Snake neurotoxin a-bungarotoxin is an antagonist at native GABAA receptors

The snake neurotoxin alpha-bungarotoxin (alpha-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, alpha-Bgtx is also used as an imaging tool for fluorophore-labelling studies, and given the structural conservation within the pentameric ligand-gated ion channel family, we assessed whether alpha-Bgtx could bind to recombinant and native gamma-aminobutyric type-A receptors (GABA(A)Rs). Applying fluorophore-linked alpha-Bgtx to recombinant alpha x beta 1/2 gamma 2 GABA(A)Rs expressed in HEK-293 cells enabled clear cell-surface labelling of alpha 2 beta 1/2 gamma 2 contrasting with the weaker staining of alpha 1/4 beta 1/2 gamma 2, and no labelling for alpha 3/5/6 beta 1/2 gamma 2. The labelling of alpha 2 beta 2 gamma 2 was abolished by bicuculline, a competitive antagonist at GABA(A)Rs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABA(A)Rs. Labelling by alpha-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor beta-alpha subunit interface forms part of the alpha-Bgtx binding site. Using whole-cell recording, high concentrations of alpha-Bgtx (20 mu M) inhibited GABA-activated currents at all alpha x beta 2 gamma 2 receptors examined, but at lower concentrations (5 mu M), alpha-Bgtx was selective for alpha 2 beta 2 gamma 2. Using alpha-Bgtx, at low concentrations, permitted the selective inhibition of alpha 2 subunit-containing GABA(A)Rs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, alpha-Bgtx can act as an inhibitor at recombinant and native GABA(A)Rs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus. (C) 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).