title: A short segment of the R domain of cystic fibrosis transmembrane conductance regulator contains channel stimulatory and inhibitory activities that are separable by sequence modification.
abstract: The regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) contains consensus phosphorylation sites for cAMP-dependent protein kinase (PKA) that are the basis for physiological regulation of the CFTR chloride channel. A short peptide segment in the R domain with a net negative charge of B9 (amino acids 817-838, NEG2) and predicted helical tendency is shown to play a critical role in CFTR chloride channel function. Deletion of NEG2 from CFTR completely eliminates the PKA dependence of channel activity. Exogenous NEG2 peptide interacts with CFTR to exert both stimulatory and inhibitory effects on the channel function. The NEG2 peptide with sequence scrambled to remove helical tendencies also inhibits channel function, but does not stimulate. Similar results are found for a NEG2 peptide whose helical structure is disrupted by a proline residue. When six of the negatively charged carboxylic acid residues are replaced by their cognate amides, reducing net negative charge to B3, but increasing helical propensity as assessed by circular dichroism, the peptide stimulates CFTR channel function, but does not inhibit. We speculate that the NEG2 region interacts with other cytosolic domains of CFTR to control opening and closing transitions of the chloride channel.
authors.affiliation: Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
authors.forename: Junxia
authors.initials: J
authors.lastname: Xie
authors.forename: Lynn M
authors.initials: LM
authors.lastname: Adams
authors.forename: Jiying
authors.initials: J
authors.lastname: Zhao
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Gerken
authors.forename: Pamela B
authors.initials: PB
authors.lastname: Davis
authors.forename: Jianjie
authors.initials: J
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D002264:Carboxylic Acids / Q000737:chemistry; D002460:Cell Line; D002713:Chlorine / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: C505032:CFTR protein, human; D002264:Carboxylic Acids; D018076:DNA, Complementary; D010455:Peptides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002713:Chlorine; D011392:Proline; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases