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Function of interfacial prolines at the transmitter-binding sites of the neuromuscular acetylcholine receptor.


ABSTRACT: The neuromuscular acetylcholine (ACh) receptor has two conserved prolines in loop D of the complementary subunit at each of its two transmitter-binding sites (?-? and ?-?). We used single-channel electrophysiology to estimate the energy changes caused by mutations of these prolines with regard to unliganded gating (?G0) and the affinity change for ACh that increases the open channel probability (?GB). The effects of mutations of ProD2 (?Pro-121/?Pro-123) were greater than those of its neighbor (?Pro-120/?Pro-122) and were greater at ?-? versus ?-?. The main consequence of the congenital myasthenic syndrome mutation ?ProD2-L was to impair the establishment of a high affinity for ACh and thus make ?GB less favorable. At both binding sites, most ProD2 mutations decreased constitutive activity (increased ?G0). LRYHQG and RL substitutions reduced substantially the net binding energy (made ?GB(ACh) less favorable) by ?2 kcal/mol at ?-? and ?-?, respectively. Mutant cycle analyses were used to estimate energy coupling between the two ProD2 residues and between each ProD2 and glycine residues (?Gly-147 and ?Gly-153) on the primary (? subunit) side of each binding pocket. The distant binding site prolines interact weakly. ProD2 interacts strongly with ?Gly-147 but only at ?-? 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 ?ProD2 and ?Gly-147. It is possible to engineer receptors having a single functional binding site by using a ?-? or ?-? 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).

PROVIDER: S-EPMC3642313 | BioStudies |

REPOSITORIES: biostudies

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