Pharmacology of GAB A ρ1 and GAB A α/β receptors expressed in Xenopus oocytes and COS cells

Abstract

1 The ρ1 protein, which we previously cloned from retina, assembles as a homooligomer that transduces the binding of γ-aminobutyric acid (GABA) into robust chloride currents. However, its insensitivity to bicuculline, pentobarbitone and benzodiazepines, all potent agents at typical GABAA receptors, suggested that it may react atypically to other GABA agonists and antagonists. 2 cDNAs for the ρ1 and the α5β1 receptors for GABA were expressed as homo- and heterooligomers, respectively, in Xenopus oocytes. The selectivities of the respective receptors for various agonists were investigated using concentration-response experiments in voltage clamped cells. 3 The most potent agonists at the ρ1 receptor were trans-4-aminocrotonic acid (TACA) > GABA > muscimol; at the α5β1 receptor the rank order was muscimol > GABA > 4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-ol (THIP). The most specific agonists were cis-(2-(aminomethyl)-cyclopropyl-carboxylic acid (CAMP) and THIP for the ρ1 and the α5β1 receptors, respectively. 4 Comparing GABA, TACA and cis-aminocrotonic acid (CACA) at ρ1 receptors expressed in COS cells gave results almost indistinguishable from those found at oocytes; the pharmacology of ρ1 seems independent of the expression system. 5 Agonists THIP, piperidine-4-sulphonic acid (P4S), and isoguvacine, whose C-C-C-N chains are constrained by rings into a folded conformation and were potent at the α5β1 receptor, were among the weakest at the ρ1 receptor. However CACA and CAMP, which align better with the extended than the folded conformation, were weakest at the α5β1 receptor but moderately potent at the ρ1 receptor. These findings suggest that the ρ1 receptor recognizes agonists in the extended conformation, in contrast to GABAA receptors, which are believed to recognize agonists in the partially folded conformation. 6 In contrast to the α5β1 receptor, gradations in maximum responses were apparent in the ρ1 receptor, suggesting various degrees of partial agonism. In particular, imidazole-4-acetic acid (I4AA), whose maximum response was only 3% of GABA's maximum, had an apparent Kd for activating the ρ1 receptor of 16 μm; but it had an apparent Kd for competitively blocking the receptor of 0.64 μm. This difference suggests that steric constraints in the activated (open channel) receptor are tighter than in the resting receptor. 7 Hill coefficients approached 2 at the ρ1 receptor, but were closer to unity at the α5β1 receptor. Thus, the ρ1 receptor displayed higher cooperativity. 8 Unlike typical GABAA receptors, the ρ1 receptor was insensitive to the competitive antagonists bicuculline, SR95531, securinine, and (+)-tubocurarine.