NMR analysis of the interaction of ethanol with the nicotinic acetylcholine receptor

Ethanol exerts its actions in the central and peripheral nervous systems through the direct interactions with several proteins, including ligand-gated ion channels such as the nicotinic acetylcholine receptor (nAChR). The binding interaction between ethanol and sodium cholate solubilized nicotinic acetylcholine receptor protein can be detected through either NMR line broadening or T 1 titration. In this paper, we examine the use of weighted Navon T 1p analysis of T 1 titration data for the estimation of the dissociation constant of ethanol for the nAChR. We show that Navon T 1p analysis underestimates binding affinity. The application of rigorous limits for confidence intervals within a nonlinear regression analysis of this data provides a best estimate of K d = 55 mM at 4 °C. within an unsymmetrical 90% confidence interval of [0.5, 440 mM]. Accordingly, the best estimate of the binding free energy is DG 0 = –5.4 Kcal/mole within a 90% confidence interval of [–8.0, -4.3 Kcal/mole], relative to conventional standard states.