Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: Cytoplasmic cation binding and release

In the preceding publication (Pintschovius and Fendler, 1999. Biophys. J. 7 6:000-000) a new technique was described that was able to produce concentra tion jumps of arbitrary ion species at the surface of a solid supported mem brane (SSM). This technique can be used to investigate the kinetics of ion translocating proteins adsorbed to the SSM. Charge translocation of the Na/K+-ATPase in the presence of ATP was investigated. Here we describe experi ments carried out with membrane fragments containing Na+/K+-ATPase from pig kidney and in the absence of ATP. Electrical currents are measured after r apid addition of Na+. We demonstrate that these currents can be explained o nly by a cation binding process on the cytoplasmic side, most probably to t he cytoplasmic cation binding site of the Na+/K+-ATPase. An electrogenic re action of the protein was observed only with Na+, but not with other monova lent cations (K+, Li+, Rb+, Cs+). Using Na+ activation of the enzyme after preincubation with K+ we also investigated the K+-dependent half-cycle of t he Na+/K+ ATPase, A rate constant for K+ translocation in the absence of AT P of 0.2-0.3 s(-1) was determined. In addition, these experiments show that K+ deocclusion, and cytoplasmic K+ release are electroneutral.