Heaulme M, Chambon J P, Leyris R, Wermuth C G, Biziere K
J Neurochem. 1987 Jun;48(6):1677-86. doi: 10.1111/j.1471-4159.1987.tb05723.x.
A synthetic derivative of gamma-aminobutyric acid (GABA), SR 95531 [2-(3'-carboxy-2'-propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microiontophoretic studies, to be a potent, selective, competitive, and reversible GABAA antagonist. In the present study, the binding of [3H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4 degrees C after 30 min of incubation. Pretreatment of the membranes with Triton X-100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [3H]SR 95531 for its binding site; Na+ had no effect. Subcellular fractionation showed that 74% of the P2 binding was in synaptosomes; 31% of the total homogenate binding was in P2 and 50% in P3. The binding of [3H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (KD of 6.34 nM and Bmax of 0.19 pmol/mg of protein; KD of 32 nM and Bmax of 0.81 pmol/mg of protein). The binding of [3H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABAA ligands were effective displacers of [3H]SR 95531. GABAA antagonists were relatively more potent in displacing [3H]SR 95531 than [3H]GABA; the inverse was true for GABAA agonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex greater than thalamus = olfactory bulb = hypothalamus = amygdala = striatum greater than pons-medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction of Bmax values at both the high- and the low-affinity binding sites. In conclusion, the present results demonstrate specific, high-affinity, saturable, and reversible binding of [3H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABAA receptor site in its antagonist conformation.
γ-氨基丁酸（GABA）的一种合成衍生物SR 95531 [2-(3'-羧基-2'-丙基)-3-氨基-6-对甲氧基苯基哒嗪溴化物]，最近根据生化和体内微离子电泳研究报道，是一种强效、选择性、竞争性和可逆的GABAA拮抗剂。在本研究中，对[3H]SR 95531与洗涤、冷冻和解冻的大鼠脑膜的结合进行了表征。特异性结合与组织浓度呈线性关系，在中性pH值时最适宜，孵育30分钟后在4℃时达到最大值。用Triton X-100预处理脑膜导致特异性结合减少50%。向孵育混合物中加入碘化物、硫氰酸盐或硝酸盐会降低[3H]SR 95531与其结合位点的亲和力；Na+没有影响。亚细胞分级分离显示，P2结合的74%存在于突触体中；总匀浆结合的31%存在于P2中，50%存在于P3中。[3H]SR 95531的结合是可饱和的；对饱和等温线的Scatchard分析揭示了两个明显的结合位点群体（KD为6.34 nM，Bmax为0.19 pmol/mg蛋白质；KD为32 nM，Bmax为0.81 pmol/mg蛋白质）。[3H]SR 95531的结合是可逆的，结合和解离动力学证实了两个结合位点的存在。只有GABAA配体是[3H]SR 95531的有效置换剂。GABAA拮抗剂在置换[3H]SR 95531方面比[3H]GABA相对更有效；GABAA激动剂则相反。结合位点的分布存在明显的区域差异：海马体 = 大脑皮层 > 丘脑 = 嗅球 = 下丘脑 = 杏仁核 = 纹状体 > 脑桥 - 延髓和小脑。小脑中结合位点密度出奇地低是由于高亲和力和低亲和力结合位点的Bmax值均显著降低。总之，本研究结果表明[3H]SR 95531与大鼠脑膜的结合具有特异性、高亲和力、可饱和性和可逆性，并强烈提示该放射性配体以其拮抗剂构象标记GABAA受体位点。
