Phosphorylated intermediates of Na,K-ATPase proteoliposomes controlled by bilayer cholesterol. Interaction with cardiac steroid

Fragmental Na,K-ATPase from the electric eel forms three phosphorylated intermediates (EP) with MgATP and Na+: ADP-sensitive K+-insensitive EP (E1P), ADP- and K+-sensitive EP (E*P), and K+-sensitive ADP-insensitive EP (E2P). The EP composition varied with the Na+ concentration. In the reconstituted Na,K-ATPase proteoliposomes (PL), the EP composition of the inside-out form was controlled not only by the intravesicular (extracellular) Na+ concentration, but also by the temperature and the cholesterol content of the lipid bilayer. When the lipid bilayer of PL contained less than 30 mol % cholesterol, the E*P content did not change significantly while the E2P content increased with an elevation in temperature (3-20 degrees C). In contrast, when the lipid bilayer contains more than 35 mol % cholesterol, the E*P content increased while the E2P content stayed less than 10% under the same temperature change. These observations suggest that a high cholesterol content in the lipid bilayer interferes with the E*P to E2P conversion. This cholesterol effect was reversed by ionophores (monensin, nigericin, and A23187). Therefore, E1P-rich EP, E*P-rich EP, or E2P-rich EP could be obtained in the PL under a constant Na+ concentration by using various concentrations of cholesterol and ionophores. The reaction between the proteoliposomal EPs and digitoxigenin (lipid-soluble cardiac steroid) occurred in a single turnover, thereby avoiding unphysiologically high Na+ concentrations. The increase in the ADP- and K+-insensitive EP, which indicated formation of the digitoxigenin-Na,K-ATPase complex, was equivalent to the decrease in the E*P under six different sets of conditions, without any significant change in the E1P and E2P contents. This result indicated that E*P is the active intermediate of the Na,K-ATPase for cardiac steroid binding. Although the E2P has been thought to be the active form for binding, it cannot bind with the cardiac steroid in the presence of Na+ and in the absence of free Mg2+.