α4β2 nAChR stoichiometry and functional effects of ACh and NS9283. X. laevis oocytes were injected with cRNA mixtures of α4 and β2 or α4VFL and β2 subunits in 10:1 ratios and subjected to two-electrode voltage-clamp electrophysiology as described in Materials and methods. The 10:1 cRNA ratios were used to ensure uniform populations of (α4)3(β2)2 and (α4VFL)3(β2)2 receptors. Data for α4 and β2 injected in a 1:4 ratio ((α4)2(β2)3 receptor) are from Harpsøe et al. (2011). (A) Functional α4β2 nAChRs can express in 2α:3β or 3α:2β stoichiometries (left and middle, respectively). The stoichiometry affects the total number of ACh-binding sites, as the 3α:2β stoichiometry contains an additional site in the α4–α4 interface. Furthermore, NS9283 binds with high selectivity in the α4–α4 site, where it behaves as an agonist. Upon mutating three amino acids in the complementary face of the α4 subunit to give α4VFL, ACh sensitivity is increased in the α4VFL–α4VFL site, and NS9283 binding is lost (right). (B) ACh CRRs. Baseline-subtracted, ACh-evoked peak current amplitudes (I) for the indicated receptors were fitted to the Hill equation by nonlinear regression and normalized to the maximal fitted values (Imax fit ACh). Normalized responses are depicted as means ± SEM as a function of the ACh concentrations, and they are fitted to biphasic equations with a fixed bottom of 0 and a Hill slope of 1. Data were obtained from n = 9–14 experiments, and regression results are presented in Table 1. Data for the (α4)2(β2)3 receptor are from Harpsøe et al. (2011). (C) NS9283 CRRs. NS9283 enhancement of ACh-evoked currents was evaluated for (α4)3(β2)2 and (α4VFL)3(β2)2 receptors by coapplication with a submaximal control concentration of ACh (10 µM). Baseline-subtracted peak current amplitudes (I) were expressed as percent change from IACh_control and are depicted as means ± SEM as a function of the NS9283 concentration. Data points were fitted by nonlinear regression to the Hill equation with a fixed bottom of 0 and a Hill slope of 1. Data were obtained from n = 13–16 experiments, and regression results are presented in Table 1. Data for the (α4)2(β2)3 receptor are from Timmermann et al. (2012). (D) Hypothetically, injection of a cRNA mixture of α4, α4VFL, and β2 into oocytes could yield eight different receptors in the 3α:2β stoichiometry. Using NS9283 as a marker, these can be divided into those that are sensitive and those that are insensitive, depending on whether the α4VFL subunit is participating in the complementary position of the α4–α4 interface.
