Defining the GABAH receptor/channels: Molecular characterization of GABAA/C receptor/channel assembly

GABA is the main inhibitory neurotransmitter in the brain. The GABA receptor/channels are therefore essential for the balance between neuronal excitation and inhibition. Several clinically important drugs modulate neuronal excitation by binding to GABA receptors. Subunit composition is the most significant determinant of GABA receptor/channel heterogeneity, and is an important determinant of the effect(s) of clinically important drugs on GABA receptor/channels. Determining subunit assembly of the GABA receptor/channel should provide a better understanding of the molecular physiology and pharmacology of the GABAergic system. The yeast two-hybrid system, a high fidelity system for determining protein-protein interactions, was used to determine whether the GABAC receptor/channel rho 1 subunit interacts with the GABAA receptor/channel alpha 1, beta2, and gamma2 subunits. The beta 2 and gamma2 subunits interacted with the rho1 subunit. There was no interaction between the alpha1 and rho 1 subunits. The Xenopus laevis expression system was used to determine whether the GABAC receptor/channel rho 1 subunit assembles with the GABAA receptor/channel alpha 1, beta2, and gamma2 subunits. Both the beta 2 and gamma2 subunits assembled with the rho1 subunit. There was no assembly between the alpha1 and rho 1 subunits. These results are in agreement with the results from the yeast two-hybrid assays. Immunoprecipitation studies were done to determine subunit assembly in the CNS. The rho1 subunit was immunoprecipitated from brain and spinal cord lysates of adult albino rats using either anti beta 2 or anti gamma2 antibody, in a high fidelity manner, showing that the rho1 subunit assembles with the beta2 and gamma 2 subunits in the CNS. Deletion and point mutations were done on the rho 1 subunit to determine the juncture of assembly with the beta 2 and gamma2 subunits. Substitution of a proline residue for a valine (P29-V), caused a disruption of assembly of the rho1 subunit with both beta2 and gamma2 subunits. Studies of recombinant GABAH receptor/channels showed that they have distinct pharmacological properties from those of the GABAA and GABA C receptor/channels. The GABAA receptor/channel antagonist, bicuculline showed no effect on recombinant GABAH receptor/channels, neither did the GABAC receptor/channel agonist/antagonist I4AA. Taken together, the results demonstrate beta2/rho1 and gamma2/rho1 assembly in the CNS to form GABA H receptor/channels, with recombinant types showing distinct properties from the GABAH and GABAC receptor/channels.