THE CFTR CHLORIDE CHANNEL - NUCLEOTIDE INTERACTIONS AND TEMPERATURE-DEPENDENT GATING

The gating cycle of CFTR (Cystic Fibrosis Transmembrane conductance Re gulator) chloride channels requires ATP hydrolysis and can be interrup ted by exposure to the nonhydrolyzable nucleotide AMP-PNP. To further characterize nucleotide interactions and channel gating, we have studi ed the effects of AMP-PNP, protein kinase C (PKC) phosphorylation, and temperature on gating kinetics. The rate of channel locking increased from 1.05 x 10(-3) sec(-1) to 58.7 x 10(-3) sec(-1) when AMP-PNP conc entration was raised from 0.5 to 5 mM in the presence of 1 mM MgATP an d 180 nM protein kinase A catalytic subunit (PKA). Although rapid lock ing precluded estimation of P-o or opening rate immediately after the addition of AMP-PNP to wild-type channels, analysis of locking rates i n the presence of high AMPPNP concentrations revealed two components. The appearance of a distinct, slow component at high [AMP-PNP] is evid ence for AMP-PNP interactions at a second site, where competition with ATP would reduce P, and thereby delay locking. All channels exhibited locking when they were strongly phosphorylated by PKA, but not when e xposed to PKC alone. AMP-PNP increased P, at temperatures above 30 deg rees C but did not cause locking, evidence that the stabilizing intera ctions between domains, which have been proposed to maintain CFTR in t he open burst state, are relatively weak. The temperature dependence o f normal CFTR gating by ATP was strongly asymmetric, with the opening rate being much more temperature sensitive (Q(10) = 9.6) than the clos ing rate (Q(10) = 3.6). These results are consistent with a cyclic mod el for gating of phosphorylated CFTR.