The antagonist beta-funaltrexamine (beta-FNA), known to bind covalently to mu-opioid receptors by a two-step, doubly discriminating sequence, was used as a sensitive gauge to compare wildtype to mutant H297Q mu-opioid receptors. We addressed whether this mutation, which enhances the intrinsic activities of alkaloid mu-receptor agents, affects both the reversible and covalent phases of beta-FNA binding. Such altered binding serves as a reporter for the dimensions and topography of the receptor's recognition site. Using the voltage-clamped Xenopus oocyte expression system with coexpressed GIRK potassium channels, we found that beta-FNA blocked the wildtype and mutant H297Q receptors both reversibly and irreversibly, indicating overall conserved tertiary structure in the mutant. The mutant H297Q receptor, however, was more resistant to both phases of blockade, indicating some disturbance of the mutant H297Q receptor recognition site. beta-FNA acted as a partial agonist at the mutant H297Q receptor expressed in both oocytes, as measured by the activation of GIRK channels, and in COS-7 cells assayed by GTPgamma(35)S binding. beta-FNA showed no activity at the wildtype receptor expressed in oocytes, but surprisingly induced binding of GTPgamma(35)S in transfected COS-7 cells. Thus, the topography of the mutant H297Q receptor recognition site is sufficiently conserved to allow the selective binding of beta-FNA, but the decrease in binding affinity and increase in efficacy in oocytes demonstrates clear differences from the wildtype receptor.
