Improvement of 5-HT_3 Receptor Binding Assay

The 5-hydroxytryptamine (5-HT)_3 receptor binding assay using [^^3 H]quipazine was examined. It was impossible to obtain specific [^^3 H]quipazine binding with the membrane fractions from rat cortex prepared by the usual procedure. When the membranes were pretreated with detergent Triton X-100, the ratio of specific [^^3 H]quipazine binding markedly increased, depending upon the concentration of Triton X-100 in the range of 0.01 - 0.1% (w/v). At a concentration of more than 0.05%, the specific binding reached a maximum of 55 to 60% of the total binding. The specific [^^3 H]quipazine binding to the Triton X-100-treated membranes was reversible and was potently inhibited by several 5-HT_3 antagonists, while 5-HT_1 , 5-HT_2 receptor antagonists and other receptor-specific ligands had no effect on the binding. Scatchard analysis indicated a single class of binding sites with a K_d of 0.62 nM and B_max of 97 fmol/mg protein. Thus, the Triton X-100-treated membranes retained the characteristics of 5-HT_3 binding sites, making it possible to use [^^3 H]quipazine for a 5-HT_3 receptor binding assay with a high ratio of specific binding.