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

Abstract

Kinetic analysis of binding of [(3)H][N-[2-[4-(2-[O-methyl-(3)H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide ([(3)H]WAY100635) to 5-HT(1A) receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding experiments revealed that [(3)H]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) x 10(-3) min(-1), means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25 degrees C. Systematic association kinetic studies of [(3)H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive cooperativity. The affinities of antagonists determined from competition with [(3)H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of [(35)S]GTPgammaS binding probably due to the ligand's kinetic peculiarities. Thus, [(3)H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies is strongly limited.

Characterization of 5-HT1A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190

“…3) with the pIC 50 values of the studied ligands being close to their previously reported values (Table 1) (Newman-Tancredi et al, 1998;Parkel and Rinken, 2006). Addition of GTPcS had limited effects on ligand binding and did not substantially alter the pIC 50 values, but it did appear to slightly lower the fluorescence anisotropy of Bodipy-FL-NAN-190 in the absence (or low concentrations) of competing ligands.…”

Overview of the detection methods for equilibrium dissociation constant KD of drug-receptor interaction

“…As shown in Table 1 , RBA can be used to determine the affinity constant, dissociation constant and the number of binding receptors [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] , [34] . The key condition of receptor binding experiments is to prepare an excellent radioligand.…”

“…The key condition of receptor binding experiments is to prepare an excellent radioligand. The basic requirements of radioligand are high radioactivity, high affinity, high specificity and stability [22] , [35] , [36] . The ability to prepare novel and selective radioligands facilitates the study of drug-receptor interaction, and RBA provides an effective tool for studying the mechanisms of drugs at molecular level [37] , [38] .…”

“…The ability to prepare novel and selective radioligands facilitates the study of drug-receptor interaction, and RBA provides an effective tool for studying the mechanisms of drugs at molecular level [37] , [38] . A variety of receptor materials can be used in the RBA method, such as the cell membrane obtained from cell and tissue [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , intact cells [29] , [30] , tissue slice [31] , [32] , [33] , [34] , and engineered protein samples [38] , [39] .…”

α2A-Adrenoceptor-specific Stimulation of [35S]GTPγS Binding to Membrane Preparations of Rat Frontal Cortex

“…Membranes of frontal cortex were prepared as described earlier [19] with slight modifications. Frontal cortices from Wistar rats were homogenized in 5 ml of homogenization buffer (50 mM Tris-HCl, 0.5 mM EDTA, 6.5 mM MgCl 2 , pH7.4) by Bandelin Sonopuls sonificator and centrifuged at 30,000g for 20 min at 4°C.…”