Electrical pump currents generated by the Ca 2+-ATPase of sarcoplasmic reticulum vesicles adsorbed on black lipid membranes

Abstract

Sarcoplasmic reticulum vesicles adsorbed on a black lipid membrane generate an electrical current after a fast increment of the concentration of ATP. This demonstrates directly that the sarcoplasmic Ca 2+-ATPase from skeletal muscle acts as an electrogenic ion pump. The increment of the concentration of ATP is achieved by the photolysis of caged ATP ( P 3-1-(2-nitro)phenylethyl adenosine 5′-triphosphate) a protected analogue of ATP (Kaplan, J.H. et al. (1978) Biochemistry 17, 1929–1935), which is split into ATP and 2-nitroso acetophenone. The release of ATP leads to a transient current flow across the lipid membrane indicating that the vesicles are capacitatively coupled to the underlying lipid membrane. In addition to this transient signal, a stationary current flow is obtained in the presence of ionophores which increase the conductance of the bilayer system and prevent the accumulation of Ca 2+ in the lumen of the vesicles. The direction of the transient and the stationary current is in accordance with the concept that Ca 2+ is pumped into the lumen of the vesicles. The transient current depends on the concentration of ATP, Ca 2+ and Mg 2+ as would be the case for a current generated by the sarcoplasmic Ca 2+-ATPase. Its amplitude is half-maximal at 10 μM ATP and 1 μM Ca 2+. At Ca 2+ concentrations above 0.1 mM the amplitude of the current signal declines again. The Mg 2+ concentration dependence of the current amplitude at a constant ATP concentration indicates that the MgATP complex is the substrate for the activation of the current. The pump current is inhibited by vanadate and ADP. No current signal is observed if caged ATP is replaced by caged ADP. However, the release of ADP from caged ADP generates a pump current in the presence of an ATP generating system such as creatine phosphate and creatine kinase.