COMPETITIVE ANTAGONISTS BRIDGE THE ALPHA-GAMMA SUBUNIT INTERFACE OF THE ACETYLCHOLINE-RECEPTOR THROUGH QUATERNARY AMMONIUM-AROMATIC INTERACTIONS

We recently demonstrated that conserved tyrosines Tyr(198) of the alph a subunit and Tyr(117) of the gamma subunit of the acetylcholine recep tor stabilize binding of the curariform antagonist dimethyl-d-tubocura rine (DMT). To test the hypothesis that DMT interacts directly with th ese tyrosines, and therefore bridges the alpha-gamma subunit interface , we introduced point mutations into these key positions and expressed one or both mutant subunits in alpha(2) beta gamma(2) acetylcholine r eceptors in 293 HEK cells. Binding of DMT, measured by competition aga inst the initial rate of I-125-alpha-bungarotoxin binding, shows high affinity for aromatic mutations, reduced affinity for polar mutations, and lowest affinity for arginine mutations. Similar side chain depend ences were observed for both Tyr(alpha 198) and Tyr(gamma 117), indica ting interaction of these residues with two symmetrical chemical group s in DMT. Two more bisquaternary antagonists, pancuronium and gallamin e, show side chain dependences similar to that of DMT, indicating that the primary stabilizing interactions are aromatic-quaternary in both subunits. For the rigid ligands DMT and pancuronium, co-expressing mut ant alpha and gamma subunits revealed independent contributions by eac h determinant, but strict independence was not observed for the flexib le ligand gallamine. The free energy contributed by each aromatic quat ernary interaction was estimated to be 2-4 kcal/mol, as determined fro m the free energy difference between aromatic and alkyl hydroxyl mutat ions. Our results suggest that bis-quaternary competitive antagonists bridge the alpha-gamma subunit interface by fitting into a pocket boun ded by tyrosines at positions 198 of the alpha subunit and 117 of the gamma subunit.