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

1471-4159

Blackwell Publishing Journal Backfiles 1879-2005

Medicine

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 was 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°C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists competed 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) 〉 MDL 72222 (23.4 nM) 〉 ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205–930 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.

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