3H]5-hydroxytryptamine binding to reconstituted fraction with sulphatides, phosphatidylserine and phosphatidylinositol

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 KD 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.