Functional Expression and Characterization of the Rat GABAA Receptor Subtype α5β2γ2 in Sf 9 Insect Cells

Abstract

The gamma amino butyric acid (GABA)A receptor in the mammalian brain is composed of different subunits (α, β, γ, δ and ρ type) and the presence of at least three different subunits (α, β, and γ) is necessary for its complete functional activity. Recombinant baculoviruses expressing rat GABAA receptor subunits were constructed. After coinfection with α5,β2and γ2 subunit producing viruses functionally active GABAA receptor complexes were obtained in Sf 9 insect cells. The pharmacological profile of these receptors produced in this heterologous system was very similar compared to that of corresponding receptors expressed in human cells. Several parameters affecting receptor expression in insect cells were tested: (1) a maximum of the receptor expression was reached after 40 h post infection (p.i.). (2) for maximal receptor production cells had to be coinfected (a) with an equal ratio of viruses and (b) at a minimal multiplicity of infection (moi.) of 3 each. A dual baculovirus expression vector containing both the β2and γ2 subunit was constructed. It was used in coinfection experiments with a virus expressing the α5 subunit. Infection with these two viruses increased the percentage of cells containing functional receptors as measured by electrophysiological analysis compared to a infection with three viruses.