Stable expression and characterisation of a human α7 nicotinic subunit chimera: a tool for functional high-throughput screening

Craig, Peter J.; Bose, Suchira; Zwart, Ruud; Beattie, Ruth E.; Folly, Elizabeth A.; Johnson, Laura R.; Bell, Emma; Evans, Non; Benedetti, Giovanni; Pearson, Kathy H.; McPhie, Gordon; Volsen, Stephen G.; Millar, Neil S.; Sher, Emanuele; Broad, Lisa M.

doi:10.1016/j.ejphar.2004.08.042

Cited by 26 publications

(22 citation statements)

References 46 publications

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“…Identification of novel PAMs that preserve native kinetics represents a significant technical hurdle for high-throughput screening methods, requiring a combination of rapid agonist application and subsecond detection of channel activity. Assay formats using ''slowed'' receptors (17,18) may not detect compounds such as compound 6. We instead used native receptors with a traditional electrophysiology format, incorporating a customized fast drug application system (28), allowing high-resolution detection of PAMs that retain rapid kinetics.…”

Section: Discussionmentioning

confidence: 99%

“…To circumvent these problems, high-throughput screening methods have used mutated or chimeric ␣7 nAChRs in which channel kinetics are slowed to assist in detection of channel activity when using plate-reader assay formats (17,18). By using this approach, which actually confounds the ability to detect compounds that preserve native desensitization kinetics, only PAMs like PNU-120596 and others that drastically alter channel kinetics have been identified to date (12,19).…”

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confidence: 99%

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Nootropic α7 nicotinic receptor allosteric modulator derived from GABA _A receptor modulators

Whittemore²,

Tran³

et al. 2007

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

162

213

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Activation of brain ␣7 nicotinic acetylcholine receptors (␣7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of ␣7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective ␣7 nAChR-positive allosteric modulator (PAM) from a library of GABA A receptor PAMs. Compound 6 (N-(4-chlorophenyl)-␣-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at ␣7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of ␣7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.cognition ͉ ion channels ͉ memory ͉ nicotine ͉ schizophrenia

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Nootropic α7 nicotinic receptor allosteric modulator derived from GABA _A receptor modulators

Whittemore²,

Tran³

et al. 2007

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

162

213

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“…A construct containing the human a7 nAChR subunit in plasmid pSP64GL was previously described (Broadbent et al, 2006). Subunit chimeras containing the extracellular domain of either the rat or human nAChR a7 subunit, fused to the transmembrane and C-terminal domains of the mouse 5-HT3A subunit, have also been previously described (Cooper and Millar, 1998;Craig et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

Activation of Human 5-Hydroxytryptamine Type 3 Receptors via an Allosteric Transmembrane Site

et al. 2014

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In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT 3 Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonistevoked responses of 5-HT 3 Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT 3 Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT 3A Rs (64 6 7% and 80 6 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT 3A Rs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT 3A Rs or are able to confer this property on mouse 5-HT 3A Rs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT 3A Rs by 5-HT. We conclude that 5-HT 3A Rs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies.

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“…The earliest reported chimera contained the N-terminal region of chick ␣7 fused to mouse 5-HT 3 (Eiselé et al, 1993). Other reported chimeras involved the N-terminal region of human ␣7 and the rest of sequence encoded by mouse 5-HT 3 (Craig et al, 2004), the N-terminal region of rat ␣7 and the remainder being mouse 5-HT 3 (Young et al, 2008), and the N-terminal sequence of human ␣7 and the remainder of human 5-HT 3A . Additional human ␣7/human 5-HT 3A chimeras were also described previously .…”

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confidence: 99%

In Vitro Pharmacological Characterization of a Novel Allosteric Modulator of α7 Neuronal Acetylcholine Receptor, 4-(5-(4-Chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), Exhibiting Unique Pharmacological Profile

Malysz¹,

Grønlien²,

Anderson³

et al. 2009

J Pharmacol Exp Ther

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Targeting ␣7 neuronal acetylcholine receptors (nAChRs) with selective agonists and positive allosteric modulators (PAMs) is considered a therapeutic approach for managing cognitive deficits in schizophrenia and Alzheimer's disease. In this study, we describe a novel type II ␣7 PAM, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), that exhibits a unique pharmacological profile. In oocytes expressing ␣7 nAChRs, A-867744 potentiated acetylcholine (ACh)-evoked currents, with an EC 50 value of ϳ1 M. At highest concentrations of A-867744 tested, ACh-evoked currents were essentially nondecaying. At lower concentrations, no evidence of a distinct secondary component was evident in contrast to 4-naphthalen-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), another type II ␣7 PAM. In the presence of A-867744, ACh concentration responses were potentiated by increases in potency, Hill slope, and maximal efficacy. When examined in rat hippocampus CA1 stratum radiatum interneurons or dentate gyrus granule cells, A-867744 (10 M) increased choline-evoked ␣7 currents and recovery from inhibition/desensitization, and enhanced spontaneous inhibitory postsynaptic current activity. A-867744, like other ␣7 PAMs tested [1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)urea (NS1738), TQS, and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596)], did not displace the binding of [ 3 H]methyllycaconitine to rat cortex ␣7* nAChRs. However, unlike these PAMs, A-867744 displaced the binding of the agonist 3 H]A-585539 in an ␣7/5-hydroxytryptamine 3 (␣7/5-HT 3 ) chimera, suggesting an interaction distinct from the ␣7 N terminus or M2-3 loop. In addition, A-867744 failed to potentiate responses mediated by 5-HT 3〈 or ␣3␤4 and ␣4␤2 nAChRs. In summary, this study identifies a novel and selective ␣7 PAM showing activity at recombinant and native ␣7 nAChRs exhibiting a unique pharmacological interaction with the receptor.Neuronal nicotinic acetylcholine receptors (nAChRs) belong to the pentameric superfamily of ligand-gated ion channels that includes 5-HT 3 , GABA A/C , and glycine receptors. These receptors are composed of either homomeric ␣ or hetThis work was supported by Abbott. All authors are employees of Abbott. Article, publication date, and citation information can be found at

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Stable expression and characterisation of a human α7 nicotinic subunit chimera: a tool for functional high-throughput screening

Cited by 26 publications

References 46 publications

Nootropic α7 nicotinic receptor allosteric modulator derived from GABA _A receptor modulators

Nootropic α7 nicotinic receptor allosteric modulator derived from GABA _A receptor modulators

Activation of Human 5-Hydroxytryptamine Type 3 Receptors via an Allosteric Transmembrane Site

In Vitro Pharmacological Characterization of a Novel Allosteric Modulator of α7 Neuronal Acetylcholine Receptor, 4-(5-(4-Chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), Exhibiting Unique Pharmacological Profile

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Stable expression and characterisation of a human α7 nicotinic subunit chimera: a tool for functional high-throughput screening

Cited by 26 publications

References 46 publications

Nootropic α7 nicotinic receptor allosteric modulator derived from GABA A receptor modulators

Nootropic α7 nicotinic receptor allosteric modulator derived from GABA A receptor modulators

Activation of Human 5-Hydroxytryptamine Type 3 Receptors via an Allosteric Transmembrane Site

In Vitro Pharmacological Characterization of a Novel Allosteric Modulator of α7 Neuronal Acetylcholine Receptor, 4-(5-(4-Chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), Exhibiting Unique Pharmacological Profile

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Nootropic α7 nicotinic receptor allosteric modulator derived from GABA _A receptor modulators

Nootropic α7 nicotinic receptor allosteric modulator derived from GABA _A receptor modulators