Proline Residues at the Nicotinic Acetylcholine Transmitter Binding Sites

Abstract

The two neuromuscular acetylcholine receptor (AChR) transmitter binding sites are located in the extracellular domain of the protein at the α-e and α-δ subunit interfaces. At each site there are two vicinal prolines in the complimentary e/δ subunit (ProD1 and ProD2). We estimated for dozens of mutations of e/δ ProD1 and ProD2 (single-channels, HEK cells, +100mV, 23 oC) the gating energies with ACh molecules bound (G2 and G1) and without any agonist (G0). From these we calculated the energy for gating arising from the affinity change for ACh (GB1+GB2=G2-G0). Mutation of ProD2 had larger effects than of ProD1. The eProD2-L mutation, which causes a congenital myasthenic syndrome, increases αGB (+3.4 kcal/mol, throughout) but does not change G0. For the same mutation in the δ subunit the change in GB was smaller (+1.9). All of the side chain substitutions at e/δProD2 increased both GB and G0 compared to the wild-type (which has the values −5.1 and +8.4). The largest increase in αGB was for eProD2-R (+4.7) and in G0 for δProD2-Q (+2.5). We used mutant cycle analysis to estimate αGB¬ coupling between side chains. The two ProD2 residues interact weakly with each other (−0.6), but there is a strong interaction between eProD2-αGlyB1 (+2.7) but not between δProD2- αGlyB2. It is possible that a concerted, Pro-Gly ‘switch' at α-e occurs near to the onset of channel-opening. We engineered AChRs having only one functional binding site and quantified GB(ACh) for either the α-e (by using the δProD2-R knockout or α-δ (by using the eProD2-R knockout) site. The sites provide +5.2 and −4.7 kcal/mol with ACh. One-site AChRs will be useful for studying the energy sources for gating by agonists at the two different binding sites.