The Two Nucleotide-binding Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Have Distinct Functions in Controlling Channel Activity

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel contains two cytoplasmic nucleotide-binding domains (NBDs). After phosphorylation of the R domain, ATP interacts with the NBDs to regulate channel activity. To learn how the NBDs regulate channel function, we used the patch-clamp technique to study CFTR and variants which contained site-directed mutations in the conserved Walker A motif lysine residues in either NBD1 (K464A), NBD2 (K1250A and K1250M), or both NBDs simultaneously (K464A/K1250A). Studies in related proteins suggest that such mutations slow the rate of ATP hydrolysis. These mutations did not alter the conductive properties of the channel or the requirement for phosphorylation and ATP to open the channel. However, all mutations decreased open state probability. Mutations in NBD1 decreased the frequency of bursts of activity, whereas mutations in NBD2 and mutations in both NBDs simultaneously prolonged bursts of activity, as well as decreased the frequency of bursts. These results could not be attributed to altered binding of nucleotide because none of the mutants studied had reduced 8-N3ATP binding. These data suggest that the two NBDs have distinct functions in channel gating; ATP hydrolysis at NBD1 initiates a burst of activity, and hydrolysis at NBD2 terminates a burst.

Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle

“…Furthermore, such prolongation of the opening burst became even more prominent at higher [ATP] (Fig. 3B), an observation not only corroborating the hypothesis that Vx-770 promotes the ATP-dependent reentry mechanism, but also distinguishing itself from the well-known increase of the open time by abolishing ATP hydrolysis (12,28,(30)(31)(32) We also measured the relaxation time constant of macroscopic current after removing different concentrations of ATP. In the presence of 200 nM Vx-770, these relaxation time constants are not significantly different over a wide range of [ATP] (Fig.…”

“…As a CFTR potentiator, Vx-770 is unique in that it increases the open time of WT-CFTR, which is mainly controlled by ATP hydrolysis (11,28,31,32) and the activity of G551D-CFTR, a mutant that does not respond to ATP (14). Indeed, the classical methods used to lengthen the open time of WT-CFTR by abolishing ATP hydrolysis with either mutations or nonhydrolyzable ATP analogs fail to work on the G551D channels (14,37).…”

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.…”

“…The methods for excised inside-out patchclamp were as described previously (5). Experiments were performed at room temperature.…”

“…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).…”