Effects of sodium and GTP on the binding kinetics of [3H]diprenorphine in NG 108-15 cell membranes.

Equilibrium binding isotherms of [3H]diprenorphine in membranes from NG 108-15 cells are consistent with a homogeneous population of binding sites. Upon addition of Na+, Mg2+ and GTP, only a 2-fold reduction in affinity with a minor decrease in the number of sites is observed. Dissociation curves of [3H]diprenorphine, however, are clearly biphasic: in the absence of Na+, Mg2+ and GTP, 80% of the bound ligand dissociates slowly with a t1/2 of 100 min, and only 20% rapidly (t1/2 4.5 min). In the presence of Mg2+, nearly all the binding is found in the slowly dissociating form. Upon the addition of either Na+ or GTP, 20-30% of the binding dissociates more rapidly. The rate constant of the rapidly dissociating form generated by Na+, however, is 2.5 times greater than that induced by the presence of GTP. Thus, the addition of both, Na+ and GTP, converts about 80% of the receptor into a very fast dissociating form (t1/2 1.7 min). Exposure of intact cells to pertussis toxin (10 ng/ml) or treatment of membranes with N-ethyl maleimide (500 microM), strongly reduces the proportion of the slowly dissociating component. Following these treatments, the effect of GTP is reduced or abolished, but that of Na+ remains unaffected. We conclude from these data that the effects of Na+ and GTP are not only distinct in site but also in mechanism of action and that there are three forms of opioid receptors that can be differentiated by their kinetic properties. The slowly dissociating receptor form requires a functional N unit.