1999 - Charge Translocation by the Na+/K+-ATPase Investigated on Solid Supported Membranes: Cytoplasmic Cation Binding and Release
SURFE²R-technology (custom-built system) publication in Biophysical Journal (1999)
Pintschovius J., Fendler K., Bamberg E.
Biophysical Journal (1999) 76(2):827-836
In the preceding publication (Pintschovius and Fendler, 1999. Biophys. J. 76:000–000) a new technique was described that was able to produce concentration jumps of arbitrary ion species at the surface of a solid supported membrane (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 experiments carried out with membrane fragments containing Na+/K+-ATPase from pig kidney and in the absence of ATP. Electrical currents are measured after rapid addition of Na+. We demonstrate that these currents can be explained only by a cation binding process on the cytoplasmic side, most probably to the cytoplasmic cation binding site of the Na+/K+-ATPase. An electrogenic reaction of the protein was observed only with Na+, but not with other monovalent cations (K+, Li+, Rb+, Cs+). Using Na+ activation of the enzyme after preincubation with K+ we also investigated the K+-dependent half-cycle of the Na+/K+-ATPase. A rate constant for K+ translocation in the absence of ATP of 0.2–0.3 s−1 was determined. In addition, these experiments show that K+ deocclusion, and cytoplasmic K+ release are electroneutral.