Molecular Basis for Differential Sensitivity of α-Conotoxin RegIIA at Rat and Human Neuronal Nicotinic Acetylcholine Receptors

alpha-Conotoxins, as nicotinic acetylcholine receptor (nAChR) antagonists, are powerful tools for dissecting biologic processes and guiding drug development. The alpha 3 beta 2 and alpha 3 beta 4 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 alpha 4/7-conotoxin RegIIA, a disulfide-bonded 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 alpha 3 beta 2 (IC50 = 10.7 nM), whereas a 70-fold lower potency was observed at human alpha 3 beta 2 nAChR (IC50 = 704.1 nM). Conversely, there were no species-specific differences in sensitivity to RegIIA at the alpha 3 beta 4 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 alpha 3 subunit corresponding to a proline on the human subunit. These findings reveal a novel species-and subunit-specific receptor-antagonist interaction.