We recently demonstrated that conserved tyrosines Tyr198 of the α subunit and Tyr117 of the γ 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 α-γ subunit interface, we introduced point mutations into these key positions and expressed one or both mutant subunits in α2βγ2 acetylcholine receptors in 293 HEK cells. Binding of DMT, measured by competition against the initial rate of 125I-α-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(α198) and Tyr(γ117), 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 α and γ 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 α-γ subunit interface by fitting into a pocket bounded by tyrosines at positions 198 of the α subunit and 117 of the γ subunit.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Oct 21 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology