The transmembrane pump N+/K+-ATPase was reconstituted within a model membrane-mimetic system of monoolein liquid-crystalline cubic phase (LCP). LCP consists of a curved lipid bilayer forming the walls of a network of aqueous channels providing the lipid environment for the hydrophobic part of N+/K+ ATPase, with the aqueous channels enabling protein contact with the aqueous environment. Electrogenic ion transport by N+/K+-ATPase was investigated by chronoamperometry (CA). The results of the electrochemical measurements were compared with the spectroscopic assay of the protein activity. The transient currents were recorded following application of potential steps to the LCP modified electrode. N+/K+-ATPase embedded in the lipidic part of the cubic phase transports cations across the mesophase film. Moreover, the lack of Na+ halted protein function because Na+ ions are required to promote the enzyme switching to P-E2 conformation. A specific N+/K+-ATPase inhibitor, ouabain, added to the solution decreased ion transport abilities of the protein. Small-angle X-ray scattering (SAXS) measurements showed that the structure of cubic phase with incorporated N+/K+-ATPase before and after the chronoamperometry experiments remained unchanged, and that the observed difference in current flowing through the cubic phase film with and without the protein was due to N+/K+-ATPase activity in this environment. (C) 2019 Elsevier Ltd. All rights reserved.