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

Eur J Pharmacol. 2002 Sep 27;452(1):21-33. doi: 10.1016/s0014-2999(02)02242-2.

Abstract

Paradoxically, the potencies (EC(50)) of agonists stimulating [35S]GTPgammaS binding are several orders of magnitude lower than their affinities in receptor binding assays. We have investigated the quantitative stoichiometry of mu-opioid receptor-G-protein coupling in postmortem human brain. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) displaced [3H]naloxone binding in a biphasic pattern. The ratio between K(i-low) and EC(50) of DAMGO stimulating [35S]GTPgammaS binding was lower than one. The K(A) of DAMGO was calculated following mu-opioid receptor alkylation by beta-funaltrexamine from [35S]GTPgammaS binding data using the "nested hyperbolic method", yielding K(A)/EC(50)>1. Thus, only 1.2 +/- 0.2% of mu-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 microM DAMGO (determined by isotopic dilution curves) and the occupied-mu-opioid receptors was 1304. In conclusion, we have determined the stoichiometric and the kinetic parameters in the mu-opioid receptor-G-protein system.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylation
  • Analgesics, Opioid / metabolism
  • Analgesics, Opioid / pharmacology
  • Baclofen / pharmacology
  • Binding, Competitive / drug effects
  • Brain Chemistry / physiology*
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / metabolism
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / pharmacology
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Ethylmaleimide / metabolism
  • Ethylmaleimide / pharmacology
  • GABA Agonists / pharmacology
  • GTP-Binding Protein Regulators / physiology
  • GTP-Binding Proteins / metabolism*
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Membranes / metabolism
  • Naloxone / metabolism
  • Naltrexone / analogs & derivatives*
  • Naltrexone / pharmacology
  • Narcotic Antagonists / pharmacology
  • Receptors, Opioid, mu / drug effects
  • Receptors, Opioid, mu / metabolism*
  • Signal Transduction / physiology

Substances

  • Analgesics, Opioid
  • Enzyme Inhibitors
  • GABA Agonists
  • GTP-Binding Protein Regulators
  • Narcotic Antagonists
  • Receptors, Opioid, mu
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Naloxone
  • Naltrexone
  • beta-funaltrexamine
  • GTP-Binding Proteins
  • Baclofen
  • Ethylmaleimide