Effects of Mutations of Proline Residues Located in the Complementary Subunit of the Acetylcholine Receptor Transmitter Binding Site

Abstract

Nicotinic acetylcholine receptor-channels (AChRs) are allosteric proteins that isomerize between a non-conducting/low-affinity and a conducting/high-affinity conformation. Each neuromuscular AChR has two transmitter binding sites located at the interface between adjacent subunits (αeor αδ.We studied the gating properties of AChRs with mutations at the vicinal prolines located at these interfaces. In both cases, mutations of the N-terminal residue had little effect but those of the C-terminal residue (eP121 or δP123) decreased substantially the open channel probability (500 μM ACh). We estimated the unliganded (E0) and diliganded (E2) gating equilibrium constants for mutations of these two prolines from single-channel currents (30 mM ACh, +100mV). All of the tested substitutions decreased both gating constants. The slope of the rate-equilibrium plot (Φ)for eP121/δP123 was ∼0.96 (diliganded) and ∼0.70 (unliganded). Some of the mutations caused a large decrease in the closed/open equilibrium dissociation constant ratio for ACh (λACh) while others had more modest effects. Probing the effects of eP121 and δP123 mutations should allow an estimation of the binding and gating properties of each of the two transmitter binding sites, separately.