Solubilisation of the 5‐Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

Abstract

5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested {1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside}, deoxycholate (0.5%) yielded the best solubilisation (54.6 .+-. 6% of receptor, 70.5 .+-. 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 .+-. 6 fmol/mg of protein; KD = 0.33 .+-. 0.09 nM; n = 4) and were similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4.degree. C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) > quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) > ICS 205-930 (0.93 nM) > GR 38032F (2.2 nM) > BRL 24924 (4.1 nM [.+-.-endo]-4-amino-5-chloro-2-methoxy-N-(1-azobicyclo[3.3.1]nonyl)benzamide monohydrochloride) > MDL 72222 (23.4 nM [1.alpha.H,3.alpha.,5.alpha.H-trypoan-3-yl-3,5-dichlorobenzoate]) > ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 [(3alpha-tropanyl)-1H-indole-3-carboxylic acid ester] binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.