Dx. Fu et Sm. Sine, COMPETITIVE ANTAGONISTS BRIDGE THE ALPHA-GAMMA SUBUNIT INTERFACE OF THE ACETYLCHOLINE-RECEPTOR THROUGH QUATERNARY AMMONIUM-AROMATIC INTERACTIONS, The Journal of biological chemistry, 269(42), 1994, pp. 26152-26157
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.