Neurotoxins from Snake Venoms and α-Conotoxin ImI Inhibit Functionally Active Ionotropic γ-Aminobutyric Acid (GABA) Receptors

Ionotropic receptors of gamma-aminobutyric acid (GABA(A)R) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of alpha-cobratoxin (alpha-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the alpha 1 beta 3 gamma 2 receptor; and at 10 mu M alpha-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nM) and less potently inhibited alpha 1 beta 2 gamma 2 approximate to alpha 2 beta 2 gamma 2 > alpha 5 beta 2 gamma 2 > alpha 2 beta 3 gamma 2 and alpha 1 beta 3 delta GABA(A)Rs. The alpha 1 beta 3 gamma 2 receptor was also inhibited by some other three-finger toxins, long alpha-neurotoxin Ls III and nonconventional toxin WTX. alpha-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive alpha-Ctx action. Fluorescent alpha-Ctx, however, could be displaced by muscimol indicating that most of the alpha-Ctx-binding sites overlap with the orthosteric sites at the beta/alpha subunit interface. Modeling and molecular dynamic studies indicated that alpha-Ctx or alpha-bungarotoxin seem to interact with GABA(A)R in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with alpha-conotoxin ImI and a chimeric Naja oxiana alpha-neurotoxin indicating that the major role in alpha-Ctx binding to GABA(A)R is played by the tip of its central loop II accommodating under loop C of the receptors.