Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels.
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.
Wang
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Department of Physiology, Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA.
Zeltwanger
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Hu
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Hwang
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D016475:3T3 Cells / Q000737:chemistry / Q000201:enzymology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D019833:Genistein / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics*; D051379:Mice; D018408:Patch-Clamp Techniques; D010649:Phenylalanine; D010766:Phosphorylation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D013873:Thionucleotides / Q000494:pharmacology
D004791:Enzyme Inhibitors; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D019833:Genistein; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases