Linking the Acetylcholine Receptor-Channel Agonist-Binding Sites with the Gate

The gating isomerization of neuromuscular acetylcholine receptors links the rearrangements of atoms at two transmitter-binding sites with those at a distant gate region in the pore. To explore the mechanism of this reversible process, we estimated the gating rate and equilibrium constants for receptors with point mutations of alpha-subunit residues located between the binding sites and the membrane domain (N95, A96, Y127, and 149). The maximum energy change caused by a side-chain substitution at alpha A96 was huge (similar to 8.6 kcal/mol, the largest value measured so far for any alpha-subunit amino acid). A Phi-value analysis suggests that alpha A96 experiences its change in energy (structure) approximately synchronously with residues alpha Y127 and alpha 149, but after the agonist molecule and other residues in loop A. Double mutant-cycle experiments show that the energy changes at alpha A96 are strongly coupled with those of alpha Y127 and alpha 149. We identify a column of mutation-sensitive residues in the alpha-subunit that may be a pathway for energy transfer through the extracellular domain in the gating isomerization.