，δ19 CFTR位于细胞内膜中，未到达细胞表面。在重组到脂质双分子层膜中后，δ19 CFTR形成了一个功能性氯离子通道，其门控特性与野生型CFTR通道几乎相同。然而，δ19 CFTR通道频繁转变为6皮安的亚电导状态，而野生型CFTR通道很少处于这种亚电导状态。这些数据表明，细胞内环参与稳定CFTR氯离子通道的全电导状态。
