[³H]5-Hydroxytryptamine Binding to Reconstituted Fraction with Sulphatides, Phosphatidylserine and Phosphatidylinositol

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As a first step toward the examination of the involvement of sulphatides, phosphatidylserine and phosphatidylinositol in 5-HT receptor mechanisms, we performed [³H]5-HT binding experiments on the various reconstituted fractions with these acidic lipids. A binding assay of [³H]5-HT to these fractions was carried out by Sephadex LH₂₀ column chromatography. Among various reconstituted fractions, only the reconstitution system with the three acidic lipids exhibited a saturable [³H]5-HT binding capacity, whereas no binding was seen with [³H]-spiperone. When the binding of [³H]5-HT to this fraction was plotted as a function of the ligand concentration, a multiple binding mode with three classes of binding components (or sites) was observed. Furthermore, the double reciprocal plot indicated that this reconstitution system had three apparent K_D values of 4.7, 15 and 59 nM. The displacement studies with various compounds indicated that only a few 5-HT agonists (5-methoxytryptamine and tryptamine) and neurotransmitters (DA and ACh) inhibited the[³H]5-HT binding to this fraction, but 5-HT antagonists, LSD analogues and neuroleptics had no effect. Moreover, GTP, GDP and Gpp(NH)p clearly inhibited the[³H]5-HT binding in spite of their weak potencies, while GMP did not have any effect.