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Deletion of phenylalanine 508 causes attenuated phosphorylation‐dependent activation of CFTR chloride channels
Author(s) -
Wang Fei,
Zeltwanger Shawn,
Hu Shenghui,
Hwang TzyhChang
Publication year - 2000
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2000.00637.x
Subject(s) - chloride channel , cystic fibrosis transmembrane conductance regulator , phosphorylation , chemistry , gating , patch clamp , cystic fibrosis , biochemistry , biophysics , microbiology and biotechnology , biology , receptor , gene , genetics
1 In cell‐attached patches stimulated with cAMP agonists, the single‐channel open probability ( P o ) of the phenylalanine 508‐deleted cystic fibrosis transmembrane conductance regulator (ΔF508‐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 ΔF508‐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 ΔF508‐CFTR channels expressed in NIH3T3 mouse fibroblasts. 2 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. 3 The time course of PKA‐dependent activation of ΔF508‐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. 4 Once ΔF508‐CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady‐state P o values, ATP dose‐response relationships and single‐channel kinetics, indicating that ΔF508‐CFTR is not defective in ATP‐dependent gating. 5 By measuring whole‐cell current density, we compared the number of functional channels in WT‐ and ΔF508‐CFTR cell membrane. Our data showed that the estimated channel density for ΔF508‐CFTR was ∼10‐fold lower than that for WT‐CFTR, but the cAMP‐dependent whole‐cell current density differed by ∼200‐fold. We thus conclude that the functional defect (a decrease in P o ) of ΔF508‐CFTR is as important as the trafficking defect (a decrease in the number of functional channels in the plasma membrane) in cystic fibrosis pathogenesis.