Na + currents generated by the purified [formula omitted] on planar lipid membranes

Abstract

Purified ( Na + + K +)-ATPase from pig kidney was attached to black lipid membranes and ATP-induced electric currents were measured as described previously by Fendler et al. ((1985) EMBO J. 4, 3079–3085). An ATP concentration jump was produced by an ultraviolet-light flash converting non-hydrolysable caged ATP to ATP. In the presence of Na + and Mg 2+ this resulted in a transient current signal. The pump current was not only ATP dependent, but also was influenced by the ATP/caged ATP ratio. It was concluded that caged ATP binds to the enzyme (and hence inhibits the signal) with a K i of approx. 30 μM, which was confirmed by enzymatic activity studies. An ATP affinity of approx. 2 μM was determined. The addition of the protonophore 1799 and the Me +/ H + exchanger monensin made the bilayer conductive leading to a stationary pump current. The stationary current was strongly increased by the addition of K + with a K 0.5 of 700 μM. Even in the absence of K + a stationary current could be measured, which showed two Na +-affinities: a high-affinity ( K 0.5 ⩽ 1 mM ) and a low-affinity ( K 0.5 ⩾ 0.2 M ). In order to explain the sustained electrogenic Na + transport during the Na +-ATPase activity, it is proposed, that Na + can replace K + in dephophorylating the enzyme, but binds about 1000-times weaker than K +. The ATP requirement of the Na +-ATPase was the same ( K 0.5 = 2 μ M ) with regard to the peak currents and the stationary currents. However, for the (Na + + K +)-ATPase the stationary currents required more ATP. The results are discussed on the basis of the Albers-Post scheme.