Reconstructing identical agonist sites in the muscle-type nicotinic acetylcholine receptor.

Abstract

Muscle-type acetylcholine receptors are heteropentameric ion channels composed of four evolutionarily related subunits. The adult form of the receptor assembles with a counter-clockwise α-ε-α-δ-β arrangement with all five subunits arranged pseudosymetrically around a central ion-conducting pore. Efficient opening is achieved upon binding of acetylcholine to its two extracellular binding sites. Acetylcholine binds at two subunit interfaces each made up of a principal α-subunit, and a complementary δ- or ε-subunit. Amino acid differences between complementary δ- or ε-subunits confer non-equivalence to the two binding sites. Using an ancestral protein reconstruction approach, we resurrect a subunit ancestral to the δ- and ε-subunits that can replace both modern-day subunits. By combining radioligand binding experiments with single-channel electrophysiology we show that acetylcholine receptors incorporating this ancestral subunit have identical binding sites, and then discuss the functional consequences of this equivalence to AChR function.