Identification of the Cystic Fibrosis Transmembrane Conductance Regulator Domains That Are Important for Interactions with ROMK2

Paula Cahill,Malcolm W Nason,Catherine Ambrose,Tong-Yi Yao,Pamela Thomas,Marie E Egan

doi:10.1074/jbc.m910205199

Abstract

In addition to functioning as a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in conferring regulatory properties on other ion channels. It is known, with respect to CFTR regulation of ROMK2 (renally derived K(ATP) channel), that the first transmembrane domain and the first nucleotide binding fold domain (NBF1) of CFTR are necessary for this interaction to occur. It has been shown that under conditions that promote phosphorylation, the ROMK2-CFTR interaction is attenuated. To elucidate the complex nature of this interaction, CFTR constructs were co-expressed with ROMK2 in Xenopus oocytes, and two microelectrode voltage clamp experiments were performed. Although the second half of CFTR can act as a functional chloride channel, our results suggest that it does not confer glibenclamide sensitivity on ROMK2, as does the first half of CFTR. The attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation is dependent on at least the presence of the R domain of CFTR. We conclude that transmembrane domain 1, NBF1, and the R domain are the CFTR domains involved in the ROMK2-CFTR interaction and that NBF2 and transmembrane domain 2 are not essential. Lastly, the R domain of CFTR is necessary for the attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation.

Highlights

The CFTR1 gene encodes for a multifunctional transmembrane protein that is both a cAMP-activated chloride channel and a regulator of other ion channels [1, 2]
The second half of CFTR can act as a functional chloride channel, our results suggest that it does not confer glibenclamide sensitivity on ROMK2, as does the first half of CFTR
We conclude that transmembrane domain 1, NBF1, and the R domain are the CFTR domains involved in the ROMK2-CFTR interaction and that NBF2 and transmembrane domain 2 are not essential

Summary

EXPERIMENTAL PROCEDURES

Preparation of Oocytes for Voltage Clamp Experiments—Stage V-VI Xenopus laevis oocytes were isolated and injected with RNA as described previously [6]. To address the possibility that the current observed, under conditions that promote phosphorylation, was stimulated ClϪ current and not Kϩ current, a subset of experiments were subjected to an alternate protocol It contained an additional 3-min barium pulse after the initial control period. These experiments were performed on oocytes expressing ROMK2/CFTR-K593X and ROMK2/CFTR-D835X and showed that the current was Ba2ϩ-sensitive Kϩ current and not stimulated ClϪ current. In addition it has been demonstrated that the second half of CFTR forms a functional chloride channel [18] These experiments were repeated, and confirmed RT2N2CFTR is expressed at membrane level with 0.278 Ϯ 0.041 ␮A of current (VHOLD ϭ Ϫ40 mV using a two-microelectrode voltage clamp) (p Ͻ 0.05 when compared with an uninjected oocyte) (Fig. 2)

RESULTS

Uninjected uA

DISCUSSION