Dissecting Kinetic Differences in Acetylcholine Receptors Incorporating an Ancestral Subunit.

Abstract

At the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) convert chemical stimuli into electrical signals. They are heteropentameric membrane protein complexes assembled from four evolutionary related subunits (two α subunits, and one each of the β-, δ-, and ε-subunits), arranged around a central ion-conducting pore, which is regulated by the neurotransmitter acetylcholine. Understanding how the binding of acetylcholine leads to channel opening is of fundamental importance. While it is known that channel opening results from a global conformational change involving the cooperative action of all five subunits, how the subunits achieve this cooperativity is unclear. Our hypothesis is that this subunit cooperation is maintained through coevolution of the subunits, and thus studies of subunit coevolution can provide insight into subunit cooperativity. Using an ancestral reconstruction approach, combined with single-molecule patch clamp electrophysiology, we have begun dissecting the mechanistic consequences of preventing coevolution of the acetylcholine receptor β-subunit. This approach has allowed us to identify new amino acid determinants of acetylcholine receptor function.