Lin Z S, Yazulla S
Department of Neurobiology and Behavior, University at Stony Brook, New York 11794-5230.
J Comp Neurol. 1994 Jul 15;345(3):429-39. doi: 10.1002/cne.903450309.
The possibility of GABAA receptor heterogeneity in goldfish retina was studied with immunocytochemical and biochemical approaches: 1) immunoblotted membrane particulates of goldfish retina with mAb 62-3G1; 2) immunoprecipitation of the detergent-solubilized membrane proteins with mAb 62-3G1 followed by the receptor binding assay; 3) photoaffinity labeling of the membrane particulates with 3H-flunitrazepam (FNZ) and visualization of the labeled receptors by SDS-PAGE and fluorography; 4) dry autoradiography of 3H-muscimol and 3H-FNZ binding sites on frozen sections. Immunoblots showed that 62-3G1 reacted with 55-57.5 kDa M(r) polypeptides, similar to the muscimol-binding subunit of the receptor complex in bovine brain; while 3H-FNZ photoaffinity labeled the 52.5 kDa and 41-43 kDa M(r) polypeptides. Immunoprecipitated receptors bound only 3H-muscimol, not 3H-FNZ. An attempt to precipitate the 3H-FNZ photolabeled polypeptides failed. Dry autoradiography showed 3H-FNZ binding only in the inner plexiform layer (IPL); the binding was enhanced with gamma-aminobutyric acid (GABA) and blocked by clonazepam. In contrast, 3H-muscimol was bound in both the outer plexiform layer (OPL) and IPL, similar to that observed with 62-3G1 immunocytochemistry. We suggest that there are two subtypes of GABAA receptor in the goldfish retina: 1) GABAA receptors that are not linked to a benzodiazepine (BZD) receptor are located in the OPL and at amacrine-to-amacrine and amacrine-to-ganglion cell synapses in the IPL and are recognized by 62-3G1; 2) GABAA receptors that are linked to a BZD receptor are located only in the IPL, largely at amacrine-to-bipolar cell synapses and are not recognized by mAb 62-3G1.
采用免疫细胞化学和生物化学方法研究了金鱼视网膜中γ-氨基丁酸A（GABAA）受体异质性的可能性：1）用单克隆抗体62-3G1对金鱼视网膜的膜微粒进行免疫印迹；2）用单克隆抗体62-3G1对去污剂溶解的膜蛋白进行免疫沉淀，随后进行受体结合测定；3）用3H-氟硝西泮（FNZ）对膜微粒进行光亲和标记，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和荧光自显影对标记的受体进行可视化；4）对冷冻切片上的3H-蝇蕈醇和3H-FNZ结合位点进行干放射自显影。免疫印迹显示，62-3G1与55-57.5 kDa相对分子质量（M(r)）的多肽发生反应，类似于牛脑中受体复合物的蝇蕈醇结合亚基；而3H-FNZ光亲和标记了52.5 kDa和41-43 kDa M(r)的多肽。免疫沉淀的受体仅结合3H-蝇蕈醇，不结合3H-FNZ。沉淀3H-FNZ光标记多肽的尝试失败。干放射自显影显示3H-FNZ仅在内网状层（IPL）有结合；γ-氨基丁酸（GABA）可增强这种结合，而氯硝西泮可阻断这种结合。相比之下，3H-蝇蕈醇在外网状层（OPL）和IPL均有结合，类似于62-3G1免疫细胞化学观察到的情况。我们认为金鱼视网膜中存在两种GABAA受体亚型：1）不与苯二氮䓬（BZD）受体相连的GABAA受体位于OPL以及IPL中无长突细胞-无长突细胞和无长突细胞-神经节细胞突触处，可被62-3G1识别；2）与BZD受体相连的GABAA受体仅位于IPL，主要在无长突细胞-双极细胞突触处，且不被单克隆抗体62-3G1识别。
