FAST TRANSIENT CURRENTS IN NA,K-ATPASE INDUCED BY ATP CONCENTRATION JUMPS FROM THE -3-[1-(3',5'-DIMETHOXYPHENYL)-2-PHENYL-5-OXO]ETHYL ESTEROF ATP

Electrogenic ion transport by Na,K-ATPase was investigated by analysis of transient currents in a model system of protein-containing membran e fragments adsorbed to planar lipid bilayers. Sodium transport was tr iggered by ATP concentration jumps in which ATP was released from an i nactive precursor by an intense near-UV light flash. The method has be en used previously with the P-3-1-(2-nitrophenyl)ethyl ester of ATP (N PE-caged ATP), from which the relatively slow rate of ATP release limi ts analysis of processes in the pump mechanism controlled by rate cons tants greater than 100 s(-1) at physiological pH. Here Na,K-ATPase was reinvestigated using the P-3-[1-(3,5-dimethoxyphenyl)-2-phenyl-2-oxo] ethyl ester of ATP (DMB-caged ATP), which has an ATP release rate of > 10(5) s(-1). Under otherwise identical conditions, photorelease of ATP from DMB-caged ATP showed faster kinetics of the transient current co mpared to that from NPE-caged ATP. With DMB-caged ATP, transient curre nts had rate profiles that were relatively insensitive to pH and the c oncentration of caged compound. Rate constants of ATP binding and of t he E-1 to E-2 conformational change were compatible with earlier studi es. Rate constants of enzyme phosphorylation and ADP-dependent dephosp horylation were 600 s(-1) and 1.5 x 10(6) M-1 s(-1) respectively, at p H 7.2 and 22 degrees C.