CONFORMATIONAL-CHANGES IN BENZODIAZEPINE RECEPTORS INDUCED BY THE ANTAGONIST RO 15-1788

  • 1 January 1983
    • journal article
    • research article
    • Vol. 23  (2) , 289-294
Abstract
The binding kinetics of [3H]Ro 15-1788, a selective benzodiazepine receptor antagonist, to synaptosomal membranes of rat cerebral cortices were studied. [3H]Ro 15-1788 binds with high affinity (dissociation constant, 0.53 nM) to a single class of binding sites (maximal binding capacity, 1.97 pmol/mg of protein). Equilibrium binding was not affected by GABA, NaCl, pentobarbital or pretreatment of the membranes at 37.degree.. Association at 0.degree. was identical whether measured in the absence or presence of GABA or bicuculline methiodide or after preincubation of the membranes at 37.degree.. The association rate under pseudo-first order conditions was curvilinear and consisted of a fast component and a slow component. Dissociation at 0.degree. with 1 .times. 10-5 M clonazepam was curvilinear and could best be fitted by 2 linear exponential components. The dissociation rate was not altered by GABA, NaCl, pentobarbital, or pretreatment of membranes at 37.degree.. The dissociation rate was similar for 0.1, 1 and 10 nM [3H]Ro 15-1788. The ratio of slow to fast dissociation component for 10 nM [3H]Ro 15-1788 was larger than that for 0.1 and 1 nM [3H]Ro 15-1788. The dissociation rate for 20 nM [3H]flunitrazepam ([3H]FNP) was much greater than that for 2 nM [3H]FNP. Using ligand concentrations occupying the same fraction of receptors, the ratio of slow to fast dissociation components was invariably greater for [3H]Ro 15-1788 than that for [3H]FNP. The rate of dissociation for [3H]Ro 15-1788 was faster under pre-equilibrium conditions than under equilibrium conditions. These results, discussed in terms of the cyclic model of interaction between receptors and benzodiazepines, suggest that [3H]Ro 15-1788 is a powerful ligand in inducing conformational changes in the initial, more labile, binary complex. Different conformational states deduced from studies of in vitro binding kinetics may not correspond to the distinct pharmacological actions of benzodiazepines. Intrinsic activities of benzodiazepines probably are determined by the step beyond the complex formation and conformational changes suggested to occur by these studies of binding kinetics.