Xie J, Drumm M L, Zhao J, Ma J, Davis P B
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Biophys J. 1996 Dec;71(6):3148-56. doi: 10.1016/S0006-3495(96)79508-5.
The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) is a splice variant of the epithelial CFTR, with lacks 30 amino acids encoded by exon 5 in the first intracellular loop. For examination of the role of exon 5 in CFTR channel function, a CFTR deletion mutant, in which exon 5 was removed from the human epithelial CFTR, was constructed. The wild type and delta exon5 CFTR were 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, whereas cells transfected with delta exon5 CFTR express only a core-glycosylated from (approximately 140 kDa). The Western blot test performed on subcellular membrane fractions showed that delta exon5 CFTR was located in the intracellular membranes. Neither incubation at lower temperature (26 degrees C) nor stimulation of 293 HEK cells with forskolin or CPT-cAMP caused improvement in glycosylation and processing of delta exon5 CFTR proteins, indicating that the human epithelial CFTR lacking exon5 did not process properly in 293 HEK cells. On incorporation of intracellular membrane vesicles containing the delta exon5 CFTR proteins into the lipid bilayer membrane, functional phosphorylation- and ATP-dependent chloride channels were identified. CFTR channels with an 8-pS full-conductance state were observed in 14% of the experiments. The channel had an average open probability (Po) of 0.098 +/- 0.022, significantly less than that of the wild type CFTR (Po = 0.318 +/- 0.028). More frequently, the delta exon5 CFTR formed chloride channels with lower conductance states of approximately 2-3 and approximately 4-6 pS. These subconductance states were also observed with wild type CFTR but to a much lesser extent. Average Po for the 2-3-pS subconductance state, estimated from the area under the curve on an amplitude histogram, was 0.461 +/- 0.194 for delta exon5 CFTR and 0.332 +/- 0.142 for wild type (p = 0.073). The data obtained indicate that deleting 30 amino acids from the first intracellular loop of CFTR affects both processing and function of the CFTR chloride channel.
囊性纤维化跨膜传导调节因子（CFTR）的心脏异构体是上皮CFTR的一种剪接变体，其在第一个细胞内环中缺少外显子5编码的30个氨基酸。为了研究外显子5在CFTR通道功能中的作用，构建了一种CFTR缺失突变体，其中从人上皮CFTR中去除了外显子5。野生型和外显子5缺失的CFTR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），而用外显子5缺失的CFTR转染的细胞仅表达一种核心糖基化形式（约140 kDa）。对亚细胞膜组分进行的蛋白质印迹试验表明，外显子5缺失的CFTR位于细胞内膜中。在较低温度（26℃）下孵育或用福斯可林或CPT - cAMP刺激293 HEK细胞均未导致外显子5缺失的CFTR蛋白的糖基化和加工得到改善，这表明缺少外显子5 的人上皮CFTR在293 HEK细胞中加工不正常。将含有外显子5缺失的CFTR蛋白的细胞内膜囊泡掺入脂质双分子层膜中时，鉴定出了功能性的磷酸化和ATP依赖性氯离子通道。在14%的实验中观察到具有8 - pS全电导状态的CFTR通道。该通道的平均开放概率（Po）为0.098±0.022，显著低于野生型CFTR（Po = 0.318±0.028）。更常见的是，外显子5缺失的CFTR形成了电导状态较低的氯离子通道，约为2 - 3 pS和约4 - 6 pS。野生型CFTR也观察到了这些亚电导状态，但程度要小得多。根据幅度直方图上曲线下的面积估计，外显子5缺失的CFTR在2 - 3 - pS亚电导状态下的平均Po为0.461±0.194，野生型为0.332±0.142（p = 0.073）。获得的数据表明，从CFTR的第一个细胞内环中删除30个氨基酸会影响CFTR氯离子通道的加工和功能。
