In liposomes with reconstituted shark Na+K+-ATPase an uncoupled Nai-efflux and a Na+/Na+ exchange can be induced on inside-out oriented pumps by the addition of external (cytoplasmic) Na+ and MgATP to liposomes that either do not contain Na+ (and other alkali cations), or include 130 mM Na+ internally (extracellular). Both modes of exchange are electrogenic and accompanied by a net hydrolysis of ATP. The coupling ratio of positive net charges translocated per ATP split is found to be close to 3:1 and 1:1, respectively, for the two modes of exchange reactions at pH 7.0. By addition of the hydrophobic anion tetraphenylboron (TPB-), which imposes a negative electrostatic membrane potential inside the lipid bilayer, the ATP hydrolysis accompanying uncoupled Na+ efflux is increased with increasing TPB- concentrations. Cholesterol which increases the inner positive dipole potential of the bilayer counteracted this activation by TPB- of uncoupled Na+ efflux. Using the structural analog tetraphenylphosphonium (TPP+), which elicits an inside positive membrane potential, ATP hydrolysis accompanying uncoupled Na+-efflux is decreased. The rate of dephosphorylation in the absence of extracellular alkali cations was affected in a similar manner, whereas the dephosphorylation in the presence of extracellular Na+ inducing Na+/Na+ exchange was unaffected by the hydrophobic ions. In both modes of exchange the phosphorylation reaction was independent of the presence of hydrophobic ions. The hydrophobic ions affected the apparent affinity for cytoplasmic Na+, indicating that binding of cytoplasmic Na+ may involve the migration of cations to binding sites through a shallow cytoplasmic access channel. The results are in accordance with the simple electrostatic model for charge translocation in which two negative charges in the cytoplasmic binding domain of the Na+,K+-ATPase co-migrate during cation transport.
