Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC.
Regulatory interactions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na(+) channel (ENaC) are readily apparent in Xenopus oocytes. However, the mechanism underlying these interactions remains controversial. CFTR's first nucleotide binding fold (NBD-1) may be important in these interactions, as dysfunctional CFTRs containing mutations within NBD-1, such as DeltaF508 and G551D, lack such functional interactions with murine ENaC (mENaC). We hypothesized that a dysfunctional CFTR containing a non-NBD-1 mutation would retain regulatory interactions with mENaC and tested this hypothesis for N1303K-CFTR, where the mutation is located in CFTR's second nucleotide binding fold (NBD-2). cRNA for alphabetagamma-mENaC and N1303K-CFTR was injected separately or together into Xenopus oocytes. ENaC and CFTR functional expression was assessed by two-electrode voltage clamp. Injection of N1303K (class II trafficking mutation) yielded low levels of CFTR function on activation with forskolin and 3-isobutyl-1-methylxanthine (IBMX). In coinjected oocytes, N1303K did not alter mENaC functional expression or surface expression before activation of N1303K. This is similar to our prior observations with DeltaF508. However, unlike our observations with DeltaF508, activation of N1303K acutely decreased mENaC functional and surface expression, and N1303K currents were enhanced by coinjection of mENaC. Furthermore, genistein only mildly enhanced the functional expression of N1303K-CFTR and did not improve regulation of ENaC by N1303K-CFTR. These data suggest that a structurally and functionally intact CFTR NBD-1 in activated CFTR can regulate mENaC surface expression independent of Cl(-) transport in Xenopus oocytes.
Suaud
Laurence
L
Division of Pulmonary Medicine, Abramson 410C, Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA 19104, USA.
Yan
Wusheng
W
Carattino
Marcelo D
MD
Robay
Amal
A
Kleyman
Thomas R
TR
Rubenstein
Ronald C
RC
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels / Q000502:physiology*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport; D051379:Mice; D009154:Mutation; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D014982:Xenopus laevis
D002712:Chlorides; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D015056:1-Methyl-3-isobutylxanthine