Dataset Information

Catch-and-hold activation of muscle acetylcholine receptors having transmitter binding site mutations.

ABSTRACT: Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of ?Y93, ?G147, ?W149, ?Y190, ?Y198, ?W55, and ?W57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (?) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations, ? ? 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90; ?G153) or lower (0.25; ?P121). The results suggest that backbone rearrangements in loop B, loop C, and the non-? surface participate in both LA binding and the LA ? HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process.

SUBMITTER: Purohit P

PROVIDER: S-EPMC4119287 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Catch-and-hold activation of muscle acetylcholine receptors having transmitter binding site mutations.

Purohit Prasad P Bruhova Iva I Gupta Shaweta S Auerbach Anthony A

Biophysical journal 20140701 1

Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, th ...[more]

PMID: 24988344

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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.

Dataset Information

Catch-and-hold activation of muscle acetylcholine receptors having transmitter binding site mutations.

Publications

Catch-and-hold activation of muscle acetylcholine receptors having transmitter binding site mutations.

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