a-Conotoxins, as nicotinic acetylcholine receptor (nAChR) antagonists, are powerful tools for dissecting biologic processes and guiding drug development. The a3b2 and a3b4 nAChR subtypes are expressed in the central and peripheral nervous systems and play a critical role in various pathophysiological conditions ranging from nicotine addiction to the development and progression of lung cancer. Here we used the a4/7-conotoxin RegIIA, a disulfidebonded peptide from the venom of Conus regius, and its analog [N11A,N12A]RegIIA to probe the specific pharmacological properties of rat and human nAChR subtypes. nAChR subtypes were heterologously expressed in Xenopus oocytes and two-electrode voltage clamp recordings used to investigate the effects of the peptides on nAChR activity. RegIIA potently inhibited currents evoked by acetylcholine (ACh) at rat a3b2 (IC50 5 10.7 nM), whereas a 70-fold lower potency was observed at human a3b2 nAChR (IC50 5 704.1 nM). Conversely, there were no speciesspecific differences in sensitivity to RegIIA at the a3b4 nAChR. Receptor mutagenesis and molecular dynamics studies revealed that this difference can be attributed primarily to a single amino acid change: Glu198 on the rat a3 subunit corresponding to a proline on the human subunit. These findings reveal a novel species- and subunit-specific receptor-antagonist interaction.
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