Function of Interfacial Prolines at the Transmitter-binding Sites of the Neuromuscular Acetylcholine Receptor

• Published in: The Journal of biological chemistry Vol. 288; no. 18; pp. 12667 - 12679
• Main Authors: Gupta, Shaweta, Purohit, Prasad, Auerbach, Anthony
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.05.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Binding Sites; Cholinergic Receptor; Cys Loop Receptors; Gating; HEK293 Cells; Humans; Ion Channels; Molecular Biophysics; Mutation, Missense; Proline - chemistry; Proline - genetics; Proline - metabolism; Receptors, Nicotinic - chemistry; Receptors, Nicotinic - genetics; Receptors, Nicotinic - metabolism; Synapses; Cholinergic Receptor; Cys Loop Receptors; Ion Channels; Gating; Synapses
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.443911

The neuromuscular acetylcholine (ACh) receptor has two conserved prolines in loop D of the complementary subunit at each of its two transmitter-binding sites (α-ϵ and α-δ). We used single-channel electrophysiology to estimate the energy changes caused by mutations of these prolines with regard to unliganded gating (ΔG0) and the affinity change for ACh that increases the open channel probability (ΔGB). The effects of mutations of ProD2 (ϵPro-121/δPro-123) were greater than those of its neighbor (ϵPro-120/δPro-122) and were greater at α-ϵ versus α-δ. The main consequence of the congenital myasthenic syndrome mutation ϵProD2-L was to impair the establishment of a high affinity for ACh and thus make ΔGB less favorable. At both binding sites, most ProD2 mutations decreased constitutive activity (increased ΔG0). LRYHQG and RL substitutions reduced substantially the net binding energy (made ΔGBACh less favorable) by ≥2 kcal/mol at α-ϵ and α-δ, respectively. Mutant cycle analyses were used to estimate energy coupling between the two ProD2 residues and between each ProD2 and glycine residues (αGly-147 and αGly-153) on the primary (α subunit) side of each binding pocket. The distant binding site prolines interact weakly. ProD2 interacts strongly with αGly-147 but only at α-ϵ and only when ACh is present. The results suggest that in the low to-high affinity change there is a concerted inter-subunit strain in the backbones at ϵProD2 and αGly-147. It is possible to engineer receptors having a single functional binding site by using a α-ϵ or α-δ ProD2-R knock-out mutation. In adult-type ACh receptors, the energy from the affinity change for ACh is approximately the same at the two binding sites (approximately −5 kcal/mol). Background: Mutation of a proline at the AChR transmitter-binding site causes a congenital myasthenic syndrome. Results: This proline interacts with a glycine across subunits but only when ACh is present. Conclusion: Proline mutations impair the establishment of a high affinity for ACh, which requires a concerted inter-subunit backbone strain. Significance: It is possible to engineer receptors having one functional binding site.