STOICHIOMETRY AND ASSEMBLY OF A RECOMBINANT GABA(A) RECEPTOR SUBTYPE

GABA(A) receptors are ligand-gated chloride ion channels that are pres umed to be pentamers composed of alpha, beta, and gamma subunits. The subunit stoichiometry, however, is controversial, and the subunit arra ngement presently is not known. In this study the ratio of subunits in recombinant alpha 1 beta 3 gamma 2 receptors was determined in Wester n blots from the relative signal intensities of antibodies directed ag ainst the N terminus or the cytoplasmic loop of different subunits aft er the relative reactivity of these antibodies had been determined wit h 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 alpha subunits, two beta subunits, and on e gamma subunit was derived. Similar experiments investigating the com position of alpha 1 beta 3 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with alpha 1 and beta 3 subunits resulted in a stoichiometry of two alpha and three beta sub units. Density gradient centrifugation studies indicated that combinat ions of alpha 1 beta 3 gamma 2 or alpha 1 beta 3 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of alp ha 1 gamma 2 or beta 3 gamma 2 subunits predominantly form heterodimer s. These results provide valuable information on the mechanism of GABA (A) receptor assembly and support the conclusion that GABA(A) receptor s are pentamers in which a total of four alternating alpha and beta su bunits are connected by a gamma subunit.