ACh and NS9283 sensitivity and potential stoichiometry of receptors from concatenated pentameric constructs with the α4–α4 site in the second to third construct positions. X. laevis oocytes were subjected to two-electrode voltage-clamp electrophysiology. Electrophysiological data were evaluated as described in Materials and methods; also see Fig. 1. (A and B) ACh (A) and NS9283 (B) CRRs were obtained from oocytes injected with the indicated pentameric constructs, where x and y denote an α4 or an α4^VFL subunit in the second and third construct positions. Data from n = 9–23 experiments are depicted as means ± SEM as a function of the ACh or NS9283 concentration, and regression results are presented in Table 2. Data for wild-type receptors from monomeric subunits in Fig. 1 are indicated as dashed lines. (C) Representative traces illustrating NS9283 responses at oocytes injected with β-21a-α-α-β-α, β-21a-α-α^VFL-β-α, or β-21a-α^VFL-α-β-α. Bars above the traces indicate the 30-s application time and concentrations of applied compounds. (D) For the β-21a-α-α^VFL-β-α receptor (α4^VFL subunit in the third construct position), NS9283-sensitive receptors originate from assembly of the linkers in a clockwise orientation (top right). However, for the β-21a-α^VFL-α-β-α receptor (α4^VFL subunit in the second construct position), NS9283-sensitive receptors are assembled with the linkers in a counterclockwise orientation (bottom left). Note that in the receptor illustrations, the first construct linkers are indicated with bold purple font, and specific linker sequences are shown in Table 3.