A functional CFTR-NBF1 is required for ROMK2-CFTR interaction

• Published in: The American journal of physiology Vol. 273; no. 5; p. F843
• Main Authors: McNicholas, C M, Nason, Jr, M W, Guggino, W B, Schwiebert, E M, Hebert, S C, Giebisch, G, Egan, M E
• Format: Journal Article
• Language: English
• Published: United States 01-11-1997
• Subjects: Amino Acid Substitution; Animals; Base Sequence; Binding Sites; Cystic Fibrosis Transmembrane Conductance Regulator - chemistry; Cystic Fibrosis Transmembrane Conductance Regulator - physiology; Female; Glyburide - pharmacology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Models, Molecular; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Oocytes - physiology; Patch-Clamp Techniques; Potassium Channels - chemistry; Potassium Channels - physiology; Potassium Channels, Inwardly Rectifying; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Xenopus laevis
• ISSN: 0002-9513
• DOI: 10.1152/ajprenal.1997.273.5.f843

In a previous study on inside-out patches of Xenopus oocytes, we demonstrated 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. Acad. Sci. USA 93: 8083-8088, 1996). In the present study, we used the two-microelectrode voltage-clamp technique to measure whole cell K+ currents in Xenopus oocytes, and we further characterized the enhanced sensitivity 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 with CFTR constructs containing mutations in or around the NBF1 domain. In oocytes coinjected with ROMK2 and a truncated construct of CFTR with an intact NBF1 (CFTR-K593X), glibenclamide inhibited K+ currents by 46%. However, in oocytes coinjected with ROMK2 and a CFTR mutant truncated immediately before NBF1 (CFTR-K370X), glibenclamide inhibited K+ currents by 12%. Also, oocytes expressing both ROMK2 and CFTR mutants with naturally occurring NBF1 point mutations, CFTR-G551D or CFTR-A455E, display glibenclamide-inhibitable K+ currents of only 14 and 25%, respectively. Because CFTR mutations that alter the NBF1 domain reduce the glibenclamide sensitivity of the coexpressed ROMK2 channel, we conclude that the NBF1 motif is necessary for the CFTR-ROMK2 interaction that confers sulfonylurea sensitivity.