Sources of energy for gating by neurotransmitters in acetylcholine receptor channels

Purohit, Prasad; Bruhova, Iva; Auerbach, Anthony

doi:10.1073/pnas.1203633109

Cited by 27 publications

(44 citation statements)

References 31 publications

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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: 57%

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: 57%

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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“…ACh is large and approximately equivalent to that observed by replacing the central loop B tryptophan (␣Trp-149) with an Ala (ϩ2.3 kcal/mol (27)). So far, for the small side chain substitutions only the mutation TyrC2-A has been shown to have a greater effect on ⌬G B ACh than ⑀ProD2-G. Recall that the Gly substitution also had a large effect on ⌬G 0 , suggesting that the gating conformational change at ⑀ProD2 may be similar in unliganded and liganded conditions.…”

Section: J D1mentioning

confidence: 85%

“…For diliganded gating, the value for ProD2 was high (Ն0.9) in both subunits, although perhaps higher in ␣-⑀ compared with ␣-␦. This indicates that these prolines, like many other residues in the vicinity of the transmitter-binding sites, change energy relatively early, and nearly synchronously, in the opening isomerization when ACh is present (27). In the absence of agonists, the ProD2 residues at both ⑀ and ␦ had slightly lower values, suggesting relatively later energy changes.…”

Section: Achmentioning

confidence: 88%

“…1c). These include four aromatic amino acids in the ␣ subunit ␣Tyr-93 (TyrA), ␣Trp-149 (TrpB), ␣Tyr-190 (TyrC1), and ␣Tyr-198 (TyrC2), two glycines in loop B ␣Gly-147 (GlyB1) and ␣Gly-153 (GlyB2), a tryptophan on the complementary D surface ⑀Trp-55/␦Trp-57 (TrpD), and now ⑀/␦ProD2 (21,24,27,33). In WT AChRs, most of the ⌬G B ACh energy comes from an "aromatic triad" composed of ␣Trp-149, ␣Tyr-190, and ␣Tyr-198 (27).…”

Section: J D1mentioning

confidence: 99%

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Function of Interfacial Prolines at the Transmitter-binding Sites of the Neuromuscular Acetylcholine Receptor

Gupta

Purohit

Auerbach

2013

Journal of Biological Chemistry

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“…In e-AChRs, three binding site groups (an "aromatic triad") in the α-subunit (αW149, αY190, and αY198) each provide ∼−2 kcal/mol to the increased stabilization of ACh in the HA vs. LA complex (33). Two other aromatic residues, αY93 and e/δW57/55 (on the non-α surface of the binding pocket), are nearby but contribute little or nothing to ΔG ACh B .…”

Section: Without Thementioning

confidence: 99%

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

Nayak

Auerbach

2013

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

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Neuromuscular acetylcholine receptors (AChRs) have two transmitter binding sites: at α−δ and either α−γ (fetal) or α-e (adult) subunit interfaces. The γ-subunit of fetal AChRs is indispensable for the proper development of neuromuscular synapses. We estimated parameters for acetylcholine (ACh) binding and gating from single channel currents of fetal mouse AChRs expressed in tissuecultured cells. The unliganded gating equilibrium constant is smaller and less voltage-dependent than in adult AChRs. However, the α−γ binding site has a higher affinity for ACh and provides more binding energy for gating compared with α−e; therefore, the diliganded gating equilibrium constant at −100 mV is comparable for both receptor subtypes. The −2.2 kcal/mol extra binding energy from α−γ compared with α−δ and α−e is accompanied by a higher resting affinity for ACh, mainly because of slower transmitter dissociation. End plate current simulations suggest that the higher affinity and increased energy from α−γ are essential for generating synaptic responses at low pulse [ACh].are ligand-gated ion channels comprised of five subunits: two α(1) and one each of β, δ, and either γ or e. In most species, the γ-subunit is replaced by e during postnatal development. Electrical activity in muscle cells induced by the neurotransmitter acetylcholine (ACh) is important for the molecular maturation of γ-to e-AChRs (1, 2).Without γ-subunit, neuromuscular synapses do not develop properly (3) and are abnormal in innervation patterns (4) and muscle fiber-type composition (5). In mice, the γ-subunit KO is lethal (6), and in humans, mutations in the cholinergic receptor, nicotinic, gamma (CHRNG) gene that encodes for γ-subunit are associated with lethal forms of multiple pterygium and Escobar syndromes (7-9).AChRs have two transmitter binding sites located in the extracellular domain at α-δ and α-γ/e-subunit interfaces. In adult AChRs, the α−δ and α−e sites are equal and independent insofar as each has the same affinity for ACh and supplies the same amount of binding energy for gating (10). In fetal AChRs, the two binding sites are asymmetric with regard to agonist affinity, with estimates for the ratio of resting equilibrium dissociation constants for ACh ranging from ∼5-(11, 12) to >100-fold (13, 14). γ-AChRs also have a longer open channel lifetime, a smaller unitary conductance, and a lower Ca 2+ permeability than e-AChRs (15-17).Fetal AChRs hold a special place in the history of ion channel biophysics. The first single channel recordings from cells were of γ-AChRs (18). They were also the first ion channels to be analyzed according to a thermodynamic cycle, which required quantifying constitutive activity in the absence of agonists (19). These pioneering studies of single channel currents from mouse myotubes led to the estimate that the unliganded gating equilibrium constant of γ-AChRs is ∼1.6 × 10 −6 . γ-AChRs were also the first channels for which the rate and equilibrium constants for closed channel binding and liganded gating were measured (13,(...

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Sources of energy for gating by neurotransmitters in acetylcholine receptor channels

Cited by 27 publications

References 31 publications

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Function of Interfacial Prolines at the Transmitter-binding Sites of the Neuromuscular Acetylcholine Receptor

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

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