Estimating Agonist Efficacy by Engineering Acetylcholine Receptor-Channels

Abstract

Nicotinic acetylcholine receptor-channels (AChRs) are allosteric proteins that spontaneously isomerize (‘gate’) between a resting, closed-channel conformation (R) and an active, open-channel one (R∗). Different agonists promote the R-R∗ conformational change to different extents because they experience different degrees of affinity change at the agonist binding site. The affinity ratio, R/R∗, is the parameter that determines the gating equilibrium constant E2 and, hence, efficacy. The most accurate way to estimate E2 is by using single-channel, patch-clamp electrophysiology. However, we wanted to examine ligand/receptor combinations that have gating rate constants either too-fast or too-slow for such analysis. In this study we describe and use a stepwise perturbation method involving mutations, depolarization and partial agonists to circumvent problems associated with the bandwidth limitations of patch electrophysiology and channel block by agonist molecules, in order to determine accurately rate constants at single channel level of seven agonists with varying efficacies. Our estimates for known agonists, obtained by using this method are in excellent agreement with previously published values. This shows that the effects on gating from the perturbations we employed are the same regardless of the activating agonist and, conversely, the effects of the agonist are independent of the perturbations. The ‘improved’ gating constant estimates for ACh on wild type, adult mouse neuromuscular AChRs (−100 mV, 23 oC, expressed in HEK cells) are: opening rate: 66,000 s−1, closing rate: 2300 s−1, and equilibrium: 29.