HYDROPHOBIC ION-INTERACTION ON NA-ATPASE( ACTIVATION AND DEPHOSPHORYLATION OF RECONSTITUTED NA+,K+)

In liposomes with reconstituted shark Na+K+-ATPase an uncoupled Nai-ef flux and a Na+/Na+ exchange can be induced on inside-out oriented pump s 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 ele ctrogenic and accompanied by a net hydrolysis of ATP. The coupling rat io of positive net charges translocated per ATP split is found to be c lose to 3:1 and 1:1, respectively, for the two modes of exchange react ions at pH 7.0. By addition of the hydrophobic anion tetraphenylboron (TPB-), which imposes a negative electrostatic membrane potential insi de the lipid bilayer, the ATP hydrolysis accompanying uncoupled Na+ ef flux is increased with increasing TPB- concentrations. Cholesterol whi ch increases the inner positive dipole potential of the bilayer counte racted this activation by TPB- of uncoupled Na+ efflux. Using the stru ctural analog tetraphenylphosphonium (TPP+), which elicits an inside p ositive membrane potential, ATP hydrolysis accompanying uncoupled Na+- efflux is decreased. The rate of dephosphorylation in the absence of e xtracellular 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 o f exchange the phosphorylation reaction was independent of the presenc e of hydrophobic ions. The hydrophobic ions affected the apparent affi nity for cytoplasmic Na+, indicating that binding of cytoplasmic Na+ m ay involve the migration of cations to binding sites through a shallow cytoplasmic access channel. The results are in accordance with the si mple electrostatic model for charge translocation in which two negativ e charges in the cytoplasmic binding domain of the Na+,K+-ATPase co-mi grate during cation transport.