[35S]GTPγS Binding Assay

The effects of the various compounds tested on [³⁵S]GTPγS binding in HeLa cells expressing the recombinant human 5-HT_1AR were evaluated according to the method of Stanton and Beer [41] with minor modifications. The stimulation experiments are as follows: Cell membranes (50–70 μg of protein) were resuspended in buffer containing 20 mM HEPES, 3 mM MgSO₄, and 120 mM NaCl (pH 7.4). The membranes were incubated with 30 μM GDP and various concentrations (from 0.1 nM to 10 μM) of test drugs or 8-OH-DPAT (reference curve) for 20 min at 30 °C in a final volume of 0.5 mL. The samples were transferred to ice, [³⁵S]GTPγS (200 pM) was added, and the samples were incubated for further 30 min at 30 °C. The preincubation with both agonist and antagonist, before initiating the [³⁵S]GTPγS binding, ensures that agonist and antagonist are at equilibrium. Nonspecific binding was determined in the presence of 10 mM GTPγS. Incubation was stopped by the addition of ice-cold HEPES buffer and rapid filtration on Unifilter B filters (PerkinElmer) using a Filtermate cell harvester (Packard). The filters were washed with ice-cold Hepes buffer, and the radioactivity retained on the filters was determined by a TopCount, PerkinElmer liquid scintillation counter at 90% efficiency.