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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Abnormal regulatory interactions of I148T-CFTR and the epithelial Na⁺ channel in Xenopus oocytes

¹Division of Pulmonary Medicine, Children's Hospital of Philadelphia, and ²Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Submitted 23 February 2006 ; accepted in final form 30 June 2006

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 -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.

cystic fibrosis transmembrane conductance regulator; genistein

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				L. Suaud, W. Yan, M. D. Carattino, A. Robay, T. R. Kleyman, and R. C. Rubenstein Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1553 - C1561. [Abstract] [Full Text] [PDF]