PHARMACOLOGY OF GABA RHO-1 AND GABA ALPHA/BETA RECEPTORS EXPRESSED IN XENOPUS OOCYTES AND COS CELLS

1 The rho1 protein, which we previously cloned from retina, assembles as a homooligomer that transduces the binding of gamma-aminobutyric acid (GABA) into robust chloride currents. However, its insensitivity to bicuculline, pentobarbitone and benzodiazepines, all potent agents at typical GABA(A) receptors, suggested that it may react atypically to other GABA agonists and antagonists. 2 cDNAs for the rho1 and the alpha,beta1 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 rho1 receptor were trans-4-aminocrotonic acid (TACA) > GABA > muscimol; at the alpha5beta1 receptor the rank order was muscimol > GABA > 4,5,6,7-tetrahydro-isoxazole[4,5-c]pyridine-3-ol (THIP). The most specific agonists were cis-(2-(aminomethyl)-cyclopropyl-carboxylic acid (CAMP) and THIP for the rho1 and the alpha5beta1 receptors, respectively. 4 Comparing GABA, TACA and cis-aminocrotonic acid (CACA) at rho1 receptors expressed in COS cells gave results almost indistinguishable from those found at oocytes; the pharmacology of rho1 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 alpha5beta1 receptor, were among the weakest at the rho1 receptor. However CACA and CAMP, which align better with the extended than the folded conformation, were weakest at the alpha5beta1 receptor but moderately potent at the rho1 receptor. These findings suggest that the rho1 receptor recognizes agonists in the extended conformation, in contrast to GABA(A) receptors, which are believed to recognize agonists in the partially folded conformation. 6 In contrast to the alpha5beta1 receptor, gradations in maximum responses were apparent in the rho1 receptor, suggesting various degrees of partial agonism. In particular, imidazole-4-acetic acid (14AA), whose maximum response was only 3% of GABA's maximum, had an apparent K(d) for activating the rho1 receptor of 16 mum; but it had an apparent K(d) for competitively blocking the receptor of 0.64 mum. 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 rho1 receptor, but were closer to unity at the alpha5beta1 receptor. Thus, the rho1 receptor displayed higher cooperativity. 8 Unlike typical GABA(A) receptors, the rho1 receptor was insensitive to the competitive antagonists bicuculline, SR95531, securinine, and (+)-tubocurarine.