A functional CFTR-NBF1 is required for ROMK2-CFTR interaction.
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.
McNicholas
C M
CM
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026, USA.
Nason
M W
MW
Guggino
W B
WB
Schwiebert
E M
EM
Hebert
S C
SC
Giebisch
G
G
Egan
M E
ME
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000502:physiology*; D005260:Female; D005905:Glyburide / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis
D009838:Oligodeoxyribonucleotides; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005905:Glyburide