Potentiation of a neuronal nicotinic receptor via pseudo-agonist site

Neuronal nicotinic receptors containing α4 and β2 subunits assemble in two pentameric stoichiometries, (α4)₃(β2)₂ and (α4)₂(β2)₃, each with distinct pharmacological signatures; (α4)₃(β2)₂ receptors are strongly potentiated by the drug NS9283, whereas (α4)₂(β2)₃ receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)₃(β2)₂ receptors, we show that mutations at either the principal face of the β2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the β2-α4 pseudo-agonist sites, the α4-α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the β2 subunit at either β2-α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4-α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the β2-α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)₃(β2)₂ receptor, the findings have implications for structure-guided drug design.