A FUNCTIONAL CFTR-NBF1 IS REQUIRED FOR ROMK2-CFTR INTERACTION

In a previous study on inside-out patches of Xenopus oocytes, we demon strated that the cystic fibrosis transmembrane conductance regulator ( CFTR) enhances the glibenclamide sensitivity of a coexpressed inwardly rectifying K+ channel, ROMK2 (C. M. McNicholas, W. B. Guggino, E. M. Schwiebert, S. C. Hebert, G. Giebisch, and M. E. Egan. Proc. Natl. Aca d. Sci. USA 93: 8083-8088, 1996). In the present study, we used the tw o-microelectrode voltage-clamp technique to measure whole cell K+ curr ents in Xenopus oocytes, and we further characterized the enhanced sen sitivity of ROMK2 to glibenclamide by CFTR. Glibenclamide inhibited K currents by 56% in oocytes expressing both ROMK2 and CFTR but only 11 % in oocytes expressing ROMK2 alone. To examine the role of the first nucleotide binding fold (NBF1) of CFTR in the ROMK2-CFTR interaction, we studied the glibenclamide sensitivity of ROMK2 when coexpressed wit h CFTR constructs containing mutations in or around the NBF1 domain. I n oocytes coinjected with ROMK2 and a truncated construct of CFTR with an intact NBF1 (CFTR-K593X), glibenclamide inhibited K+ currents by 4 6%. However, in oocytes coinjected with ROMK2 and a CFTR mutant trunca ted immediately before NBF1 (CFTR-K370X), glibenclamide inhibited K+ c urrents by 12%. Also, oocytes expressing both ROMK2 and CFTR mutants w ith naturally occurring NBF1 point mutations, CFTR-G551D or CFTR-A455E , display glibenclamide-inhibitable K+ currents of only 14 and 25%, re spectively. Because CFTR mutations that alter the NBF1 domain reduce t he glibenclamide sensitivity of the coexpressed ROMK2 channel, we conc lude that the NBF1 motif is necessary for the CFTR-ROMK2 interaction t hat confers sulfonylurea sensitivity.