Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

Kouvatsos, Nikolaos; Giastas, Petros; Chroni-Tzartou, Dafni; Poulopoulou, Cornelia; Tzartos, Socrates J.

doi:10.1073/pnas.1602619113

Cited by 57 publications

(74 citation statements)

References 70 publications

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“…In the muscle-type nAChR, the three triad residues cooperate as a unit, as demonstrated via triple mutant cycle analysis. This corroborates previous functional and crystallographic work that suggested a salt bridge between two members of the triad is involved in receptor gating (10,12,15,17,22,28). We show here that the salt bridge between K145 and D200 depends on T202, which may coordinate this interaction.…”

Section: Discussionsupporting

confidence: 93%

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

Mosesso

Dougherty

2018

Journal of Biological Chemistry

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Cys-loop receptors are pentameric ligandgated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cysloop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT 3A receptors (5-HT 3A Rs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT 3A R can be exchanged between the homomeric 5-HT 3A R and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Further, nAChR-type mutations of the 5-HT 3A R affect the affinity of nicotine, a competitive antagonist of 5-HT 3A Rs, in a cooperative manner. Using mutant cycle analyses between the 5-HT 3A triad, loop A residues Asn-101 and Glu-102, ß9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

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Section: Discussionsupporting

confidence: 93%

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

Mosesso

Dougherty

2018

Journal of Biological Chemistry

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“…1D and E), is next to F119, a residue that interacts with the epibatidine chloropyridine ring and stabilizes the epibatidine chlorine atom through its backbone carboxyl group. Moreover, the A110V replacement is next to V111, another AA residue that interacts with epibatidine via van der Waals forces (21, 28, 29). These replacements are located in β-sheets that are involved in epibatidine binding, but are less involved in ACh binding (21, 28, 30).…”

Section: Aa Replacements In Poison Frog Nachr Are Proximal To the Epimentioning

confidence: 99%

“…Moreover, the A110V replacement is next to V111, another AA residue that interacts with epibatidine via van der Waals forces (21, 28, 29). These replacements are located in β-sheets that are involved in epibatidine binding, but are less involved in ACh binding (21, 28, 30). The β2 − side of the binding pocket is further from ACh than is the α4 + side, and thus forms looser interactions with ACh, such that AA replacements in the β − region that allow changes in epibatidine binding may be less likely to affect ACh sensitivity.…”

Section: Aa Replacements In Poison Frog Nachr Are Proximal To the Epimentioning

confidence: 99%

“…α4 subunits are in light grey, β2 subunits are in gold, and the ligand-binding sites are indicated by grey spheres. AA residues identified with grey and gold arrows are known to be involved in ACh and/or epibatidine binding (21, 28, 29). Closer view of the binding site from ( D ) extracellular space and ( E ) viewpoint indicated by labeled arrow in ( C ).…”

Section: Figmentioning

confidence: 99%

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Interacting amino acid replacements allow poison frogs to evolve epibatidine resistance

Tarvin

Borghese

Sachs

et al. 2017

Science

113

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Animals that wield toxins face self-intoxication. Poison frogs have a diverse arsenal of defensive alkaloids that target the nervous system. Among them is epibatidine, a nicotinic acetylcholine receptor (nAChR) agonist that is lethal at microgram doses. Epibatidine shares a highly conserved binding site with acetylcholine, making it difficult to evolve resistance yet maintain nAChR function. Electrophysiological assays of human and frog nAChR revealed that one amino acid replacement, which evolved three times in poison frogs, decreased epibatidine sensitivity but at a cost of acetylcholine sensitivity. However, receptor functionality was rescued by additional amino acid replacements that differed among poison frog lineages. Our results demonstrate how resistance to agonist toxins can evolve and that such genetic changes propel organisms towards an adaptive peak of chemical defense.

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“…The role of pLGICs is extremely important in signaling mechanisms and functioning of the nervous system, and disruptions to their function lead to a wide variety of diseases and disorders [9][10][11]13]. According to existing structures of several pLGICs, such as 5-HT 3 , several nAChRs, GABA A β 3 and Glyα1 receptors, there is a high conservation in composition and structure of the ECD and TMD of these receptors [26-28, [35][36][37][38]. The commonality of most of these structures is that intracellular domains (ICD) have been removed, shortened, or in case of full-length receptor structures, the ICD remained partially resolved [26,27].…”

Section: Discussionmentioning

confidence: 99%

Delineating the site of interaction on the intracellular domain of 5-HT3A receptors with the chaperone protein RIC-3

Pirayesh

Stuebler

Jansen

2019

Preprint

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Statement of Significance:The chaperone protein RIC-3 is known to modulate the functional surface expression of cationconducting pentameric ligand-gated ion channels. Previously we have demonstrated that the intracellular domain of serotonin channels mediates this effect. Here we provide experimental evidence for a 24-amino acid long segment within the 115-amino acid long intracellular domain as a determinant for RIC-3 interaction. Recently it was found experimentally that the identified segment contains an alpha helix that has been observed or predicted to be present in other cation-conducting channels. The present work provides novel insights into protein-protein interactions that are likely also relevant for other cation-conducting members of this large ion channel family that includes nACh and 5-HT3 receptors. ABSTRACTThe serotonin type 3A (5-HT 3A ) receptor is a homopentameric cation-selective member of the pentameric ligand-gated ion channel (pLGIC) superfamily. Members of this superfamily assemble from five subunits, each of which consists of three domains, extracellular (ECD), transmembrane (TMD), and intracellular domain (ICD). Previously, we have also demonstrated that 5-HT 3A -ICD is required and sufficient for the interaction between 5-HT 3A and RIC-3.Additionally, we have shown that 5-HT 3A -ICD fused to maltose binding protein (MBP) directly interacts with the chaperone protein resistance to inhibitors of choline esterase (RIC-3), without the involvement of other protein(s). To elucidate the molecular determinants of this interaction we developed different MBP-fused 5-HT 3A -ICD constructs by deletion of large portions of its amino acid sequence. We have expressed seven mutants in Escherichia coli and purified them to homogeneity. Using a RIC-3 affinity pull-down assay, the interaction of MBP-5HT 3A -ICD constructs and RIC-3 is investigated. Furthermore, we co-expressed 5-HT 3A and 5-HT 3AB , a heteromeric form of 5-HT 3 Rs, with RIC-3 in Xenopus oocytes to compare their interaction with RIC-3 in-vivo by two electrode voltage clamp (TEVC) recordings. Full-length 5-HT 3A -and 5-HT 3AB mediated currents are significantly reduced when RIC-3 is co-expressed in either condition. In summary, we identify a 24-amino acid long segment of the 5-HT 3A -ICD as a molecular determinant for the interaction between the 5-HT 3A -ICD and RIC-3.

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Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

Cited by 57 publications

References 70 publications

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

Interacting amino acid replacements allow poison frogs to evolve epibatidine resistance

Delineating the site of interaction on the intracellular domain of 5-HT3A receptors with the chaperone protein RIC-3

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