F. Cornelius, HYDROPHOBIC ION-INTERACTION ON NA-ATPASE( ACTIVATION AND DEPHOSPHORYLATION OF RECONSTITUTED NA+,K+), Biochimica et biophysica acta. Biomembranes, 1235(2), 1995, pp. 183-196
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.