Schwiebert E M, Flotte T, Cutting G R, Guggino W B
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Am J Physiol. 1994 May;266(5 Pt 1):C1464-77. doi: 10.1152/ajpcell.1994.266.5.C1464.
From whole cell patch-clamp recordings at 35 degrees C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)-activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP-activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP-activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in delta F508-CFTR-containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.
在35摄氏度下，利用制霉菌素穿孔法或在移液管溶液中加入5 mM MgATP的传统方法进行全细胞膜片钳记录，结果表明，囊性纤维化跨膜电导调节因子（CFTR）氯离子（Cl-）通道和外向整流Cl-通道（ORCC）均对培养的人气道上皮细胞中3',5'-环磷酸腺苷（cAMP）激活的全细胞Cl-电流有贡献。无论记录是在两种正常人细胞系上进行，还是在稳定补充有野生型CF基因的两种囊性纤维化（CF）细胞系上进行，这些结果都是相似的。这些结果是通过利用CFTR和ORCC电流不同的生物物理特性获得的，例如电流-电压关系的整流程度、对Cl-通道阻断药物（如4,4'-二异硫氰酸芪-2,2'-二磺酸（DIDS）、杯芳烃和二苯胺羧酸（DPC））的敏感性差异，以及两个通道相对于I-的相反Cl-通透性。在正常细胞或补充CF的细胞中，8-（4-氯苯硫基）腺苷3',5'-环磷酸刺激外向整流全细胞Cl-电流。在cAMP存在的情况下加入DIDS会抑制cAMP激活的Cl-电流的外向整流部分。剩余的cAMP激活的、DIDS不敏感的线性CFTR Cl-电流被DPC完全抑制。其他结果表明，不仅ORCC和CFTR Cl-通道对表达野生型CFTR的气道上皮细胞中cAMP激活的Cl-电流有贡献，而且在含有ΔF508-CFTR的CF气道细胞中，这两种通道均对cAMP无反应。我们得出结论，CFTR不仅在气道上皮细胞中作为cAMP调节的Cl-通道发挥作用，而且还控制ORCC的调节。
