Quantitative stoichiometry of G-proteins activated by μ-opioid receptors in postmortem human brain

Paradoxically, the potencies (EC₅₀) of agonists stimulating [³⁵S]GTPγS binding are several orders of magnitude lower than their affinities in receptor binding assays. We have investigated the quantitative stoichiometry of μ-opioid receptor-G-protein coupling in postmortem human brain. [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin (DAMGO) displaced [³H]naloxone binding in a biphasic pattern. The ratio between K_i-low and EC₅₀ of DAMGO stimulating [³⁵S]GTPγS binding was lower than one. The K_A of DAMGO was calculated following μ-opioid receptor alkylation by β-funaltrexamine from [³⁵S]GTPγS binding data using the "nested hyperbolic method", yielding K_A/EC₅₀>1. Thus, only 1.2±0.2% of μ-opioid receptors was needed to be occupied to achieve the half-maximal effect of DAMGO. The estimated ratio between the G-proteins activated by 10 μM DAMGO (determined by isotopic dilution curves) and the occupied-μ-opioid receptors was 1304. In conclusion, we have determined the stoichiometric and the kinetic parameters in the μ-opioid receptor-G-protein system.