Invariant aspartic acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding

We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant aD89 forms a highly conserved interdomain contact near αT148, αW149, and αT150. Patch-clamp recordings show that the mutation αD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither αT148L, αT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor αD89. However substituting a negative charge at αT148, but not at αT150, counteracts the effect of αD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligaiid binding site, these results implicate main chain amide groups in the domain harboring αW149 as principal hydrogen bond donors for αD89. The specific effect of αD89N on ACh association suggests that interdomain hydrogen bonding positions αW149 for optimal interaction with ACh.