Energy measurements from electrophysiology. The α−δ site of the adult-type human AChRs was studied in isolation after disabling the α−ε site by adding the mutation εP121R. (a) Gating with CCh. Top: Gating with CCh. [CCh] = 20 mM (to fully saturate the α−δ site) and V_m = +70 mV (to reduce channel block by CCh). Openings (top) are clustered; intercluster gaps reflect desensitization and intracluster intervals mainly reflect ^AR⇄^AR* gating. Intracluster interval duration histograms (bottom) and an example cluster. (b) CCh binding. Association and dissociation rate constants were estimated by fitting across [CCh] (see Materials and methods). (c and d) Ebx gating and binding. Free energies were calculated from the equilibrium constants estimated from the forward/backward rate constant ratios.