Characteristics of Binding of [3H]WAY100635 to Rat Hippocampal Membranes

Kinetic analysis of binding of ³H]N-2-4-(2-O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide (³H]WAY100635) to 5-HT_1A receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding experiments revealed that ³H]WAY100635 binds to a single class of receptors with very high apparent affinity (K _D = 87 ± 4 pM, B _max = 15.1 ± 0.2 fmol/mg protein). The binding was almost irreversible, as the dissociation rate constant obtained k _off = (7.8 ± 1.1) × 10⁻³ min⁻¹, means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25°C. Systematic association kinetic studies of ³H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive cooperativity. The affinities of antagonists determined from competition with ³H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of ³⁵S]GTPγS binding probably due to the ligand’s kinetic peculiarities. Thus, ³H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies is strongly limited.