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J. Biol. Chem., Vol. 269, Issue 42, 26152-26157, Oct, 1994
DX Fu and SM Sine
We recently demonstrated that conserved tyrosines Tyr198 of the alpha
subunit and Tyr117 of the gamma subunit of the acetylcholine receptor
stabilize binding of the curariform antagonist dimethyl-d-tubocurarine
(DMT). To test the hypothesis that DMT interacts directly with these
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 receptors in 293
HEK cells. Binding of DMT, measured by competition against the initial rate
of 125I-alpha-bungarotoxin binding, shows high affinity for aromatic
mutations, reduced affinity for polar mutations, and lowest affinity for
arginine mutations. Similar side chain dependences were observed for both
Tyr alpha 198 and Tyr gamma 117, indicating interaction of these residues
with two symmetrical chemical groups in DMT. Two more bisquaternary
antagonists, pancuronium and gallamine, 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 mutant alpha and gamma subunits revealed
independent contributions by each determinant, but strict independence was
not observed for the flexible ligand gallamine. The free energy contributed
by each aromatic- quaternary interaction was estimated to be 2-4 kcal/mol,
as determined from the free energy difference between aromatic and alkyl
hydroxyl mutations. Our results suggest that bis-quaternary competitive
antagonists bridge the alpha-gamma subunit interface by fitting into a
pocket bounded by tyrosines at positions 198 of the alpha subunit and 117
of the gamma subunit.
Competitive antagonists bridge the alpha-gamma subunit interface of the acetylcholine receptor through quaternary ammonium-aromatic interactions
Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905.
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