Functional characterization of mutations on PKA putative sites in the α4 subunit with nicotine used as agonist. (A) Family of nicotine-induced macroscopic currents for mutation α4S471Dβ2 and the wild-type α4β2 nAChR. Calibration bars are shown for all family of currents, horizontal bars indicate time (5 s) and vertical bars indicate the inward current (500 nA). (B) Dose-response relationships obtained by voltage-clamp using nicotine as agonist. Nicotine dose-response curves were determined using seven nicotine concentrations (0.1, 1, 3, 10, 30, 100 and 300 M). The responses were normalized to the maximum response (I/Imax). (C) Comparison of the nicotine-induced macroscopic peak currents of the mutations and wild-type receptor shown in nA (n=6-17) (** p<0.005).