Absalom_2013_J.Biol.Chem_288_26521

The alpha4beta2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the alpha4beta2 nAChR, (alpha4)2(beta2)3 and (alpha4)3(beta2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and alpha4beta2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the alpha4-alpha4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an alpha4beta2 receptor containing an alpha4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (alpha4)3(beta2)2 stoichiometry, indicating that MLA binds to the alpha4-alpha4 interface of the (alpha4)3(beta2)2 and providing direct evidence of ligand binding to the alpha4-alpha4 interface. Consistent with other studies, we propose that the alpha4-alpha4 interface is a structural target for potential therapeutics that modulate (alpha4)3(beta2)2 nAChRs.