Current transients generated by the Na+/K+-ATPase after an ATP concentration jump: dependence on sodium and ATP concentration.

Planar membrane fragments containing a high density of oriented Na+/K+-ATPase molecules are bound to planar lipid bilayers. ATP is released in the aqueous solution within milliseconds from an inactive, photolabile precursor ('caged ATP') by an intense light flash. By this ATP-concentration jump a large number of pump molecules is activated almost simultaneously. Charge translocation in the pump molecule results in a voltage transient which is recorded in the external measuring circuit. From the voltage signal, the intrinsic pump current Ip(t) can be evaluated using information on the circuit parameters of the compound membrane system. The pump current Ip(t) is compared with the results of numerical simulations of a reaction cycle derived from the Post-Albers reaction scheme combined with the photochemical release reaction of caged ATP. The time course of Ip can be satisfactorily fitted using kinetic parameters of the Na+/K+-ATPase from the literature. The dependence of Ip on sodium concentration cNa can be described using a single set of kinetic parameters in which only cNa is varied. Ip as a function of cNa is well fitted by a first-order Michaelis-Menten type equation with Km approximately equal to 4 mM. This finding is consistent with the assumption that two sodium binding sites have a high affinity and that a third site of lower affinity is rate limiting. The ATP concentration dependence of Ip is studied by varying the concentration of caged ATP in the solution and the yield of photochemical release of ATP.