Stoichiometry and assembly of a recombinant GABA(A) receptor subtype

Abstract

GABA(A) receptors are ligand-gated chloride ion channels that are presumed to be pentamers composed of α, β, and γ, subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of subunits in recombinant α1β3γ2 receptors was determined in Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been determined with GABA(A) receptor subunit chimeras composed of the N- terminal domain of one and the remaining part of the other subunit. Via this method a subunit stoichiometry of two α subunits, two β subunits, and one γ subunit was derived. Similar experiments investigating the composition of α1β3 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with α1 and β3 subunits resulted in a stoichiometry of two α and three subunits. Density gradient centrifugation studies indicated that combinations of α1β3γ2 or α1β3 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of α1γ2 or β3γ2 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABA(A) receptor assembly and support the conclusion that GABA(A) receptors are pentamers in which a total of four alternating α and β subunits are connected by a γ subunit.