Wang F, Zeltwanger S, Hu S, Hwang T C
Department of Physiology, Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA.
J Physiol. 2000 May 1;524 Pt 3(Pt 3):637-48. doi: 10.1111/j.1469-7793.2000.00637.x.
In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of DeltaF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and DeltaF508-CFTR channels expressed in NIH3T3 mouse fibroblasts. For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites. The time course of PKA-dependent activation of DeltaF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel. Once DeltaF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that DeltaF508-CFTR is not defective in ATP-dependent gating. By measuring whole-cell current density, we compared the number of functional channels in WT- and DeltaF508-CFTR cell membrane. Our data showed that the estimated channel density for DeltaF508-CFTR was approximately 10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by approximately 200-fold. We thus conclude that the functional defect (a decrease in Po) of DeltaF508-CFTR is as important as the trafficking defect (a decrease in the number of functional channels in the plasma membrane) in cystic fibrosis pathogenesis.
在用环磷酸腺苷（cAMP）激动剂刺激的细胞贴附式膜片中，苯丙氨酸508缺失的囊性纤维化跨膜传导调节因子（DeltaF508 - CFTR）通道（囊性纤维化中最常见的疾病相关突变）的单通道开放概率（Po）异常低（一种功能缺陷）。为了研究DeltaF508 - CFTR对cAMP刺激反应不佳的机制，我们在切除的内面向外膜片中，检测了在NIH3T3小鼠成纤维细胞中表达的野生型（WT）和DeltaF508 - CFTR通道的蛋白激酶A（PKA）依赖性磷酸化激活和ATP依赖性门控。对于WT - CFTR，添加PKA和ATP后CFTR通道电流的激活时间进程遵循S形函数，其时间常数随着[PKA]的增加而减小。[PKA]与表观激活速率之间的曲线关系表明，CFTR在多个磷酸化位点存在增量的磷酸化依赖性激活。DeltaF508 - CFTR通道电流的PKA依赖性激活时间进程也遵循S形函数，但激活速率比WT通道慢至少7倍。这一结果表明，苯丙氨酸508的缺失导致CFTR氯离子通道的PKA依赖性磷酸化减弱。一旦DeltaF508 - CFTR通道被PKA最大程度激活，突变通道和WT通道的稳态Po值、ATP剂量反应关系和单通道动力学无法区分，这表明DeltaF508 - CFTR在ATP依赖性门控方面没有缺陷。通过测量全细胞电流密度，我们比较了WT - 和DeltaF508 - CFTR细胞膜中功能性通道的数量。我们的数据显示，DeltaF508 - CFTR的估计通道密度比WT - CFTR低约10倍，但cAMP依赖性全细胞电流密度相差约200倍。因此，我们得出结论，在囊性纤维化发病机制中，DeltaF508 - CFTR的功能缺陷（Po降低）与转运缺陷（质膜中功能性通道数量减少）同样重要。
