The Na+, K+-Pump, Part B: Cellular Aspects, pages 429-436 © 1988 Alan R. Liss, Inc. TWO-SIDED FUNCTIONAL Na,K-ATPase-LIPOSOMES FOR CHARACTERIZING THE PERMEABILITY AND SIDE 0F ACTION 0F PUMP INHIBITORS H.G. Rey. P. Meda*, and B.M. Armer Departments of Pharmacology and Histology*, Geneva University Médical Center (CMU), CH-1211 Geneva 4, Switzerland INTRODUCTION The molecular mechanism of action, thé specificity and thé side of action of many substances interacting directly or indirectly with thé Na,K-ATPase of thé cell membrane are often uncertain. On one band, thé target site and thé selectivity of a potential pump ligand are difficult to define precisely in intact cells. On thé other hand, when broken membrane fragments containing purified Na,K-ATPase molécules are used as test-system, thé side at which thé ligands interact with Na,K-ATPase is hardly determinable. Therefore, we developed thé ATP-filled, bi-functional Na,K-ATPase-liposomes containing co-reconstituted, randomly oriented and transport-active Na,K-ATPase molécules (Rey et al., 1987; Armer et al., 1988) into a miniaturized test-system for examining thé membrane permeability and target-site of inhibitors interacting with Na,K-ATPase. It is well known that thé transmembrane Na,K-ATPase molécule is asymmetric: thé ATP, Mg and Na binding sites are located on thé intracellular protein protusion whereas thé cardioactive steroid receptor as well as thé K or Rb binding sites on thé extracellular protein surface. In conséquence, thé time required by a drug added externally to interact with thé right-side-out (r-s-o) and thé inside-out (i-s-o) oriented Na,K-ATPase provides information about its membrane permeability and site of action.