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Modal affinities of endplate acetylcholine receptors caused by loop C mutations.

ABSTRACT: 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 ?P197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the ??-binding site. (f) Substituting four ?-subunit residues into the ? subunit (three in loop E and one in the ?5-?5' 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 ?W57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except ?Y93. The results suggest that, at the ?? agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of ?W57 with respect to the aromatic core and, hence, affinity.

SUBMITTER: Vij R

PROVIDER: S-EPMC4621750 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Modal affinities of endplate acetylcholine receptors caused by loop C mutations.

Vij Ridhima R Purohit Prasad P Auerbach Anthony A

The Journal of general physiology 20151101 5

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 ...[more]

PMID: 26503719

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Project description:The interaction of a small molecule made in one cell with a large receptor made in another is the signature event of cell signaling. Understanding the structure and energy changes associated with agonist activation is important for engineering drugs, receptors and synapses. The nicotinic acetylcholine receptor (AChR) is a ?300kD ion channel that binds the neurotransmitter acetylcholine (ACh) and other cholinergic agonists to elicit electrical responses in the central and peripheral nervous systems. This mini-review is in two sections. First, general concepts of skeletal muscle AChR operation are discussed in terms of energy landscapes for conformational change. Second, adult vs. fetal AChRs are compared with regard to interaction energies between ACh and agonist-site side chains, measured by single-channel electrophysiology and molecular dynamics simulations. The five aromatic residues that form the core of each agonist binding site can be divided into two working groups, a triad (led by ?Y190) that behaves similarly at all sites and a coupled pair (led by ?W55) that has a large influence on affinity only in fetal AChRs. Each endplate AChR has 5 homologous subunits, two of ?(1) and one each of ?, ?, and either ? (fetal) or ? (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at ?? and either ?? or ?? subunit interfaces. The receptor undergoes a reversible, global isomerization between structures called C and O. The C shape does not conduct ions and has a relatively low affinity for ACh, whereas O conducts cations and has a higher affinity. When both agonist sites are empty (filled only with water) the probability of taking on the O conformation (PO) is low, <10(-6). When ACh molecules occupy the agonist sites the C?O opening rate constant and C?O gating equilibrium constant increase dramatically. Following a pulse of ACh at the nerve-muscle synapse, the endplate current rises rapidly to reach a peak that corresponds to PO ?0.96.

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Modal affinities of endplate acetylcholine receptors caused by loop C mutations.

Publications

Modal affinities of endplate acetylcholine receptors caused by loop C mutations.

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