title: Abnormal regulatory interactions of I148T-CFTR and the epithelial Na+ channel in Xenopus oocytes.
abstract: The mechanisms underlying regulatory interactions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na(+) channel (ENaC) in Xenopus oocytes are controversial. CFTR's first nucleotide binding domain (NBD-1) may be important in these interactions, because mutations within NBD-1 impair these functional interactions. We hypothesized that an abnormal CFTR containing a non-NBD-1 mutation and able to transport chloride would retain regulatory interactions with murine ENaC (mENaC). We tested this hypothesis for I148T-CFTR, where the mutation is located in CFTR's first intracellular loop. I148T-CFTR has been associated with a severe CF phenotype, perhaps because of defects in its regulation of bicarbonate transport, but it transports chloride similarly to wild-type CFTR in model systems (Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. Nature 410: 94-97, 2001). cRNAs encoding alphabetagamma-mENaC and I148T-CFTR were injected separately or together into Xenopus oocytes. mENaC and CFTR functional expression were assessed by two-electrode voltage clamp. mENaC whole oocyte expression was determined by immunoblotting, and surface expression was quantitated by surface biotinylation. Injection of I148T-CFTR cRNA alone yielded high levels of CFTR functional expression. In coinjected oocytes, mENaC functional and surface expression was not altered by activation of I148T-CFTR with forskolin/ IBMX. Furthermore, the CFTR potentiator genistein both enhanced functional expression of I148T-CFTR and restored regulation of mENaC surface expression by activated I148T-CFTR. These data suggest that the ability to transport chloride is not a critical determinant of regulation of mENaC by activated CFTR in Xenopus oocytes and provide further evidence that I148T-CFTR is dysfunctional despite maintaining the ability to transport chloride.
authors.affiliation: Division of Pulmonary Medicine, Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA 19104, USA.
authors.forename: Laurence
authors.initials: L
authors.lastname: Suaud
authors.forename: Wusheng
authors.initials: W
authors.lastname: Yan
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Rubenstein
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004594:Electrophysiology; D053503:Epithelial Sodium Channels / Q000378:metabolism* / Q000502:physiology; D005260:Female; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007267:Injections; D007532:Isoleucine; D051379:Mice; D009154:Mutation; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D018075:RNA, Complementary / Q000008:administration & dosage; D013912:Threonine; D014981:Xenopus
chemical_list: D002712:Chlorides; D053503:Epithelial Sodium Channels; D018075:RNA, Complementary; D007532:Isoleucine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D013912:Threonine; D019833:Genistein; D015056:1-Methyl-3-isobutylxanthine