Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity
Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity
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DOI：
10.1074/jbc.271.44.27493
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发表时间：
1996-11-01
影响因子：
4.8
通讯作者：
Clarke, DM
中科院分区：
文献类型：
--
作者：
Seibert, FS;Linsdell, P;Clarke, DM
为了研究囊性纤维化跨膜传导调节因子（CFTR）的大胞质环对通道活性的贡献，在囊性纤维化患者的第三胞质环（CL3，残基933-990）中检测到三个点突变（S945L, H949Y, G970R），稳定表达野生型CFTR或突变型G970R-CFTR的中国仓鼠卵巢细胞系中，产生的主要产物为表观质量为170 kDa的多肽。而突变体S945L-CFTR和H949Y-CFTR的主要产物表观质量为150 kDa。150 kDa形式的CFTR对内糖苷酶H消化敏感，表明这些突变干扰了蛋白质的成熟。突变体H949Y在较低温度（26℃）下生长或在10%甘油存在下孵育时，成熟CFTR水平（170 kDa）增加。对于所有突变体，CFTR通道的开放概率（P-0）显著改变，S945L-CFTR和G970R-CFTR显示P-0严重降低。而H949Y突变的P-0是野生型的两倍。P-0的变化主要是由于平均爆发持续时间的改变，这表明CL3参与了获得和/或维持开放状态的稳定性。此外，突变体S945L和G970R在+/-80 mV范围内的电流-电压关系不是完全线性的，但表现出轻微的向外整流。CL3突变对通道电导的影响很微妙，这表明该区域可能在物理上接近孔的内口。
To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis, Chinese hamster ovary cell lines stably expressing wildtype CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein, Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 degrees C) or incubated in the presence of 10% glycerol, For all mutants, the open probability (P-0) of the CFTR channels was significantly altered, S945L-CFTR and G970R-CFTR showed a severe reduction in the P-0, whereas the H949Y mutation doubled the P-0 relative to wild type. The changes in P-0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range +/-80 mV, but showed slight outward rectification, The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.