Energetics of gating at the apo-acetylcholine receptor transmitter binding site

Acetylcholine receptor channels switch between conformations that have a low versus high affinity for the transmitter and conductance for ions (R<-->R*; gating). The forward isomerization, which begins at the transmitter binding sites and propagates approximately 50 A to the narrow region of the pore, occurs by approximately the same sequence of molecular events with or without agonists present at the binding sites. To pinpoint the forces that govern the R versus R* agonist affinity ratio, we measured single-channel activation parameters for apo-receptors having combinations of mutations of 10 transmitter binding site residues in the alpha (Y93, G147, W149, G153, Y190, C192, and Y198), epsilon (W55 and P121), or delta (W57) subunit. Gating energy changes were largest for the tryptophan residues. The alphaW149 energy changes were coupled with those of the other aromatic amino acids. Mutating the aromatic residues to Phe reduces the R/R* equilibrium dissociation constant ratio, with alphaY190 and alphaW149 being the most sensitive positions. Most of the mutations eliminated long-lived spontaneous openings. The results provide a foundation for understanding how ligands trigger protein conformational change.