Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance.
Cystic fibrosis transmembrane conductance regulator (CFTR) apparently forms Cl- channels in apical membranes of secretory epithelial cells. A detailed model describes molecular structure and biophysical properties of CFTR and the impact of various mutations as they occur in cystic fibrosis. In the present report mutations were introduced into the putative 6th alpha-helical transmembrane pore forming domain of CFTR. The mutants were subsequently expressed in Xenopus oocytes by injection of the respective cRNAs. Whole cell (wc) conductances could be reversibly activated by IBMX (1 nmol/l) only in oocytes injected with wild-type (wt) or mutant CFTR but not in oocytes injected with water or antisense CFTR. The activated conductance was partially inhibited by (each 100 mumol/l) DIDS (27%) and glibenclamide (77%), but not by 10 mumol/l NPPB. The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H. They did not measurably change the wt-CFTR anion permeability (P) and we conductance (G) sequence of: PCl- > PBr- > P1- and GCl- > GBr- > G1-, respectively. Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR. Various mutants for which positively charged amino acids were replaced by histidines (K335H, R347H, R334H) did not show pH sensitivity of the IBMX activated wc conductance. We, therefore, cannot confirm previous results. CFTR might have a different molecular structure than previously suggested or it might act as a regulator of ion conductances.
Hipper
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Physiologisches Institut, Albert-Ludwigs-Universität Freiburg, Germany.
Mall
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Greger
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Kunzelmann
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D000838:Anions; D018118:Chloride Channels / Q000502:physiology*; D000242:Cyclic AMP / Q000494:pharmacology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004553:Electric Conductivity; D005260:Female; D015870:Gene Expression; D006639:Histidine; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010539:Permeability; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013861:Thiocyanates / Q000378:metabolism; D014162:Transfection; D014982:Xenopus laevis
D000838:Anions; D018118:Chloride Channels; D013861:Thiocyanates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006639:Histidine; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine