Glutamate 172, essential for modulation of L 247 T α 7 ACh receptors by Ca 2+ , lines the extracellular vestibule

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␣-7 Nicotinic acetylcholine receptors (AChRs) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick ␣7 AChR, this modulation involves a conserved glutamate in loop 9 (Glu 172 ) that undergoes agonistdependent movements during activation. From these observations, we hypothesized that movements of the nearby ␤-sheet formed by the ␤7, ␤9, and ␤10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis, we examined functional properties of cysteine mutations of the ␤7 and ␤10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the ␤7/

Section: Single Cysteine Mutations Insupporting

confidence: 72%

Section: Methodsmentioning

confidence: 99%

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

McLaughlin¹,

Fu²,

Sproul³

et al. 2006

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“…Mutation at the L9Ј position enhances our ability to measure modification kinetics for cysteine replacements in which the ACh-evoked current amplitudes are attenuated (Beene et al, 2002). All mutations were introduced by site-directed mutagenesis using the QuikChange method (Stratagene, La Jolla, CA) as described previously (Eddins et al, 2002) and were confirmed by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…Currents were low pass-filtered at 50 Hz and sampled at 100 Hz. Agonist dose-response curves were obtained as described previously (Eddins et al, 2002), and data were fit to the Hill equation using Origin software (OriginLab Corp, Northampton, MA).…”

mentioning

confidence: 99%

Agonist-Driven Conformational Changes in the Inner β-Sheet of α7 Nicotinic Receptors

McLaughlin

Fu²,

Rosenberg

2007

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Cys-loop ligand-gated ion channels assemble as pentameric proteins, and each monomer contributes two structural elements: an extracellular ligand-binding domain (LBD) and a transmembrane ion channel domain. Models of receptor activation include rotational movements of subunits leading to opening of the ion channel. We tested this idea using substituted cysteine accessibility to track conformational changes in the inner ␤ sheet of the LBD. Using a nondesensitizing chick ␣7 background (L 247 T), we constructed 18 consecutive cysteine replacement mutants (Leu 36 to Ile 53 ) and tested each for expression of acetylcholine (ACh)-evoked currents and functional sensitivity to thiol modification. We measured rates of modification in the presence and absence of ACh to identify conformational changes associated with receptor activation. Resting modification rates of eight substituted cysteines in the ␤1 and ␤2 strands and the sequence between them (loop 2) varied over several orders of magnitude, suggesting substantial differences in the accessibility or electrostatic environment of individual side chains. These differences were in general agreement with structural models of the LBD. Eight of 18 cysteine replacements displayed ACh-dependent changes in modification rates, indicating a change in the accessibility or electrostatic environment of the introduced cysteine during activation. We were surprised that the effects of agonist exposure were difficult to reconcile with rotational models of activation. Acetylcholine reduced the modification rate of M 40 C but increased it at N 52 C despite the close physical proximity of these residues. Our results suggest that models that depend strictly on rigid-body rotation of the LBD may provide an incomplete description of receptor activation.

“…The amino acid position 172 in the extracellular domain was initially identified by Galzi et al (1996) as the site of action for the calcium modulation, an observation further supported by subsequent sitedirected mutagenesis studies (Eddins et al, 2002;McLaughlin et al, 2006). In contrast, little information is available about the site of modulation by molecules such as IVM, 5-HI, NS-1738, PNU-120596, or other PAMs.…”

mentioning

confidence: 96%

Positive Allosteric Modulation of the α7 Nicotinic Acetylcholine Receptor: Ligand Interactions with Distinct Binding Sites and Evidence for a Prominent Role of the M2-M3 Segment

Bertrand¹,

Bertrand²,

Cassar³

et al. 2008

103

The ␣7 nicotinic acetylcholine receptor (nAChR), a homopentameric, rapidly activating and desensitizing ligand-gated ion channel with relatively high degree of calcium permeability, is expressed in the mammalian central nervous system, including regions associated with cognitive processing. Selective agonists targeting the ␣7 nAChR have shown efficacy in animal models of cognitive dysfunction. Use of positive allosteric modulators selective for the ␣7 receptor is another strategy that is envisaged in the design of active compounds aiming at improving attention and cognitive dysfunction. The recent discovery of novel positive allosteric modulators such as 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethylphenyl)urea (NS-1738) and 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea (PNU-120596) that are selective for the ␣7 nAChRs but display significant phenotypic differences in their profile of allosteric modulation, suggests that these molecules may act at different sites on the receptor. Taking advantage of the possibility to obtain functional receptors by the fusion of proteins domains from the ␣7 and the 5-HT 3 receptor, we examined the structural determinants required for positive allosteric modulation. This strategy revealed that the extracellular N-terminal domain of ␣7 plays a critical role in allosteric modulation by NS-1738. In addition, ␣7-5HT 3 chimeras harboring the M2-M3 segment showed that spontaneous activity in response to NS-1738, which confirmed the critical contribution of this small extracellular segment in the receptor gating. In contrast to NS-1738, positive allosteric modulation by PNU-120596 could not be restored in the ␣7-5HT 3 chimeras but was selectively observed in the reverse 5HT 3 -␣7 chimera. All together, these data illustrate the existence of distinct allosteric binding sites with specificity of different profiles of allosteric modulators and open new possibilities to investigate the ␣7 receptor function.The ␣7 nicotinic acetylcholine receptor (nAChR) belongs to the family of ionotropic receptors that share four transmembrane domains as a common structural feature (for review, see Hogg et al., 2005). By virtue of its high expression levels in brain regions involved in learning and memory, such as hippocampus and cerebral cortex, and its unique physiological properties, partly attributed to a high permeability to Ca 2ϩ , the ␣7 nAChR has received considerable attention as drug target for development of drugs intended to treat cognitive/attention disorders underlying neuropsychiatric and neurodegenerative diseases (for review, see Dani and Bertrand, 2007). Furthermore, despite its low sensitivity to acetylcholine, the ␣7 nAChR has been shown to modulate the release of other neurotransmitters and, in some cases, to contribute directly to signal transmission (for review, see Wonnacott et al., 2006).Gene knock-out and antisense studies together with pharmacological studies using small-molecule selective agonists and positive allosteric modulators have demon...