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

Academic Article

Abstract

  • 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.
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    Keywords

  • Amino Acid Substitution, Animals, Base Sequence, Binding Sites, Cystic Fibrosis Transmembrane Conductance Regulator, Female, Glyburide, Membrane Potentials, Models, Molecular, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides, Oocytes, Patch-Clamp Techniques, Potassium Channels, Potassium Channels, Inwardly Rectifying, Protein Structure, Secondary, Recombinant Proteins, Xenopus laevis
  • Digital Object Identifier (doi)

    Author List

  • McNicholas CM; Nason MW; Guggino WB; Schwiebert EM; Hebert SC; Giebisch G; Egan ME
  • Start Page

  • F843
  • End Page

  • F848
  • Volume

  • 273
  • Issue

  • 5