Multiple affinity binding states of the sigma receptor: effect of GTP-binding protein-modifying agents

The sigma receptor, which is labeled with (+)-[3H]3-(3-hydroxyphenyl)-N- 1-(propyl)piperidine [(+)-[3H]3-PPP], is a site that binds several psychotomimetic opiate benzomorphans and certain antipsychotics, such as haloperidol. In order to elucidate the mechanisms involved in sigma receptor ligand binding, equilibrium binding analysis and kinetics of association and dissociation of the relatively selective sigma receptor ligand (+)-[3H]3-PPP were determined in rat brain membranes in the absence and presence of 5'-guanylylimidodiphosphate [Gpp(NH)p]. In the absence of Gpp(NH)p, (+)-3-PPP, cyclazocine, pentazocine, and (+)-SKF 10047 bind to high and low affinity sites (KH = 1.3-7.5 nM; KL = 84-500 nM), as determined by computer assisted analysis of the inhibition of (+)-[3H]3-PPP binding by the sigma ligands. The antipsychotics haloperidol and chlorpromazine inhibit (+)-[3H]3-PPP binding in a manner indicating interaction with a single state of the receptor. Gpp(NH)p (0.1 mM) abolished the high affinity binding component of the sigma agonist-like compounds tested but had no effect on the affinities of the antipsychotics for the receptor. Gpp(NH)p decreased the association rate of (+)-[3H]3-PPP binding 5-fold and also converted the biexponential dissociation kinetics of the ligand, observed in the absence of Gpp(NH)p, to a rapid monophasic dissociation process. Pretreatment of membranes with N-ethylmaleimide and pertussis toxin inhibited (+)-[3H]3-PPP binding and abolished the effect of Gpp(NH)p on the sigma ligand binding. These findings indicate of the sigma receptor is capable of existing in two discrete states, having high and low affinity for sigma agonist-like drugs. The regulation of the high affinity binding state by GTP-binding protein-modifying agents suggests its coupling to GTP-binding protein(s).