Characterization of irreversible binding of β-funaltrexamine to the cloned rat μ opioid receptor

Abstract

Binding of β-funaltrexamine (β-FNA) to the cloned rat μ opioid receptor expressed in COS-1 cells or Chinese hamster ovary cells was examined. β-FNA bound to the μ receptor with high affinity. Irreversible binding of [3H]β- FNA was defined as the binding that could not be dissociated by trichloroacetic acid. Na+ greatly enhanced the specific irreversible binding of [3H]β-FNA to the μ receptor, which was concentration- and time- dependent. Specific irreversible binding of [3H]β-FNA was potently inhibited by CTAP (a μ ligand), but not by ICI174,864 (a δ ligand) or U50,488H (a κ ligand). These results indicate that [3H]β-FNA binds irreversibly to the cloned μ opioid receptor. SDS-polyacrylamide gel electrophoresis and fluorography showed that [3H]β-FNA-labeled receptors migrated as one broad and diffuse hand with a mass of 80 kDa in Chinese hamster ovary or COS cells and as one band with a mass of 67 kDa in the rat brain preparation. Upon removal of N-linked carbohydrates, labeled receptors became a sharper band with a mass of ~40 kDa. [3H]β-FNA did not bind irreversibly to the cloned rat κ receptor. [3H]β-FNA binding to four chimeric μ/κ receptors was examined. The region from the middle of the third intracellular loop to the C terminus of the μ receptor is necessary for irreversible binding of β-FNA.