The recently-identified GABA Rho1 receptor allows the examination of GABA receptor structure/function, since it expresses well as a homooligomer. This receptor is insensitive to bicuculline, baclofen and modulators of GABA A receptor, and desensitizes very slowly facilitating characterization of this receptor's binding site for agonists and competitive antagonists. The agonist profile of the GABA Rho1 receptor was strikingly different from that of the typical GABA A receptor in the Xenopus oocyte expression system. Studies of cyclic agonists, such as THIP and isonipecotic acid, active at GABA A receptors but virtually inert at GABA Rho1 receptors, indicate that the recognition of site of the GABA Rho1 receptor selects for a more extended conformation of agonists. The GABA Rho2 receptor, while closely resembling GABA Rho1 in primary sequence, showed 20% difference in the N-terminal region. Like Rho1, it functioned as a homooligomer, but the currents responded to GABA more slowly and their maxima were conspicuously smaller. Concentration response curves yielded similar agonist profiles for Rho1 and Rho2, though Rho2 generally had lower Kd values by a factor of about two-fold. Among the 9 agonists tested, the outstanding exception was imidazole-4- acetic acid. Its Kd was 16-times smaller, its maximum current (with respect to the maximum produced by GABA) 7-times greater, and its Hill coefficient double at Rho2 in comparison to Rho1. Sixteen mutations in the Rho1 sequence were constructed. Mutations in residue 141H diminished the affinity of GABA or abolished the responses, whereas most other mutations increased affinity. Most mutations, however, seemed to diminish the maximum current. The mutations had variable effects on the Hill coefficient, Nh = 2.26 in Rho 1. Some decreased Nh to as little as 1.24, but mutations at residue 316, in the extracellular domain between membrane spanning regions 2 and 3, increased it to as high as 3.9 The maximum responses to THIP remained low in all mutations, indicating that the receptors retained Rho1 -like selectivity for agonists. These studies suggest regions in the receptor's structure that may be crucial in agonist recognition or transduction, and that may facilitate cooperative interaction among subunits.
