Site Specificity of Agonist-Induced Opening and Desensitization of the<i>Torpedo</i><i>californica</i>Nicotinic Acetylcholine Receptor

Andreeva, Iraida E.; Nirthanan, Selvanayagam; Cohen, Jonathan B.; Pedersen, Steen E.

doi:10.1021/bi0516024

Cited by 19 publications

(18 citation statements)

References 40 publications

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“…This concept was extended to suggest that each agonist-binding subunit pair also has only one desensitized structure, and that fast and slow desensitization differ merely in the number of desensitized pairs (Prince and Sine, 1999). Rapid kinetics studies with a fluorescent agonist provide supporting evidence that the αδ agonist site desensitizes more slowly than the αγ site (Andreeva et al, 2006; Song et al, 2003). Thus, αγ subunit pair rearrangement should accompany fast desensitization, while αδ rearrangement accompanies slow desensitization.…”

Section: Asymmetry and Allosteric Mechanisms In Hetero-oligomersmentioning

confidence: 95%

Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors

2015

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General anesthetics are a heterogeneous group of small amphiphilic ligands that interact weakly at multiple allosteric sites on many pentameric ligand gated ion channels (pLGICs), resulting in either inhibition, potentiation of channel activity, or both. Allosteric principles imply that modulator sites must change configuration and ligand affinity during receptor state transitions. Thus, general anesthetics and related compounds are useful both as state-dependent probes of receptor structure and as potentially selective modulators of pLGIC functions. This review focuses on general anesthetic sites in nicotinic acetylcholine receptors, which were among the first anesthetic-sensitive pLGIC experimental models studied, with particular focus on sites formed by transmembrane domain elements. Structural models place many of these sites at interfaces between two or more pLGIC transmembrane helices both within subunits and between adjacent subunits, and between transmembrane helices and either lipids (the lipid-protein interface) or water (i.e. the ion channel). A single general anesthetic may bind at multiple allosteric sites in pLGICs, producing a net effect of either inhibition (e.g. blocking the ion channel) or enhanced channel gating (e.g. inter-subunit sites). Other general anesthetic sites identified by photolabeling or crystallography are tentatively linked to functional effects, including intra-subunit helix bundle sites and the lipid-protein interface.

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Section: Asymmetry and Allosteric Mechanisms In Hetero-oligomersmentioning

confidence: 95%

Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors

2015

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“…These transitions occur in at least three phases (21). A fast fluorescence change at 25-50 ms reflects agonist binding to the small population of receptors that already exist in the desensitized state ("predesensitized" nAChRs), and also includes a smaller component of binding to the ␣-␦ site in its resting state (47). This component is followed by intermediate (Ͻ1 s) and slow steps of desensitization, reflecting intermediate transitions of the nAChR as it desensitizes.…”

Section: The Functional Interaction Between the Nachr And The Nak-atmentioning

confidence: 99%

“…1), 3 thereby excluding possible nonspecific effects of ouabain. In addition, the amplitude of the initial rapid phase, which reflects the initial equilibrium between predesensitized and resting states (47), is unaltered. Thus, ouabain does not alter the intrinsic equilibrium between resting and desensitized states in the absence of nicotinic agonist.…”

Section: The Functional Interaction Between the Nachr And The Nak-atmentioning

confidence: 99%

The Nicotinic Acetylcholine Receptor and the Na,K-ATPase α2 Isoform Interact to Regulate Membrane Electrogenesis in Skeletal Muscle

Heiny

Кравцова

Mandel

et al. 2010

Journal of Biological Chemistry

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116

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The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase ␣2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase ␣ subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membranedelimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation. The nicotinic acetylcholine receptor (nAChR)2 and the Na,K-ATPase are integral membrane proteins that play key roles in membrane excitation. We previously identified a regulatory mechanism, termed acetylcholine (ACh)-induced hyperpolarization, whereby the nAChR and the Na,K-ATPase functionally interact to modulate the membrane potential of rat skeletal muscle (1-4). In this interaction, the binding of nanomolar concentrations of ACh to the nAChR stimulates electrogenic transport by the Na,K-ATPase ␣2 isozyme, causing a membrane hyperpolarization of about Ϫ4 mV. This effect requires prolonged exposure to nanomolar concentrations of nicotinic agonist. This property distinguishes it from the more well characterized, rapid action of micromolar concentrations of ACh, which open the nAChR and produce membrane depolarization (5). This finding suggested that a non-conducting conformation of the nAChR, rather than the open state, is involved in signaling to the Na,K-ATPase. In addition, it was shown that the nAChR and Na,K-ATPase can reciprocally interact in a membrane preparation from the Torpedo electric organ (1), a muscle-derived tissue that is rich in muscle nAChRs and Na,K-ATPase. This finding suggested that the nAChR and Na,K-ATPase may interact as part of a membrane-associated regulatory complex.Importantly, this regulation of Na,K-ATPase activity by the nAChR operates under the physiological conditions of normal muscle use. Its ACh concentration dependence is in the range of the residual ACh concentrations that remain in the muscle interstitial spaces for some time following nerve excitation, and to the ACh concentrations that arise at the neuromuscular junction (NMJ) from non-quantal ACh release. The later have also been shown to activate the Na,K-ATPase and hyperpolarize the end plate membrane (6, 7). Notably, this hyperpolariza-* This work was supported, in whole or in part, by National Institutes of Health

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“…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)(16)(17).…”

mentioning

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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Site Specificity of Agonist-Induced Opening and Desensitization of theTorpedocalifornicaNicotinic Acetylcholine Receptor

Cited by 19 publications

References 40 publications

Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors

Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors

The Nicotinic Acetylcholine Receptor and the Na,K-ATPase α2 Isoform Interact to Regulate Membrane Electrogenesis in Skeletal Muscle

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

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