Gating of CFTR chloride channels: distinct closed states revealed by the action of AMP-PNP

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The CFTR chloride channel is gated by ATP through an irreversible gating cycle driven by an input of free energy of ATP hydrolysis. The open state of the CFTR chloride channel is associated with the dimerization of its two nucleotide binding domains (NBDs). Mutations of the glutamate 1371 residue (e.g., E1371S), the catalytic base for hydrolysis of ATP at CFTR's NBD2, dramatically prolong the lifetime of the open state ([tau]=~110 s) by abolishing the hydrolytic pathway for channel closing. Interestingly, when the E1371S mutant is opened by AMP-PNP, a non-hydrolysable ATP analog, the mean open time is less than 1 s, indicating that AMP-PNP alone cannot support a stable dimer configuration. Surprisingly, however, the macroscopic E1371S-CFTR channel currents do not decay upon ATP washout in the presence of AMP-PNP. Furthermore, when AMP-PNP was applied during the current decay after ATP washout, it not only reopened the closed E1371S channels, the AMP-PNP reopened channels have a lifetime of 113 s. These results indicate the presence of at least two different closed states that can be differentiated by their distinct responses to AMP-PNP. The relative amount of current induced by AMP-PNP during the ATP washout phase is decreased as the washout time increases. Quantitative analysis of these results using a simplified kinetic scheme yields a lifetime of ~85 s for the closed state, C2, which can be re-locked open by AMP-PNP. The lifetime of this closed state is prolonged when the channel is opened by N6 - phenylethyl-ATP, a high-affinity hydrolysable ATP analog, suggesting that at least one nucleotide remains bound in this closed state. WT-CFTR channels exhibiting a fast gating behavior ([tau]=0.45 s) presumably due to the hydrolysis of ATP were also examined. Consistently once the channels are locked-open by ATP and AMP-PNP, providing AMP-PNP during the current relaxation after the removal of all nucleotides derives significant numbers of channels to enter into the re-locked open state. This result hints that the different closed state is also present in WT-CFTR channels when they are closed through non-hydrolytic pathway.