Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes.
Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes.
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没有外显子 5 的人上皮囊性纤维化跨膜电导调节器维持细胞内膜中的部分氯离子通道功能。
DOI：
10.1016/s0006-3495(96)79508-5
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Davis,PB
中科院分区：
文献类型：
--
作者：
Xie,J;Drumm,ML;Zhao,J;Ma,J;Davis,PB
囊性纤维化跨膜传导调节因子(CFTR)的心脏亚型是上皮CFTR的剪接变体，在第一细胞内环中缺少由外显子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，构建了一个从人上皮CFTR中去除外显子5的CFTR缺失突变体。野生型和Delta外显子5在人胚胎肾细胞系(293HEK)中表达。免疫共沉淀法检测到野生型CFTRc DNA可表达完全成熟的糖基化cftR(约170 kDa)，而转Delta外显子5 cftr的细胞仅表达约140 kDa的核心糖基化cftr。对亚细胞膜进行的Western印迹实验表明，Delta外显子5 CFTR定位于细胞内膜。在较低温度(26℃)下孵育或用Forsklin或CPT-cAMP刺激293 HEK细胞，都不能改善其糖基化和处理外显子5的CFTR蛋白，表明缺失外显子5的人上皮性CFTR不能在293 HEK细胞中正常加工。当含有外显子5的CFTR蛋白的细胞内膜囊泡被掺入脂双层膜时，功能依赖的磷酸化和ATP依赖的氯通道被鉴定出来。在14%的实验中观察到具有8-ps全电导状态的CFTR通道。通道的平均开放概率(Po)为0.098+/-0.022，显著低于野生型(Po=0.318+/-0.028)。更常见的是，增量外显子5形成的氯离子通道具有大约2-3和大约4-6ps的低电导状态。野生型CFTR也观察到了这些亚导状态，但程度要小得多。根据幅值直方图上曲线下面积估计的2-3-pS亚导状态的平均Po为0.461+/-0.194，而野生型为0.332+/-0.142(p=0.073)。所获得的数据表明，从CFTR的第一个细胞内环中删除30个氨基酸会影响CFTR氯通道的加工和功能。
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.