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

• Published in: The Journal of biological chemistry Vol. 290; no. 37; pp. 22747 - 22758
• Main Authors: Kudryavtsev, Denis S., Shelukhina, Irina V., Son, Lina V., Ojomoko, Lucy O., Kryukova, Elena V., Lyukmanova, Ekaterina N., Zhmak, Maxim N., Dolgikh, Dmitry A., Ivanov, Igor A., Kasheverov, Igor E., Starkov, Vladislav G., Ramerstorfer, Joachim, Sieghart, Werner, Tsetlin, Victor I., Utkin, Yuri N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.09.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Binding Sites; Cell Line, Tumor; Cobra Neurotoxin Proteins - chemistry; Cobra Neurotoxin Proteins - pharmacology; Conotoxins - chemistry; Conotoxins - pharmacology; Cys-loop receptor; Elapidae; electrophysiology; fluorescence; GABAA receptor; Mice; molecular dynamics; Molecular Dynamics Simulation; molecular modeling; neurotoxin; Protein Structure, Secondary; Receptors, GABA-A - chemistry; Receptors, GABA-A - genetics; Receptors, GABA-A - metabolism; Signal Transduction; snake venom; three finger toxin; Cys-loop receptor; three finger toxin; electrophysiology; fluorescence; molecular modeling; neurotoxin; snake venom; molecular dynamics; GABAA receptor
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.648824

Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the α1β3γ2 receptor; and at 10 μm α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nm) and less potently inhibited α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABAARs. The α1β3γ2 receptor was also inhibited by some other three-finger toxins, long α-neurotoxin Ls III and nonconventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx-binding sites overlap with the orthosteric sites at the β/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR 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 α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accommodating under loop C of the receptors. Background: Different snake venom three-finger toxins interact with various receptors, channels, and membranes. Results: Here, we demonstrate that GABAA receptors are inhibited by α-cobratoxin, other long chain α-neurotoxins, nonconventional toxin from Naja kaouthia, and α-conotoxin ImI. Conclusion: Some toxin blockers of nicotinic acetylcholine receptors also inhibit GABAA receptors. Significance: Three-finger toxins offer new scaffolds for the design of GABAA receptor effectors.