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Address correspondence to Steven M. Sine, Department of Physiology and Biophysics, Mayo Clinic College of Medicine, 200 First St., SW, MSB 1-35, Rochester, MN 55905. Fax: (507) 284-9420; email: sine{at}mayo.edu
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 
D89 forms a highly conserved interdomain contact near T148, W149, and T150. Patch-clamp recordings show that the mutation D89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither T148L, T150A, 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 D89. However substituting a negative charge at T148, but not at T150, counteracts the effect of D89N, 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 ligand binding site, these results implicate main chain amide groups in the domain harboring W149 as principal hydrogen bond donors for D89. The specific effect of D89N on ACh association suggests that interdomain hydrogen bonding positions W149 for optimal interaction with ACh.
Key Words: acetylcholine receptor • ligand binding site • single channel kinetics • hydrogen bond • structural model
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