Heterogeneity of GABAA receptor in goldfish retina

The possibility of GABA_A 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 Mr 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 Mr polypeptides. Immunoprecipitated receptors bound only ³H-muscimol, not ³H-FNZ. An attempt to precipitate the ³H-FNZ photolabeled polypeptides failed. Dry autoradiography showed ³H-PNZ binding only in the inner plexiform layer (IPL); the binding was enhanced with γ-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 GABA_A receptor in the goldfish retina: (1) GABA_A 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) GABA_A 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. © 1994 Wiley-Liss, Inc.