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

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.The CFTR 1 gene encodes for a multifunctional transmembrane protein that is both a cAMP-activated chloride channel and a regulator of other ion channels (1, 2). It is a member of the ATP binding cassette superfamily, and like other members of this group it is comprised of transmembrane spanning domains and nucleotide binding folds (NBFs); however, it has a regulatory domain (R domain), which is unique to CFTR (3). The domains of CFTR involved in its regulation of other ion channels may be functionally distinct from those involved in its Cl Ϫ channel function.CFTR expression has been linked to protein kinase A regulation of the outwardly rectifying chloride channel (ORCC), the epithelial sodium channel (ENaC), and a K ATP channel, ROMK2 (4, 5, 6). The first nucleotide binding domain of CFTR and the R domain are important for it's regulation of ORCC and ENaC (7,8). For CFTR to regulate ROMK2 channels, in vitro, an intact NBF1 is required (9). Although many groups are investigating the role of NBF1 and NBF2 with regard to channel gating, their role with regard to regulation of other ion channels has yet to be elucidated. For instance, it is not known if NBF2 can have the same effect as NBF1 with respect to regulation of ROMK2 channels.Many of the disease-producing mutations are located in the NBF domains of CFTR, predominantly within NBF1. These mutations are often associated with abnormal CFTR channel activity, as well as abnormal kinase regulation of ORCC and ENaC. For example, the protein kinase activation of ORCCs depends on CFTR expression and is affected by mutations in NBF1, as is the cAMP-dependent protein kinase-dependent inhibition o...

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca 2+ -dependent potassium channels

“…Several recent studies have provided insights into the molecular aspects of the CFTR regulation of outwardly rectify- (45), and ROMK2 potassium channels (36). Interestingly, these channels colocalize with CFTR to the apical membrane of the epithelia where they are expressed.…”

CFTR is required for PKA-regulated ATP sensitivity of Kir1.1 potassium channels in mouse kidney

“…In support of this latter possibility, gating of the 30 pS K channel in TAL or principal cells requires Mg-ATP, rather than Na-ATP (62, 63), suggesting that ATP hydrolysis may be involved in K channel gating by nucleotides. Moreover, NBD1 on CFTR has been found to be essential for glibenclamide sensitivity, and by inference for ATP sensitivity (20), of ROMK expressed in X. laevis oocytes (23,24).…”

“…These in vitro effects of CFTR on ROMK function are likely mediated through binding of both proteins via PSD95/DLG/ZO-1 (PDZ) interactions to the scaffolding protein Na + /H + exchange regulatory factor (NHERF; ref. 22) and require the transmembrane domain 1, the first nucleotide-binding fold, and the regulatory domain on CFTR (23,24). In addition, CFTR-ROMK functional interaction can be regulated by cAMP-dependent protein kinase A (PKA), since increasing cAMP abrogates the effect of CFTR on glibenclamide sensitivity of ROMK in Xenopus laevis oocytes (21).…”

ΔF508 Mutation Results in Impaired Gastric Acid Secretion

“…Initially thought to be merely a chloride conductance channel, the cystic fibrosis transmembrane conductance regulator, as the name implies, has been shown to regulate several other cellular processes. CFTR interacts and plays a regulatory role in the activity of the outwardly rectifying chloride channel (11)(12)(13)(14), epithelial sodium channel (15,16), as well as the renal outer medullary K ϩ channel type 2 (ROMK2 or Kir1.1b) (17)(18)(19)(20)(21).…”