ELIC-α7 Nicotinic Acetylcholine Receptor (α7nAChR) Chimeras Reveal a Prominent Role of the Extracellular-Transmembrane Domain Interface in Allosteric Modulation

Tillman, Tommy S.; Seyoum, Edom; Mowrey, David D.; Xu, Yan; Tang, Pei

doi:10.1074/jbc.m113.524611

Cited by 25 publications

(42 citation statements)

References 57 publications

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“…Single cysteine mutations were introduced using the same method used for the NMR samples. Preparation of RNA, expression in Xenopus laevis oocytes, and electrophysiology experiments were performed as previously reported (Pan et al, 2012a; Pan et al, 2012b; Tillman et al, 2013; Tillman et al, 2014). In order to ensure TET labeling does not destroy channel function, the purified TET-labeled ELIC was reconstituted into lipid vesicles as previously reported (Bondarenko et al, 2014) and injected into oocytes (50 nL; 2 mg/mL protein) for functional tests.…”

Section: Methodsmentioning

confidence: 99%

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

et al. 2015

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SUMMARY Structural rearrangements underlying functional transitions of pentameric ligand-gated ion channels (pLGICs) are not fully understood. Using 19F NMR and ESR spectroscopy, we found that ELIC, a pLGIC from Erwinia chrysanthemi, expanded the extracellular end and contracted the intracellular end of its pore during transition from the resting to an apparent desensitized state. Importantly, the contraction at the intracellular end of the pore likely forms a gate to restrict ion transport in the desensitized state. This gate differs from the hydrophobic gate present in the resting state. Conformational changes of the TM2-TM3 loop were limited to the N-terminal end. The TM4 helices and the TM3-TM4 loop appeared relatively insensitive to agonist-mediated structural rearrangement. These results indicate that conformational changes accompanying functional transitions are not uniform among different ELIC regions. This work also revealed the co-existence of multiple conformations for a given state and suggested asymmetric conformational arrangements in a homomeric pLGIC.

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Section: Methodsmentioning

confidence: 99%

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

et al. 2015

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“…The early observation that the α 7 nAChR(ECD)-5HT 3 R(TMD+ICD) chimera was functional was the first evidence that the ECD and TMD may constitute independent tertiary modules (26). The α 7 nAChR(ECD)-α 1 GlyR(TMD+ICD) (18), the AChBP-5HT 3 (TMD+ICD) (29), and recently the ELIC(ECD)-α 7 nAChR(TMD) chimera (30) were subsequently reported to be functional, the ECD retaining "native-like" pharmacology for agonists and the TMD native-like channel selectivity. It is noteworthy that extensive mutations at the ECD-TMD interface were required to produce properly folded and/or functional AChBP-5HT 3 (TMD+ICD) and ELIC (ECD)-α 7 nAChR(TMD) chimeras.…”

Section: Discussionmentioning

confidence: 99%

Allosteric and hyperekplexic mutant phenotypes investigated on an α ₁ glycine receptor transmembrane structure

Moraga-Cid

Sauguet

Huon

et al. 2015

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

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The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α 1 GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α 1 GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α 1 GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a poreopening mechanism for the α 1 GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α 1 GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor.glycine receptor | hyperekplexia | allostery

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“…These residues (L60, M63, E67, N75, N193, and E195) were evaluated along with several others in the surrounding region (Table 2). Figure 5 shows the residues in the gating interface with |ΔΔG| ≥ 0.5 kcal/mol (Hanek, et al, 2008; Tillman, et al, 2014). Tillman et al showed through chimera analysis that specific loops and linkers were necessary for PNU-120596 potentiation of α7 receptors (Tillman, et al, 2014).…”

Section: Resultsmentioning

confidence: 99%

“…Figure 5 shows the residues in the gating interface with |ΔΔG| ≥ 0.5 kcal/mol (Hanek, et al, 2008; Tillman, et al, 2014). Tillman et al showed through chimera analysis that specific loops and linkers were necessary for PNU-120596 potentiation of α7 receptors (Tillman, et al, 2014). Here, we were able to isolate specific residues on some of these identified regions: in Loop 2, E67; in Loop 9, E195; and in the M2-M3 Linker, S287.…”

Section: Resultsmentioning

confidence: 99%

An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors

Marotta

Lester

Dougherty

2015

Chemistry & Biology

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Summary Nicotinic acetylcholine receptors (nAChRs) are vital to neuronal signaling, are implicated in important processes such as learning and memory, and are therapeutic targets for neural diseases. The α7 nAChR has been implicated in Alzheimer’s disease and schizophrenia, and allosteric modulators have become one focus of drug development efforts. We investigate the mode of action of the α7-selective positive allosteric modulator, PNU-120596, and show that the higher potency of acetylcholine in the presence of PNU-120596 is not due to an altered agonist binding site. In addition, we propose several residues in the gating interface and transmembrane region that are functionally important to transduction of allosteric properties and link PNU-120596, the acetylcholine binding region, and the receptor’s gate. These results suggest global protein stabilization from a communication network through several key residues that alter the gating equilibrium of the receptor while leaving the agonist binding properties unperturbed.

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ELIC-α7 Nicotinic Acetylcholine Receptor (α7nAChR) Chimeras Reveal a Prominent Role of the Extracellular-Transmembrane Domain Interface in Allosteric Modulation

Cited by 25 publications

References 57 publications

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

Allosteric and hyperekplexic mutant phenotypes investigated on an α ₁ glycine receptor transmembrane structure

An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors

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ELIC-α7 Nicotinic Acetylcholine Receptor (α7nAChR) Chimeras Reveal a Prominent Role of the Extracellular-Transmembrane Domain Interface in Allosteric Modulation

Cited by 25 publications

References 57 publications

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

Allosteric and hyperekplexic mutant phenotypes investigated on an α 1 glycine receptor transmembrane structure

An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors

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Allosteric and hyperekplexic mutant phenotypes investigated on an α ₁ glycine receptor transmembrane structure