Calcium alters the properties of [3H]diazepam binding sites in rat brain membranes.

[3H]diazepam [( 3H]DZ) was used as a ligand to study the effects of Ca2+ on benzodiazepine binding to rat brain membranes. [3H]DZ bound at a single class of binding sites showing KD and Bmax values of 5.4 nM and 852 fmol/mg protein respectively. These values are consistent with previous reports. Amongst the various divalent cations tested Mg2+, Fe2+, Cd2+ and Sr2+ had no significant effect on [3H]DZ binding. Mn2+, Ba2+, Co2+, Ni2+ and La3+ enhanced radioligand binding, whereas Ca2+, Zn2+ and Pb2+ inhibited of [3H]DZ binding by Ca2+ was concentration-dependent. 50% inhibition occurred at a Ca2+ concentration of 5.6 mM. The Hill coefficient for the inhibition was 1.03, displaying noncooperativity. The effect of Ca2+ on [3H]DZ binding could be prevented by La3+ but was not reversed by EGTA. A kinetic analysis of Ca2+ inhibition of [3H]DZ binding indicates that Ca2+ inhibited competitively. Analysis of binding isotherms indicates that both KD and Bmax were altered at the [3H]DZ binding sites. The marked increase in KD value in the presence of Ca2+ (5 mM) can be explained by a drastic increase in the dissociation rate constant. It was suggested that Ca2+ may induce a conformational change in the diazepam binding sites on rat brain membranes. The unchanged Hill coefficient in the presence or absence of Ca2+ indicates that a single population of binding sites was labeled by [3H]DZ. The calmodulin antagonists, trifluoperazine and W-7 were weak inhibitors of [3H]DZ binding.