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Multiple interconvertible affinity states for the delta opioid agonist-receptor complex.

J W Spain, C J Coscia

    The Journal of Biological Chemistry
    |July 5, 1987
    PubMed
    Summary

    Guanine nucleotide affects opioid receptor binding kinetics. It converts a high-affinity state to a lower-affinity state, suggesting a multistep binding process involving at least three steps for DADL to bind synaptic membranes.

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    Area of Science:

    • Neuroscience
    • Pharmacology
    • Biochemistry

    Background:

    • Previous studies indicated that [3H-D-Ala2-D-Leu5]enkephalin ([3H]DADL) binding to bovine hippocampal synaptic plasma membranes (SPMs) is time-dependent, suggesting a multistep process.
    • Understanding the kinetic intermediates of opioid receptor binding is crucial for elucidating receptor function and drug interactions.

    Purpose of the Study:

    • To investigate the role of guanine nucleotide in modulating the dissociation kinetics of [3H]DADL from SPMs.
    • To characterize the different kinetic intermediates involved in the opioid ligand-receptor binding process.

    Main Methods:

    • Radioligand binding assays using [3H]DADL on bovine hippocampal SPMs.
    • Measurement of dissociation rates (off-rates) of [3H]DADL under various conditions.

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  • Inclusion of guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) to assess guanine nucleotide effects.
  • Use of unlabeled [D-Ala2-Me-Phe4-Gly-Ol5]enkephalin to ensure delta (δ) site selectivity.
  • Pretreatment of membranes with N-ethylmaleimide (NEM) to probe specific binding steps.

    • Main Results:

    • Gpp(NH)p significantly increased the off-rate of [3H]DADL, particularly after prolonged association times, converting a high-affinity state to a lower-affinity one.
    • When Gpp(NH)p was present during association, the slowly dissociating high-affinity state was abolished, and the off-rate was faster and linear.
    • NEM treatment eliminated time-dependent dissociation rates, suggesting it prevents the formation of the high-affinity state, possibly involving GTP-binding proteins.

    Conclusions:

    • The association of DADL to bovine hippocampal SPMs involves at least three distinct kinetic steps.
    • Guanine nucleotides play a critical role in modulating opioid receptor affinity and kinetics, likely by interacting with an associated GTP-binding protein.
    • NEM-sensitive components are essential for the time-dependent formation of a high-affinity opioid receptor-ligand complex.