Human alpha 6 beta 4 Nicotinic Acetylcholine Receptor: Heterologous Expression and Agonist Behavior Provide Insights into the Immediate Binding SiteS

Study of a6#4 nicotinic acetylcholine receptors (nAChRs) as a pharmacological target has recently gained interest because of their involvement in analgesia, control of catecholamine secretion, dopaminergic pathways, and aversive pathways. However, an ex-tensive characterization of the human a6#4 nAChRs has been viti-ated by technical difficulties resulting in poor receptor expression. In 2020, Knowland and collaborators identified BARP (#-anchoring and regulatory protein), a previously known voltage-gated cal-cium channel suppressor, as a novel human a6#4 chaperone. Here, we establish that co-expression of human BARP with hu-man a6#4 in Xenopus oocytes, resulted in the functional expres-sion of human a6#4 receptors with acetylcholine-elicited currents that allow an in-depth characterization of the receptor using two electrode voltage-clamp electrophysiology together with diverse agonists and receptor mutations. We report: 1) an extended pharmacological characterization of the receptor, and 2) key resi-dues for agonist-activity located in or near the first shell of the binding pocket. SIGNIFICANCE STATEMENTThe human a604 nicotinic acetylcholine receptor has attained in-creased interest because of its involvement in diverse physiological processes and diseases. Although recognized as a pharmacologi-cal target, development of specific agonists has been hampered by limited knowledge of its structural characteristics and by challenges in expressing the receptor. By including the chaperone #-anchoring and regulatory protein for enhanced expression and employing dif-ferent ligands, we have studied the pharmacology of a604, provid-ing insight into receptor residues and structural requirements for ligands important to consider for agonist-induced activation.