Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator.
CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.
Schwiebert
E M
EM
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Morales
M M
MM
Devidas
S
S
Egan
M E
ME
Guggino
W B
WB
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001980:Bronchi / Q000502:physiology / Q000503:physiopathology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D018076:DNA, Complementary; D004847:Epithelial Cells / Q000502:physiology; D005260:Female; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection; D014982:Xenopus laevis
C505032:CFTR protein, human; D018118:Chloride Channels; D002712:Chlorides; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid