ACh and NS9283 sensitivity and potential stoichiometry of receptors from concatenated pentameric constructs with short first linkers. 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 in which x denotes an α4 or an α4^VFL subunit in the fifth construct position. Data from n = 5–15 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-3a-α-β-α-α (top) or β-3a-α-β-α-α^VFL (bottom). Bars above the traces indicate the 30-s application time and concentrations of applied compounds. (D) Based on the NS9283 responses observed with x = α4^VFL in B, the receptor pool consists mainly of pentamers with the linkers assembled in the counterclockwise orientation when viewed from the synaptic cleft. 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.