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A functional CFTR-NBF1 is required for ROMK2-CFTR interaction
C M McNicholas1, M W Nason, W B Guggino
1Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026, USA.
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.
Insights
The cystic fibrosis transmembrane conductance regulator (CFTR) enhances ROMK2 channel sensitivity to glibenclamide. The first nucleotide-binding fold (NBF1) of CFTR is crucial for this interaction, as mutations in NBF1 reduce glibenclamide sensitivity.
Area of Science:
- * Molecular physiology
- * Ion channel function
Background:
- * Previous studies showed cystic fibrosis transmembrane conductance regulator (CFTR) enhances glibenclamide sensitivity of the ROMK2 channel.
- * The precise mechanism of this interaction requires further characterization.
Purpose of the Study:
- * To investigate the role of CFTR's first nucleotide-binding fold (NBF1) in mediating the enhanced glibenclamide sensitivity of ROMK2.
- * To determine if mutations within NBF1 affect the interaction between CFTR and ROMK2.
Main Methods:
- * Two-microelectrode voltage-clamp technique in Xenopus oocytes.
- * Coexpression of ROMK2 with wild-type and mutant CFTR constructs.
- * Measurement of whole-cell potassium (K+) currents and glibenclamide inhibition.
Main Results:
- * Glibenclamide inhibited K+ currents by 56% in oocytes expressing both ROMK2 and CFTR, versus 11% in oocytes with ROMK2 alone.
- * Truncated CFTR (CFTR-K593X) with intact NBF1 retained partial enhancement (46% inhibition).
- * CFTR mutants with altered or absent NBF1 (CFTR-K370X, CFTR-G551D, CFTR-A455E) significantly reduced glibenclamide inhibition of ROMK2 currents (12-25%).
Conclusions:
- * The NBF1 domain of CFTR is essential for the interaction with ROMK2 that confers glibenclamide sensitivity.
- * Mutations affecting NBF1 disrupt this interaction, leading to reduced sulfonylurea sensitivity.