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Characterization of irreversible binding of beta-funaltrexamine to the cloned rat mu opioid receptor
1Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Abstract:
Binding of beta-funaltrexamine (beta-FNA) to the cloned rat mu opioid receptor expressed in COS-1 cells or Chinese hamster ovary cells was examined. beta-FNA bound to the mu receptor with high affinity. Irreversible binding of [3H]beta-FNA was defined as the binding that could not be dissociated by trichloroacetic acid. Na+ greatly enhanced the specific irreversible binding of [3H]beta-FNA to the mu receptor, which was concentration- and time-dependent. Specific irreversible binding of [3H]beta-FNA was potently inhibited by CTAP (a mu ligand), but not by ICI174,864 (a delta ligand) or U50,488H (a kappa ligand). These results indicate that [3H]beta-FNA binds irreversibly to the cloned mu opioid receptor. SDS-polyacrylamide gel electrophoresis and fluorography showed that [3H]beta-FNA-labeled receptors migrated as one broad and diffuse band 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 approximately 40 kDa. [3H]beta-FNA did not bind irreversibly to the cloned rat kappa receptor. [3H]beta-FNA binding to four chimeric mu/kappa receptors was examined. The region from the middle of the third intracellular loop to the C terminus of the mu receptor is necessary for irreversible binding of beta-FNA.
Insights
Beta-funaltrexamine (beta-FNA) irreversibly binds to the mu opioid receptor with high affinity. This binding is specific and influenced by sodium ions, indicating its utility in studying mu opioid receptor pharmacology.
Area of Science:
- Pharmacology
- Neuroscience
- Molecular Biology
Background:
- The mu opioid receptor is a key target for pain management.
- Understanding the precise binding characteristics of opioid ligands is crucial for drug development.
Purpose of the Study:
- To investigate the irreversible binding of beta-funaltrexamine (beta-FNA) to the cloned rat mu opioid receptor.
- To characterize the molecular properties and binding requirements of beta-FNA.
Main Methods:
- Utilized cloned rat mu opioid receptors expressed in COS-1 and Chinese hamster ovary cells.
- Employed radioligand binding assays with [3H]beta-FNA, including trichloroacetic acid precipitation to define irreversible binding.
- Analyzed receptor characteristics using SDS-polyacrylamide gel electrophoresis and fluorography, including deglycosylation experiments.
- Examined binding to chimeric mu/kappa receptors.
Main Results:
- [3H]beta-FNA demonstrated high-affinity, irreversible binding to the mu opioid receptor, enhanced by Na+.
- Binding was specifically inhibited by mu opioid ligands (CTAP) but not delta (ICI174,864) or kappa (U50,488H) ligands.
- SDS-PAGE revealed beta-FNA-labeled mu receptors with apparent masses of 80 kDa (CHO/COS cells) and 67 kDa (rat brain), reduced to ~40 kDa after deglycosylation.
- The third intracellular loop to C-terminus region of the mu receptor was essential for irreversible beta-FNA binding.
Conclusions:
- [3H]beta-FNA serves as a specific, irreversible radioligand for the mu opioid receptor.
- The binding site for irreversible interaction is localized to the intracellular domain of the mu opioid receptor.
- These findings provide valuable insights into mu opioid receptor structure-function relationships and ligand interactions.