Differential Interactions of Nucleotides at the Two Nucleotide Binding Domains of the Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5 -adenylyl-␤,␥-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing.The cystic fibrosis transmembrane conductance regulator (CFTR) 1 has two nucleotide binding domains (NBDs) that are believed in some manner to regulate permeation of chloride ions (1). Whereas the two NBDs of another ABC protein, the P-glycoprotein multidrug transporter, are highly homologous and believed to be functionally equivalent (2), the domains in members of the ABCC subfamily to which CFTR belongs show less sequence similarity (3). This asymmetry is reflected functionally in two members of this subfamily, SUR1 (4, 5) and MRP1 (6 -8). Both ATP and ADP act on SUR1 to regulate the Kir6.2 potassium channel; ATP is bound at NBD1 and also bound and hydrolyzed at NBD2, and the ADP produced stabilizing ATP binding to NBD1 (9). Similarly in the case of MRP1, which transports conjugated anions, ATP binding was detected exclusively at NBD1 and was enhanced allosterically by the trapping of ADP produced by hydrolysis at NBD2 (6).Schemes of ATP binding and hydrolysis by CFTR have been proposed solely on the basis of channel-gating responses (10 -12). Implicit in these models is the idea that channel opening is driven by ATP binding and hydrolysis at one NBD and closing at the other (13). Intact CFTR (14) and bacterial fusion proteins containing either of the NBDs (15, 16) hydrolyze ATP. It has been reported also that photoaffinity labeling of CFTR by 8-azido-ATP at NBD1 occurred...

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

“…4ab). This behavior, noted by others in studies of crude membranes [14,15], has not been reported for other ABC transporters [14,15,33] and may be a distinctive feature of CFTR.We also evaluated recovery of functional CFTR in a quantitative manner by comparing nucleotide trapping of CFTR in parallel with that of its well-characterized relative, Pgp, which had been expressed in Sf9 cells and purified and reconstituted using conditions optimized for its own recovery [33,[35][36][37]38] (Table I, Fig. 4c-e).…”

“…4ab). This behavior, noted by others in studies of crude membranes [14,15], has not been reported for other ABC transporters [14,15,33] and may be a distinctive feature of CFTR.…”

“…And by use of a vanadate (Vi)-trapping assay, CFTR-specific ATP binding and hydrolysis has also been observed in the crude membranes of such cells [14,15]. Indeed, because of its high sensitivity, we used this latter method to evaluate conditions suited to purification of human CFTR expressed in these cells.…”

“…Current evidence suggests that the TMDs define the CFTR chloride channel, while the NBDs and the R domain mediate channel gating [3][4][5][6][7][8][9][10][11] Although CFTR is glycosylated, there is currently no evidence indicating that the presence of carbohydrate affects CFTR structure or function [12]. Consistent with this presumption, expression of human CFTR in Sf9 insect cells results in appearance of the 140 kD core polypeptide -containing little or no glycosylation -that mediates a newly acquired anion permeability with the electrophysiological signature of CFTR [13] [14,15]. In this study, we focused on purification of human CFTR from this heterologous expression system as a way to provide amounts of CFTR that might support extended biochemical and biophysical work with purified material.…”

“…a functional competence for the CFTR NBDs [14,15]. In this study, we focused on purification of human CFTR from this heterologous expression system as a way to provide amounts of CFTR that might support extended biochemical and biophysical work with purified material.…”