Use of 2-[¹²⁵I]iodomelatonin to characterize melatonin binding sites in chicken retina

2-[¹²⁵I]Iodomelatonin binds with high affinity to a site possessing the pharmacological characteristics of a melatonin receptor in chicken retinal membranes. The specific binding of 2-[¹²⁵I]iodomelatonin is stable, saturable, and reversible. Saturation experiments indicated that 2-[¹²⁵I]iodomelatonin labeled a single class of sites with an affinity constant (K(d)) of 434 ± 56 pM and a total number of binding sites (B(max)) of 74.0 ± 13.6 fmol/mg of protein. The affinity constant obtained from kinetic analysis was in close agreement with that obtained in saturation experiments. Competition experiments showed a monophasic reduction of 2-[¹²⁵I]iodomelatonin binding with a pharmacological order of indole amine affinities characteristic of a melatonin receptor: 2-iodomelatonin > 6-chloromelatonin ≥ melatonin ≥ 6,7-dichloro-2-methylmelatonin > 6-hydroxymelatonin ≥ 6-methoxymelatonin > N-acetyltryptamine > N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine >>> 5-hydroxytryptamine (inactive). The affinities of these melatonin analogs in competing for 2-[¹²⁵I]iodomelatonin binding sites were correlated closely with their potencies for inhibition of the calcium-dependent release of [³H]dopamine from chicken and rabbit retinas, indicating association of the binding site with a functional response regulated by melatonin. The results indicate that 2-[¹²⁵I]iodomelatonin is a selective, high-affinity radioligand for the identification and characterization of melatonin receptor sites.