A Kinetic Analysis of k‐Opioid Agonist Binding Using the Selective Radioligand [3H]U69593

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Abstract
The interaction of the nonselective opioid ligand [3H]bremazocine and of the k-opioid [3H]U69593 with the k-receptor was investigated in guinea-pig cortical membranes. Each radioligand bound to a single population of high-affinity sites, although [3H]U69593 apparently recognised only 70% of those sites labelled by [3H]bremazocine. Naloxone and the selective ligands U69593 and PD117302 exhibited full inhibition of the binding of both radioligands. Kinetic analysis demonstrated biphasic rates of association and dissociation for both [3H]bremazocine and [3H]U69593. Detailed analysis of the binding of [3H]U69593 revealed that the fast rate of association was dependent on radioligand concentration, in contrast to the slow rate, which was independent of ligand concentration. Guanylyl-5′-imidodiphosphate (GppNHp) inhibited binding of [3H]U69593; saturation analysis demonstrated that the inhibitory effects of GppNHp resulted in a decrease in affinity without any significant change in binding capacity. GppNHp attenuated the formation of the slow component of [3H]U69593 binding, while accelerating the fast component. The data are consistent with the formation of a high-affinity complex between the k-receptor and a guanine nucleotide binding protein. Guanine nucleotides promote the dissociation of this ternary complex and the stabilisation of a lower-affinity state of the receptor.

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