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Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target
1Department of Neuropharmacology, Max-Planck-Institut für Psychiatrie, Planegg-Martinsreid, Federal Republic of Germany.
Abstract:
The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex. The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa.
Insights
This study investigated opioid receptor binding, finding that G-proteins do not influence the receptor
Area of Science:
- Neuropharmacology
- Molecular Biology
- Biochemistry
Background:
- Opioid receptors are crucial in pain modulation and addiction.
- Their interaction with GTP-binding regulatory proteins (G-proteins) is key to signal transduction.
- Previous studies suggested G-protein involvement in opioid receptor complex structure.
Purpose of the Study:
- To determine the target size of opioid receptors.
- To investigate the role of G-proteins and sodium ions in opioid receptor complex structure and function.
- To elucidate the nature of sodium's allosteric effect on opioid receptors.
Main Methods:
- Irradiation target size analysis of opioid receptors in NG 108-15 cells and rat brain membranes.
- Radioligand binding assays with and without GTP analogs, pertussis toxin, N-ethylmaleimide, and sodium ions.
- Analysis of agonist and partial agonist binding kinetics under varying conditions.
Main Results:
- Opioid receptor target size was consistently ~100 kDa, unaffected by G-protein manipulations (GTP, pertussis toxin, N-ethylmaleimide).
- Sodium ions induced biphasic decay and flattened binding curves, suggesting loss of a low-affinity state upon irradiation.
- Sodium's effect on target size was independent of G-proteins, indicating a distinct regulatory component.
Conclusions:
- The 100 kDa opioid receptor species does not appear to include an active G-protein subunit.
- Sodium ions regulate opioid receptors via an allosteric inhibitor, distinct from G-proteins, potentially a novel receptor complex component.
- This sodium-binding unit is estimated to be 168 kDa, representing a new facet of opioid receptor regulation.