title: Snake neurotoxin α-bungarotoxin is an antagonist at native GABA(A) receptors.
abstract: The snake neurotoxin α-bungarotoxin (α-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, α-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 α-Bgtx could bind to recombinant and native γ-aminobutyric type-A receptors (GABAARs). Applying fluorophore-linked α-Bgtx to recombinant αxβ1/2γ2 GABAARs expressed in HEK-293 cells enabled clear cell-surface labelling of α2β1/2γ2 contrasting with the weaker staining of α1/4β1/2γ2, and no labelling for α3/5/6β1/2γ2. The labelling of α2β2γ2 was abolished by bicuculline, a competitive antagonist at GABAARs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABAARs. Labelling by α-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor β-α subunit interface forms part of the α-Bgtx binding site. Using whole-cell recording, high concentrations of α-Bgtx (20 μM) inhibited GABA-activated currents at all αxβ2γ2 receptors examined, but at lower concentrations (5 μM), α-Bgtx was selective for α2β2γ2. Using α-Bgtx, at low concentrations, permitted the selective inhibition of α2 subunit-containing GABAARs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, α-Bgtx can act as an inhibitor at recombinant and native GABAARs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus.
authors: Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors: Saad
authors: S
authors: Hannan
authors: Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors: Martin
authors: M
authors: Mortensen
authors: Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom. Electronic address: t.smart@ucl.ac.uk.
authors: Trevor G
authors: TG
authors: Smart
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology*; D002038:Bungarotoxins / Q000378:metabolism / Q000494:pharmacology*; D004622:Embryo, Mammalian; D018756:GABA Antagonists / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D053444:Inhibitory Postsynaptic Potentials / Q000187:drug effects; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects*; D018733:Nicotinic Antagonists / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism; D014403:Tubocurarine / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D002038:Bungarotoxins; D018756:GABA Antagonists; D018733:Nicotinic Antagonists; D011963:Receptors, GABA-A; D011978:Receptors, Nicotinic; D005680:gamma-Aminobutyric Acid; D014403:Tubocurarine
keywords: Dentate gyrus; Electrophysiology; GABA receptor; Immunofluorescence; Nicotinic acetylcholine receptor; α-bungarotoxin
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