Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels.
Gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels requires intermolecular or interdomain interactions, but the exact nature and physiological significance of those interactions remains uncertain. Subconductance states of the channel may result from alterations in interactions among domains, and studying mutant channels enriched for a single conductance type may elucidate those interactions. Analysis of CFTR channels in inside-out patches revealed that mutation of cysteine residues in NBD1 and NBD2 affects the frequency of channel opening to the full-size versus a 3-pS subconductance. Mutating cysteines in NBD1 resulted in channels that open almost exclusively to the 3-pS subconductance, while mutations of cysteines in NBD2 decreased the frequency of subconductance openings. Wild-type channels open to both size conductances and make fast transitions between them within a single open burst. Full-size and subconductance openings of both mutant and wild-type channels are similarly activated by ATP and phosphorylation. However, the different size conductances open very differently in the presence of a nonhydrolyzable ATP analog, with subconductance openings significantly shortened by ATPgammaS, while full-size channels are locked open. In wild-type channels, reducing conditions increase the frequency and decrease the open time of subconductance channels, while oxidizing conditions decrease the frequency of subconductance openings. In contrast, in the cysteine mutants studied, altering redox potential has little effect on gating of the subconductance.
Harrington
Melissa A
MA
Department of Biology, Delaware State University, Dover, Delaware 19901, USA. mharring@dsc.edu
Kopito
Ron R
RR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics; D004553:Electric Conductivity; D004594:Electrophysiology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors
C505032:CFTR protein, human; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D003545:Cysteine