Modal affinities of endplate acetylcholine receptors caused by loop C mutations.
The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability ("modes"). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5-β5' linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity.
Neuroscience Program and Department of Physiology, State University of New York at Buffalo, Buffalo, NY 14214.
Ridhima
R
Vij
Neuroscience Program and Department of Physiology, State University of New York at Buffalo, Buffalo, NY 14214.
Prasad
P
Purohit
Neuroscience Program and Department of Physiology, State University of New York at Buffalo, Buffalo, NY 14214 auerbach@buffalo.edu.
Anthony
A
Auerbach
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D018679:Cholinergic Agonists / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D018679:Cholinergic Agonists; D011950:Receptors, Cholinergic