Asymmetric transmitter binding sites of fetal muscle acetylcholine receptors shape their synaptic response

Nayak, Tapan K.; Auerbach, Anthony

doi:10.1073/pnas.1308247110

Cited by 16 publications

(20 citation statements)

References 36 publications

(42 reference statements)

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“…Previously, estimates of the ACh-binding free energy difference in mouse adult-type receptors after mutations indicated that only three of the mentioned aromatics (αY190, αY198, and αW149) are important (18), and other experiments showed that the free energy difference from both agonist sites combined is greater in fetal vs. adult AChRs (19). Here, we extend and refine these estimates.…”

supporting

confidence: 65%

“…From the relationship ΔG B1 = +0.59lnK d (SI Appendix, Methods), we estimate that at αγ, αe, and αδ K d ACh = 5, 175, and 200 μM, respectively. Simulations of synaptic responses show that fetal AChRs produce a substantially larger response to the neurotransmitter in the concentration range 10-100 μM (19). Second, the more favorable ΔG B1 for choline at αγ enables fetal AChRs to respond to lower concentrations of this physiological ligand.…”

Section: Discussionmentioning

confidence: 99%

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Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Nayak

Bruhova

Chakraborty

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αe (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔG B1 ). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔG B1 is ∼−2 kcal/mol more favorable at αγ compared with at αe and αδ. Only three of the aromatics contribute significantly to ΔG B1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔG B1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αe and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αe and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse.allosteric protein | ion channel | ligand binding sites | single-channel electrophysiology | synaptic maturation R eceptors at synapses respond to specific chemical signals in the extracellular environment because the active conformation of the protein has a higher affinity for the ligand compared with the resting conformation (1, 2). The active vs. resting difference in binding free energy increases the relative stability of the active state and, hence, the probability of a cellular response. In this report, we describe and distinguish sources of ligandbinding free energy in three kinds of agonist site present in mouse muscle nicotinic acetylcholine receptors (AChRs). Our goal was to use single-channel electrophysiology to assess the relative contribution of significant functional groups to the overall free energy generated by the affinity change at each type of site.At cholinergic synapses, the main chemical signals are ACh released from nerve terminals and choline, which is an ACh precursor, hydrolysis product, and stable component of serum (3). The muscle AChR has central pore surrounded by five subunits of composition α 2 βδe in adult-type and α 2 βδγ in fetal-type (Fig. 1A) (4). The fetal, γ, subunit is essential for proper synapse maturation, and the adult, e, subunit is necessary for proper function of mature synapses (5-7). Each AChR pentamer has two agonist binding sites in the extracellular domain, at αδ and either αe (adult) or αγ (fetal) subunit interfaces.The change in agonist affinity occurs within the global, resting↔active...

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confidence: 65%

Section: Discussionmentioning

confidence: 99%

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Nayak

Bruhova

Chakraborty

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The large, uphill energy gap between unliganded C and O becomes downhill because of the energy deposit arising from new, favorable interactions at two agonist sites with ACh molecules. Similar measurements with fetal-type AChRs, in which a γ subunit replaces ε, show that ΔG B2 ACh =−12.2 kcal (22). …”

Section: 2 Energy From the Agonistmentioning

confidence: 54%

“…In the third approach, j on and j off for ACh were estimated directly from cross-concentration fitting of single-channel shut current interval durations, much as is done for measuring k on and k off (22, 38). Here, background mutations that increase intrinsic gating and low [ACh] were used to make activation by the anti-clockwise pathway more probable than by the clockwise route.…”

Section: 3 Catch and Holdmentioning

confidence: 99%

Agonist activation of a nicotinic acetylcholine receptor

Auerbach

2015

Neuropharmacology

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How does an agonist activate a receptor? In this article I consider the activation process in muscle nicotinic acetylcholine receptors (AChRs), a prototype for understanding the energetics of binding and gating in other ligand-gated ion channels. Just as movements that generate gating currents activate voltage-gated ion channels, movements at binding sites that generate an increase in affinity for the agonist activate ligand-gated ion channels. The main topics are: i) the schemes and intermediate states of AChR activation, ii) the energy changes of each of the steps, iii) the sources of the energies, iv) the three kinds of AChR agonist binding site and v) the correlations between binding and gating energies. The binding process is summarized as sketches of different conformations of an agonist site. The results suggest that agonists lower the free energy of the active conformation of the protein in stages, by establishing favorable, local interactions at each binding site independently.

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“…It is possible to estimate the amount of favorable binding energy each ACh molecule provides [8–10]. Another way to describe the above scenario of agonist action is to say that the O state has a higher affinity for ACh than the C state (in Fig.…”

Section: Energy Landscapementioning

confidence: 99%

Activation of endplate nicotinic acetylcholine receptors by agonists

Auerbach

2015

Biochemical Pharmacology

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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 ~300 kD 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 αY190) that behaves similarly at all sites and a coupled pair (led by γW55) that has a large influence on affinity only in fetal AChRs. Each endplate AChR has 5 homologous subunits, two of α(1) and one each of β, δ and either γ (fetal) or ε (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at αδ and either αγ or αε 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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Asymmetric transmitter binding sites of fetal muscle acetylcholine receptors shape their synaptic response

Cited by 16 publications

References 36 publications

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Agonist activation of a nicotinic acetylcholine receptor

Activation of endplate nicotinic acetylcholine receptors by agonists

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