Acetylcholine receptors (AChRs) are ligand-gated ion channels involved in vertebrate neuromuscular transmission. Binding of acetylcholine (ACh, the endogenous neurotransmitter) increases the equilibrium constant of the ‘gating’ isomerization and the probability that ion channel domain adopts an ion-permeable conformation. The ratio of the isomerization equilibrium constants with two and zero bound agonist molecules (E2/E0) is equal to the ratio of agonist affinities in the inactive vs. active conformations of the protein, at two identical binding sites [(Kd/Jd)2]. We define R=Kd/Jd. Different agonists have different efficacies because they have different R values (E0=6.5E-7). For ACh (=100 mV, 23 oC, mouse α2βδe), E2=28.2 and R=6500. For choline, E2=0.05 and R=270. For tetramethylammonium and carbachol, E2=6.8 and R=3300. As part of a larger project to engineer the AChR transmitter binding site, we have measured E2 and R values for a series of small molecular fragments. The goal is to generate an atomic map of the isomerization-induced energy changes at the ligand [kcal/mol=0.59ln(R)]. We first studied a series of five- and six- membered N-containing rings (fragments of nicotine). In the nicotine-bound AChBP structure, the pyrrolidine ring of the ligand is at the center of an aromatic ‘box’. Systematic substitutions of the atoms of this ring are being made and the corresponding R-values are being determined. Preliminary work suggests that the following small molecules activate wild-type AChRs: 1,1-Dimethyl pyrrolidin-1-ium (E2∼0.26, R∼630), 1,1-Dimethyl Thiazolidin-1-ium (E2∼0.68, R∼1016) and 4,4-Dimethyl morpholinium (E2∼0.3, R∼675).