Role of the Outer β-Sheet in Divalent Cation Modulation of α7 Nicotinic Receptors

McLaughlin, James T.; Fu, Jie; Sproul, Adrian D.; Rosenberg, Robert L.

doi:10.1124/mol.106.023259

Cited by 21 publications

(27 citation statements)

References 28 publications

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“…All receptors contained a cysteine-to-alanine mutation at position 115. Cys115 is the only unpaired cysteine in the LBD, and the C115A mutation simplifies the interpretation of thiol modification experiments without affecting responses to ACh (McLaughlin et al, 2006) or PNU-120596. The utility of the L247T mutation is described under Results.…”

Section: Methodsmentioning

confidence: 99%

“…A model of the chick ␣7 nicotinic receptor extracellular domain, based on the coordinates of the Lymnea stagnalis ACh Binding Protein (Brejc et al, 2001) was constructed as described previously (Lyford et al, 2003;McLaughlin et al, 2006). Images of the model were generated with Pymol (DeLano Scientific, South San Francisco, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Benzodiazepines, the archetypal positive allosteric modulators of GABA A receptors, induce conformational changes in the ligand-binding domain of GABA A receptors (Sharkey and Czajkowski, 2008). We have found that agonists of ␣7 receptors induce structural transitions in the LBD, as measured by the substituted cysteine accessibility method (SCAM) (Lyford et al, 2003;McLaughlin et al, 2006McLaughlin et al, , 2007. Based on the existing data, we hypothesize that PAMs and agonists cause similar but nonidentical conformational changes.…”

mentioning

confidence: 98%

“…All mutants also contained the C115A mutation, in which the single unpaired cysteine in the LBD is mutated to an alanine. This mutation, which has no effect on activation kinetics, ligand sensitivity, or ion permeation, simplifies interpretation of cysteine modification experiments (McLaughlin et al, 2006).…”

Section: Conformational Effects Of Pnu-120596 On ␣7 Nachrs 255mentioning

confidence: 99%

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An Allosteric Modulator of α7 Nicotinic Receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), Causes Conformational Changes in the Extracellular Ligand Binding Domain Similar to Those Caused by Acetylcholine

Barron

McLaughlin²,

See³

et al. 2009

Molecular Pharmacology

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Nicotinic acetylcholine receptors are implicated in several neuropsychiatric disorders, including nicotine addiction, Alzheimer's, schizophrenia, and depression. Therefore, they represent a critical molecular target for drug development and targeted therapeutic intervention. Understanding the molecular mechanisms by which allosteric modulators enhance activation of these receptors is crucial to the development of new drugs. We used the substituted cysteine accessibility method to study conformational changes induced by the positive allosteric modulator N- (5-chloro-2,4-dimethoxyphenyl)-NЈ-(5-methyl-3-isoxazolyl)-urea (PNU-120596) in the extracellular ligand binding domain of ␣7 nicotinic receptors carrying the L247T mutation. PNU-120596 caused changes in cysteine accessibility at the inner beta sheet, transition zone, and agonist binding site. These changes in accessibility are similar to but not identical to those caused by ACh alone. In particular, PNU-120596 induced changes in MTSEA accessibility at N170C (in the transition zone) that were substantially different from those evoked by acetylcholine (ACh). We found that PNU-120596 induced changes at position E172C in the absence of allosteric modulation. We identified a cysteine mutation of the agonist binding site (W148C) that exhibited an unexpected phenotype in which PNU-120596 acts as a full agonist. In this mutant, ACh-evoked currents were more sensitive to thiol modification than PNUevoked currents, suggesting that PNU-120596 does not bind at unoccupied agonist-binding sites. Our results provide evidence that binding sites for PNU-120596 are not in the agonist-binding sites and demonstrate that positive allosteric modulators such as PNU-120596 enhance agonist-evoked gating of nicotinic receptors by eliciting conformational effects that are similar but nonidentical to the gating conformations promoted by ACh.Nicotinic acetylcholine receptors (nAChRs) are the prototypical member of the Cys-loop family of ligand-gated ion channels that also includes GABA A , serotonin type 3 (5-HT 3 ), and glycine receptors. This family of receptors assembles as heteromeric or homomeric pentamers around a central pore (Karlin, 2002). Each subunit contains an extracellular ligand-binding domain (LBD), an ␣-helical transmembrane domain (TMD), a transition zone that couples the LBD to the TMD, and an intracellular domain (Gay and Yakel, 2007).Neuronal nAChRs are expressed diffusely throughout most of the central nervous system; ␣7-containing receptors show the highest levels of expression (Orr-Urtreger et al., 1997). Of the neuronal nicotinic receptors, the homomeric ␣7 receptor is implicated in neurological diseases such as schizophrenia, Alzheimer's Disease, and anxiety disorders (Gotti and Clementi, 2004). Therefore, the ␣7 nicotinic receptor represents an important therapeutic target.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 98%

Section: Conformational Effects Of Pnu-120596 On ␣7 Nachrs 255mentioning

confidence: 99%

See 2 more Smart Citations

An Allosteric Modulator of α7 Nicotinic Receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), Causes Conformational Changes in the Extracellular Ligand Binding Domain Similar to Those Caused by Acetylcholine

Barron

McLaughlin²,

See³

et al. 2009

Molecular Pharmacology

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“…Influence of single amino acid substitution in the ␣7 nicotinic receptors has been intensively studied using site-directed mutagenesis (Placzek et al, 2005;McLaughlin et al, 2006). Yet, little is known about the polymorphisms located in the coding region of the human CHRNA7 gene, because the existence of a duplicated ␣7 receptor subunit (CHRFAM7A) gene, in which exons 5 to 10 overlap with those in the CHRNA7 gene (Gault et al, 1998), complicates polymorphism screening.…”

mentioning

confidence: 99%

Novel G423S Mutation of Human α7 Nicotinic Receptor Promotes Agonist-Induced Desensitization by a Protein Kinase C-Dependent Mechanism

Tsuneki

Kobayashi²,

Takagi³

et al. 2006

Mol Pharmacol

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The ␣7 nicotinic acetylcholine receptor subunit (CHRNA7) gene harbors a high degree of polymorphism. In this study, we found a novel variant (1267 G to A) in exon 10 of the CHRNA7 gene in a Japanese population. This variant results in glycine-to-serine substitution at position 423 (G423S) located in the large cytoplasmic loop of the protein. To clarify the possibility that the G423S mutation alters the pharmacological properties of ␣7 receptors, acetylcholine (ACh)-elicited current through ␣7-G423S mutant receptors expressed in Xenopus laevis oocytes was measured using the two-electrode voltage-clamp technique. We found that the current elicited by ACh (1 mM, 5 s) through ␣7-G423S receptors, but not through ␣7 receptors, was significantly decreased by treatment with a protein kinase C activator, phorbol-12-myristate-13-acetate (PMA, 10 -30 nM). In addition, PMA (10 nM) selectively promoted a progressive decrease in ␣7-G423S current induced by repetitive application of ACh pulses (1 mM, 0.1 s, 0.17-0.33 Hz) compared with ␣7 current. PMA also enhanced the inactivation of ␣7-G423S mutant receptors induced by a prolonged application of choline (30 M) without affecting ␣7 receptor responses. Western blot analysis showed that the treatment with PMA (30 nM) increased the serine phosphorylation level of the ␣7-G423S mutant receptors but not that of the wild-type receptors. These findings demonstrate that the G423S mutation promotes receptor desensitization by a protein kinase C-dependent mechanism. Thus, we provide the first evidence that a variant in the human CHRNA7 gene alters the function of ␣7 nicotinic receptors.

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Probing the Role of Backbone Hydrogen Bonding in a Critical β Sheet of the Extracellular Domain of a Cys‐Loop Receptor

2009

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Long-range communication is essential for the function of members of the Cys-loop family of neurotransmitter-gated ion channels. The involvement of the peptide backbone in binding-induced conformational changes that lead to channel gating in these membrane proteins is an interesting, but unresolved issue. To probe the role of the peptide backbone, we have incorporated a series of α-hydroxy acid analogues into the β-sheet rich extracellular domain of the muscle subtype of the nicotinic acetylcholine receptor, the prototypical Cys-loop receptor. Specifically, mutations were made in β-strands 7 and 10 of the α-subunit. A number of single backbone mutations in this region were well-tolerated. However, simultaneous introduction of two proximal backbone mutations led to surface-expressed, non-functional receptors. Together, these data suggest that while the receptor is remarkably robust in its ability to tolerate single amide-to-ester mutations throughout these β-strands, more substantial perturbations to this region have a profound effect on the protein. These results support a model in which backbone movements in the outer β-sheet are important for receptor function.

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Role of the Outer β-Sheet in Divalent Cation Modulation of α7 Nicotinic Receptors

Cited by 21 publications

References 28 publications

An Allosteric Modulator of α7 Nicotinic Receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), Causes Conformational Changes in the Extracellular Ligand Binding Domain Similar to Those Caused by Acetylcholine

An Allosteric Modulator of α7 Nicotinic Receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), Causes Conformational Changes in the Extracellular Ligand Binding Domain Similar to Those Caused by Acetylcholine

Novel G423S Mutation of Human α7 Nicotinic Receptor Promotes Agonist-Induced Desensitization by a Protein Kinase C-Dependent Mechanism

Probing the Role of Backbone Hydrogen Bonding in a Critical β Sheet of the Extracellular Domain of a Cys‐Loop Receptor

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