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 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 dramatically prolong the lifetime of the open state ([equivalent to]110s) by abolishing the hydrolytic pathway for channel closing. Surprisingly, the macroscopic E1371S-CFTR channel currents do not decay upon ATP washout in the presence of AMP-PNP. These results indicate the presence of at least two different closed states that can be differentiated by their distinct responses to AMP-PNP. Quantitative analysis of these results using a simplified kinetic scheme yields a lifetime of 8̃5 s for the closed state that can be re-locked open by AMP-PNP. WT-CFTR channels exhibiting a fast gating behavior 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 nonhydrolytic pathway.