NA-ATPASE PUMP CURRENTS IN GIANT EXCISED PATCHES ACTIVATED BY AN ATP CONCENTRATION JUMP(,K+)

The giant-patch technique was used to study the Na+,K+-ATPase in excis ed patches from rat or guinea pig ventricular myocytes. Na+,K+-pump cu rrents showed a saturable ATP dependence with a K-m of similar to 150 mu M at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absenc e of extracellular Na+ was slow, with a k(off) of 3 . 10(-4) s(-1) (K- 1 approximate to 0.5 mu M, at 24 degrees C). Stationary currents were markedly dependent on intracellular pH, with a maximum at pH 7.9. Temp erature-dependence measurements of the stationary pump current yielded an activation energy of similar to 100 kJ mol(-1). Partial reactions in the transport cycle were investigated by generating ATP concentrati on jumps through photolytic release of ATP from caged ATP at pH 7.4 an d 6.3, Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slower decay to a stationary current. It wa s concluded that the fast rate constant of similar to 200 s(-1) at 24 degrees C (pH 6.3) reflects a step rate-limiting the electrogenic Narelease. Simulating the data with a simple three-state model enabled u s to estimate the turnover rate under saturating substrate concentrati ons, yielding rates(at pH 7.4) of similar to 60 s(-1) and 200 s(-1) at 24 degrees C and 36 degrees C, respectively.