Studies on kinetics of [3H]beta-funaltrexamine binding to mu opioid receptor

LY Liu-Chen, SX Li and RJ Tallarida

Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.

beta-Funaltrexamine (beta-FNA) was shown to be a reversible kappa agonist and an irreversible mu antagonist. [3H]beta-FNA at low concentrations (less than 10 nM) covalently binds to mu but not delta or kappa opioid receptors in brain membranes. The interaction between beta-FNA and mu opioid receptors was thought to involve two steps; a reversible ligand-receptor complex is formed before the formation of an irreversible complex, based on observations in bioassays in vitro. In this study, we investigated whether such a two-step process occurred in binding using bovine striatal membranes and determined the kinetic parameters by examining the time courses of both reversible and irreversible binding of [3H]beta-FNA to mu opioid receptors. Specific binding was defined as the difference between binding in the presence of levorphanol and dextrorphan (1 microM). Reversible binding was determined as the difference between membrane (reversible and irreversible) binding and irreversible binding. At 25 degrees, the rate of formation of irreversible [3H]beta-FNA-receptor complex increased as the concentration increased and reached a plateau at 2 nM; further increase in [3H]beta-FNA concentration did not enhance the rate of formation, indicating that the rate saturation effect exists for irreversible binding of [3H]beta-FNA to mu opioid receptors. At 10 degrees and low concentrations (less than 1 nM) of [3H]beta-FNA, appreciable reversible binding to opioid receptors occurred before any irreversible [3H]beta-FNA-receptor complex could be detected. These observations support the notion that reversible binding occurs before alkylation of the receptor. The binding of [3H]beta-FNA to mu opioid receptors was thus modeled to allow for such a two-step process: (formula; see text) A mathematical analysis method was derived to allow determination of all kinetic parameters (k+1, k-1, k2, and Kd) of such a two-step reaction. Values of k2, k+1, k-1, and Kd were determined at 10 degrees for 0.5, 0.25, and 0.125 nM [3H]beta-FNA and were found to be very similar among these three concentrations. Raising the incubation temperature from 10 degrees to 37 degrees greatly enhanced the values of k+1, k-1, and k2 without affecting Kd. At 37 degrees incubation without 200 mM NaCl significantly decreased the values of k+1, k-1, and k2 without affecting Kd. NaCl increased the irreversible binding, probably by shifting the equilibrium towards a conformation that binds more easily with beta-FNA. Under all conditions examined, the value of k-1 was found to be at least 5-fold greater than k2, indicating that the majority of the reversible complex dissociates and only a small portion proceeds to form irreversible complex. This finding is consistent with published observations that only a portion of beta-FNA binding to mu opioid receptors is irreversible. In conclusion, [3H]beta-FNA binds reversibly to mu opioid receptors before forming covalent bonding...

Volume 37, Issue 2, pp. 243-250, 02/01/1990
Copyright © 1990 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				A. C. Barrett, E. S. Smith, and M. J. Picker Use of Irreversible Antagonists to Determine the Relative Efficacy of {micro}-Opioids in a Pigeon Drug Discrimination Procedure: Comparison of {beta}-Funaltrexamine and Clocinnamox J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 1061 - 1070. [Abstract] [Full Text] [PDF]

				J. P. McLaughlin, K. P. Hill, Q. Jiang, A. Sebastian, S. Archer, and J. M. Bidlack Nitrocinnamoyl and Chlorocinnamoyl Derivatives of Dihydrocodeinone: In Vivo and In Vitro Characterization of µ-Selective Agonist and Antagonist Activity J. Pharmacol. Exp. Ther., April 1, 1999; 289(1): 304 - 311. [Abstract] [Full Text]

				C. Chen, J. Yin, J. K. d. Riel, R. L. DesJarlais, L. F. Raveglia, J. Zhu, and L.-Y. Liu-Chen Determination of the Amino Acid Residue Involved in [3H]beta -Funaltrexamine Covalent Binding in the Cloned RatµOpioid Receptor J. Biol. Chem., August 30, 1996; 271(35): 21422 - 21429. [Abstract] [Full Text] [PDF]

				J. Zhu, J. Yin, P.-Y. Law, P. A. Claude, K. C. Rice, C. J. Evans, C. Chen, L. Yu, and L.-Y. Liu-Chen Irreversible Binding of cis-(+)-3-Methylfentanyl Isothiocyanate to the [IMAGE] Opioid Receptor and Determination of Its Binding Domain J. Biol. Chem., January 19, 1996; 271(3): 1430 - 1434. [Abstract] [Full Text] [PDF]

				C. Chen, J.-C. Xue, J. Zhu, Y.-W. Chen, S. Kunapuli, J. K. d. Riel, L. Yu, and L.-Y. Liu-Chen Characterization of Irreversible Binding of [IMAGE]-Funaltrexamine to the Cloned Rat µ Opioid Receptor J. Biol. Chem., July 28, 1995; 270(30): 17866 - 17870. [Abstract] [Full Text] [PDF]

				D. R. Groebe and S. N. Abramson Lophotoxin Is a Slow Binding Irreversible Inhibitor of Nicotinic Acetylcholine Receptors J. Biol. Chem., January 6, 1995; 270(1): 281 - 286. [Abstract] [Full Text] [PDF]