title: Kinetics of beta-funaltrexamine binding to wild-type and mutant mu-opioid receptors expressed in Chinese hamster ovary cells.
abstract: The two-stage reaction whereby the antagonist beta-funaltrexamine (beta-FNA) binds covalently to micro opioid receptors makes it a highly discriminating probe into the tertiary structure of the receptor's recognition pocket. To obtain a quantitative measure of how well this pocket is preserved in a mutated form of the receptor, in which His-297 is substituted with glutamine, we employed [3H]-beta-FNA to evaluate the kinetic rate constants for both the reversible as well as the irreversible stages of its binding to wild-type and mutant H297Q micro receptors stably expressed in Chinese hamster ovary cells. The expression levels of the wild-type and mutant H297Q receptors were matched by exploiting the variation in receptor density as a function of plating day and by raising the expression level by pretreatment with naloxone. We found that all of the kinetic rate constants for [3H]-beta-FNA were diminished by about one-half at the mutant H297Q micro receptors with respect to wild-type receptors. By comparison, the association rate constant of [3H]-naloxone likewise decreased by one-half; however, the dissociation rate constant increased 5-fold at the mutant H297Q receptor. We conclude that the mutation has had only minor influence on the recognition site and that the function of position 297 is more likely as a link in the transduction chain.
authors.affiliation: Cellular Neurobiology Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, DHHS, Baltimore, Maryland 21224, USA. cspivak@intra.nida.nih.gov
authors.forename: Charles E
authors.initials: CE
authors.lastname: Spivak
authors.forename: Carol L
authors.initials: CL
authors.lastname: Beglan
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005786:Gene Expression Regulation / Q000502:physiology; D009154:Mutation; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D011485:Protein Binding / Q000502:physiology; D017450:Receptors, Opioid, mu / Q000235:genetics* / Q000378:metabolism*
chemical_list: D017450:Receptors, Opioid, mu; D009271:Naltrexone; C024524:beta-funaltrexamine