Chromatographic Analyses of the Serotonin 5-HT1AReceptor Solubilized from the Rat Hippocampus

Abstract

Serotonin 5-HT_1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopro-pyl)dimethylammonio]-l-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylami-no)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [³H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT_1A sites led to a buffer composed of 50 mM Tris-HCl, 50 μM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mA/ MnCl₂, and 50 μg/ml of cholesleryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT_1A sites, compatible with chromatographic analyses for 2–4 days at 4°C. Adsorption and subsequent elution of [³H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethyl-aminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT_1Abinding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT_1A sites. Sodium do-decyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with M_r close to 60,000.