On the Mechanism of MgATP-dependent Gating of CFTR Cl− Channels

Abstract

CFTR, the product of the gene mutated in cystic fibrosis, is an ATPase that functions as a Cl− channel in which bursts of openings separate relatively long interburst closed times (τib). Channel gating is controlled by phosphorylation and MgATP, but the underlying molecular mechanisms remain controversial. To investigate them, we expressed CFTR channels in Xenopus oocytes and examined, in excised patches, how gating kinetics of phosphorylated channels were affected by changes in [MgATP], by alterations in the chemical structure of the activating nucleotide, and by mutations expected to impair nucleotide hydrolysis and/or diminish nucleotide binding affinity. The rate of opening to a burst (1/τib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR's nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Burst duration of neither wild-type nor mutant channels was much influenced by [MgATP]. Poorly hydrolyzable nucleotide analogs, MgAMPPNP, MgAMPPCP, and MgATPγS, could open CFTR channels, but only to a maximal rate of opening ∼20-fold lower than attained by MgATP acting on the same channels. NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Corresponding NBD1 mutations did not affect timing of burst termination in normal, hydrolytic conditions. However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. In light of recent biochemical and structural data, the results suggest that: nucleotide binding to both NBDs precedes channel opening; at saturating nucleotide concentrations the rate of opening to a burst is influenced by the structure of the phosphate chain of the activating nucleotide; normal, rapid exit from bursts occurs after hydrolysis of the nucleotide at NBD2, without requiring a further nucleotide binding step; if hydrolysis at NBD2 is prevented, exit from bursts occurs through a slower pathway, the rate of which is modulated by the structure of the NBD1 catalytic site and its bound nucleotide. Based on these and other results, we propose a mechanism linking hydrolytic and gating cycles via ATP-driven dimerization of CFTR's NBDs.

ATP-sensitive K channel channel/enzyme multimer: Metabolic gating in the heart

“…Thus, the probability that NBD2 will adopt the ADP-bound state through the ATPase cycle will be higher than the probability that this state will form through simple competitive binding in the absence of ATPase activity [14]. Hence, the SUR catalytic activity provides conformational changes that would otherwise occur only rarely, as has also been suggested for gating of CFTR channels [51] with analogy to GTP hydrolysis by G-proteins [56].…”

“…Rather, in the SUR/Kir6.2 complex, similarly to CFTR that also functions as a channel [45,[49][50][51], an intrinsic catalysis is not required for passive ion permeation down the elecrtochemical gradient but could be involved in allosteric regulation of pore gating. Specifically, coupling of discrete conformations in the SUR catalytic cycle with channel gating was solved using nucleotide trapping procedures in conjunction with on-line channel recording when intrinsic enzymatic activity was enhanced by elevating temperature [9].…”

“…In the structure of the NBD2 monomer of SUR2A [14] as well as other ABC members, the ATP-binding site is exposed, suggesting that the catalytic site can be completed by interaction with another domain [51,66,67]. Evidence of physical proximity of NBDs [68] has hinted that one NBD monomer could complete the binding pocket of the adjacent one.…”

Structure and function of ABC transporters: the ATP switch provides flexible control

“…Two ATP molecules are normally bound at the dimer interface in the 'closed dimer'-two bound nucleotides have been observed in crystallographic dimers of mutant LolD [10] and HlyB-NBD [51], MalK [42] and Rad50 [9]; have been detected biochemically in the Mdl1p dimer [55]; and have been shown, biochemically, to be bound by the intact MRP1 and P-gp transporters [34,52]. For CFTR, there is indirect evidence that ATP must bind to both sites to open the channel [59,60]. Finally, co-operative ATP binding suggests a role for two ATP molecules [50,[61][62][63][64].…”

“…For some ABC transporters, the 'choice' of which NBD hydrolyses first may be stochastic [65]. For others, there is a clear asymmetry: for MRP1, NBD2 appears to hydrolyse ATP initially [66,74], and in CFTR, hydrolysis of one ATP is much slower than the other [84]. Nevertheless, the net result is the same: destabilisation of the 'closed dimer' albeit with different kinetic control.…”