Snake neurotoxin α-bungarotoxin is an antagonist at native GABA(A) receptors

Neuropharmacology. 2015 Jun:93:28-40. doi: 10.1016/j.neuropharm.2015.01.001. Epub 2015 Jan 26.

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.

Keywords: Dentate gyrus; Electrophysiology; GABA receptor; Immunofluorescence; Nicotinic acetylcholine receptor; α-bungarotoxin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Brain / cytology*
  • Bungarotoxins / metabolism
  • Bungarotoxins / pharmacology*
  • Embryo, Mammalian
  • GABA Antagonists / pharmacology*
  • Humans
  • In Vitro Techniques
  • Inhibitory Postsynaptic Potentials / drug effects
  • Male
  • Membrane Potentials / drug effects
  • Mice
  • Mice, Inbred C57BL
  • Neurons / drug effects*
  • Nicotinic Antagonists / pharmacology
  • Patch-Clamp Techniques
  • Protein Binding / drug effects
  • Rats
  • Receptors, GABA-A / metabolism
  • Receptors, Nicotinic / genetics
  • Receptors, Nicotinic / metabolism
  • Tubocurarine / pharmacology
  • gamma-Aminobutyric Acid / pharmacology

Substances

  • Bungarotoxins
  • GABA Antagonists
  • Nicotinic Antagonists
  • Receptors, GABA-A
  • Receptors, Nicotinic
  • gamma-Aminobutyric Acid
  • Tubocurarine