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

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.

The first-nucleotide binding domain of the cystic-fibrosis transmembrane conductance regulator is important for inhibition of the epithelial Na + channel

“…Moreover, it was shown in other reports that CFTR is also able to replace the sulfonyl urea receptor (SUR) to some degree and is thus able to confer glybenclamide sensitivity to K ATP channels (13,24,25). Using truncated versions of CFTR that were similar to those described here, the authors arrived at the conclusion that NBF-1 is essential for the interaction of CFTR with K ATP (14).…”

“…CFTR also has been shown to bind sulfonyl urea compounds and to confer glybenclamide sensitivity to ATP-gated K ϩ channels (13). Both regulation of outwardly rectifying Cl Ϫ channels by CFTR and interaction with K ATP channels essentially depend on the presence of the first-nucleotide binding domain (NBF-1) of CFTR (12,14). In the present article, we show that the ability of CFTR to inhibit ENaC in a cAMP-dependent manner requires the NBF-1, which cannot be replaced by NBF-1 of other ABC proteins.…”

Assembly and Trafficking of a Multiprotein ROMK (Kir 1.1) Channel Complex by PDZ Interactions

“…It should be pointed out, however, that we cannot exclude the possibility that similar direct interactions do not occur between ROMK and CFTR. In fact, previous studies, documenting functional modification of ROMK by CFTR (14,78,79) even in the excised patch-clamp configuration (15), coupled with present observations that ROMK coimmunoprecipitates with CFTR in the absence of exogenous NHERF, imply that ROMK is capable of interacting directly with CFTR. Perhaps by simultaneously binding ROMK and CFTR, NHERF proteins simply bring channel components into close proximity to promote low affinity intersubunit interactions.…”

Wild type but not ΔF508 CFTR inhibits Na+ conductance when coexpressed in Xenopus oocytes

“…Very recent reports also suggest an interaction of CFTR with the outwardly rectifying C1-channel [17], with the multidrug resistant protein [8], and even ROM-K + channels [14]. In addition evidence has accumulated that CFTR may be a regulator of exocytosis [4,10,18].…”