Walker Mutations Reveal Loose Relationship between Catalytic and Channel-Gating Activities of Purified CFTR (Cystic Fibrosis Transmembrane Conductance Regulator)

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATPase and as a chloride channel. It has been hypothesized, on the basis of electrophysiological findings, that the catalytic activity of CFTR is tightly coupled to the opening and closing of the channel gate. In the present study, to determine the structural basis for the ATPase activity of CFTR, we assessed the effect of mutations within the "Walker A" consensus motifs on ATP hydrolysis by the purified, intact protein. Mutation of the lysine residue in the "Walker A" motif of either the first nucleotide binding fold (CFTRK464A) or the second nucleotide binding fold (CFTRK1250A) inhibited the ATPase activity of the purified intact CFTR protein significantly, by greater than 50%. This finding suggests that the two nucleotide binding folds of CFTR are functioning cooperatively in catalysis. However, the rate of channel gating was only significantly inhibited in one of these purified mutants, CFTRK1250A, suggesting that ATPase activity may not be tightly coupled to channel gating as previously hypothesized.

A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle

“…Earlier studies of the effects of ATP concentration, nucleoside analogs, site-directed mutations, and chemical modifications have established the central role of the NBDs and ATP in controlling CFTR channel gating (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). However, the molecular mechanisms of gating, including the function of the individual NBDs and their interactions remain uncertain.…”

“…Consequently, investigators have proposed several models to describe CFTR gating (1, 2, 4-6, 10-12, 20). Most recent models account for the following features of ATP-dependent gating: (i) ATP binding alone is able to open the channel (9,12,20,23,24); (ii) ATP hydrolysis initiates conformational changes that ultimately lead to channel closure (the channel can also close with ATP dissociation) (1, 2, 6, 10-12); (iii) ATP binding and hydrolysis by either NBD enables channel gating (4,12); (iv) the two NBDs interact functionally (9,11,12,19,25).…”

“…Earlier studies have revealed much about how ATP interacts with the two NBDs to control gating. For example, both NBDs contribute to ATP-dependent gating, and their interactions with ATP both open and close the channel (3)(4)(5)(6)(7)(8)(9)(10)(11)(12).…”

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…Moreover, this locked-open phenotype is also observed with ATP as a ligand when hydrolysis is abolished by mutations at residue K1250 or glutamate 1371 (E1371), both essential for effective ATP hydrolysis (Gunderson and Kopito 1995;Ramjeesingh et al 1999;Zeltwanger et al 1999;Powe et al 2002;Bompadre et al 2005b;Vergani et al 2005;Stratford et al 2007). But is hydrolysis per se closing the gate as suggested by a thermodynamic study of CFTR gating (Csanády et al 2006)?…”

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

“…In CFTR, unlike other ABC transporters, a third domain, termed the regulatory (R) domain, is located between the two half molecules. 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].…”