Subunit arrangement of gamma-aminobutyric acid type A receptors.

The GABA(A) receptors are ligand-gated chloride channels. The subunit stoichiometry of the receptors is controversial; four, five, or six subunits per receptor molecule have been proposed for alphabeta receptors, whereas alphabetagamma receptors are assumed to be pentamers. In this study, alpha-beta and beta-alpha tandem cDNAs from the alpha1 and beta2 subunits of the GABA(A) receptor were constructed. We determined the minimal length of the linker that is required between the two subunits for functional channel expression for each of the tandem constructs. 10- and 23-amino acid residues are required for alpha-beta and beta-alpha, respectively. The tandem constructs either alone or in combination with each other failed to express functional channels in Xenopus oocytes. Therefore, we can exclude tetrameric or hexameric alphabeta GABA(A) receptors. We can also exclude proteolysis of the tandem constructs. In addition, the tandem constructs were combined with single alpha, beta, or gamma subunits to allow formation of pentameric arrangements. In contrast to the combination with alpha subunits, the combination with either beta or gamma subunits led to expression of functional channels. Therefore, a pentameric arrangement containing two alpha1 and three beta2 subunits is proposed for the receptor composed of alpha and beta subunits. Our findings also favor an arrangement betaalphagammabetaalpha for the receptor composed of alpha, beta, and gamma subunits.

Interaction between GABA(A) receptor beta subunits and the multifunctional protein gC1q-R.

gamma-Aminobutyric acid type A (GABA(A)) receptors were immunopurified from bovine brain using a monoclonal antibody directed against the alpha1 subunit. Of the several proteins that copurified, a 34-kDa protein was analyzed further. After enrichment and tryptic proteolysis, the resulting fragments were sequenced, and the protein was identified as gC1q-R. Using anti-gC1q-R and anti-GABA(A) receptor antibodies, mutual coimmunoprecipitation could be demonstrated from solubilized rat brain membranes. The stability of this interaction was estimated to be very high. Using the yeast two-hybrid system, various GABA(A) receptor subunit intracellular loop constructs were tested for an interaction with gC1q-R. All beta subunits, but not alpha 1 and gamma 2 subunits, were found to bind to gC1q-R. NH(2)- and COOH-terminally truncated beta 2 subunit loops were used to find the region responsible for the interaction with gC1q-R. A stretch of 15 amino acids containing 7 positively charged residues was identified (amino acids 399--413). This region contains residue Ser-410, which is a protein kinase substrate, and it is known that phosphorylation of this residue leads to an alteration in receptor activity. Localization studies suggested a predominantly intracellular localization. Our observations therefore suggest a tight interaction between gC1q-R and the GABA(A) receptor which might be involved in receptor biosynthesis or modulation of the mature function.