Abstract
The gamma-aminobutyric acid type A receptor (GABA(A)R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent alphabetagamma GABA(A)R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing delta subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of delta-containing GABA(A)Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed delta-containing GABA(A)Rs, we inserted the alpha-bungarotoxin binding site tag in the alpha(4), beta(2), and delta subunit N termini. An enhanced green fluorescent protein tag was also inserted into the beta(2) subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged alpha(4)beta(2)delta GABA(A)Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent alpha-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA(A)Rs. The results demonstrate that the subunit stoichiometry of alpha(4)beta(2)delta GABA(A)Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of delta subunit cDNA transfected increased delta subunit incorporation into surface receptors with a concomitant decrease in beta(2) subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA(A)R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed alpha(4)beta(2)delta GABA(A)Rs. Successful bungarotoxin binding site tagging of GABA(A)R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA(A)R trafficking in live cells.
Highlights
GABAARs play a key role, mediating tonic inhibition and the molecular actions of endogenous neuroactive steroids, alcohol, and several anesthetic agents [1]
We demonstrate that the subunit stoichiometry of heterologously expressed ␣42␦ GABAARs is highly influenced by the ratio of subunit cDNAs used in a transfection
As the amount of ␦ cDNA transfected increased, ␦ subunit incorporation into surface GABAARs increased with a concomitant decrease in 2 subunit incorporation (Fig. 5C)
Summary
GABAARs play a key role, mediating tonic inhibition and the molecular actions of endogenous neuroactive steroids, alcohol, and several anesthetic agents [1]. We examined whether the ratio of subunit cDNA in a transfection influences the subunit stoichiometry of cell surface ␣42␦ GABAARs expressed in HEK293T cells. BBS-tagged ␣42␦ GABAARs were heterologously expressed in HEK293T cells using a variety of subunit cDNA ratios.
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