Collins T et al. (2011), Competitive binding at a nicotinic receptor tra...

XB-ART-45752

Neuropharmacology 2011 Dec 01;618:1306-13. doi: 10.1016/j.neuropharm.2011.07.035.

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Competitive binding at a nicotinic receptor transmembrane site of two α7-selective positive allosteric modulators with differing effects on agonist-evoked desensitization.

Collins T , Young GT , Millar NS .

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Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as a novel area of therapeutic drug discovery. Two types of α7-selective PAMs have been identified (type I and type II). Whilst both potentiate peak agonist-induced responses, they have different effects on the rate of agonist-induced receptor desensitization. Type I PAMs have little or no effect on the rapid rate of desensitization that is characteristic of α7 nAChRs, whereas type II PAMs cause dramatic slowing of receptor desensitization. Previously, we have obtained evidence indicating that PNU-120596, a type II PAM, causes potentiation by interacting with an allosteric transmembrane site. In contrast, other studies have demonstrated the importance of the 'M2-M3 segment' in modulating the effects of the type I PAM NS1738 and have led to the proposal that NS1738 may interact with the extracellular N-terminal domain. Here, our aim has been to compare the mechanism of allosteric potentiation of α7 nAChRs by NS1738 and PNU-120596. Functional characterization of a series of mutated α7 nAChRs indicates that mutation of amino acids within a proposed intrasubunit transmembrane cavity have a broadly similar effect on these two PAMs. In addition, we have employed a functional assay designed to examine the ability of ligands to act competitively at either the orthosteric or allosteric binding site of α7 nAChRs. These data, together with computer docking simulations, lead us to conclude that both the type I PAM NS1738 and the type II PAM PNU-120596 bind competitively at a mutually exclusive intrasubunit transmembrane site.

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Species referenced: Xenopus laevis
Genes referenced: chrna4 pam tpm3

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	Fig. 1. Chemical structure of α7-selective potentiators NS1738 and PNU-120596. Both NS1738 (a type I PAM) and PNU-120596 (a type II PAM) are urea compounds containing a halogenated aromatic group.
	Fig. 2. Positive allosteric modulation of α7 nAChRs by NS1738 and PNU-120596. Representative traces are shown illustrating agonist-evoked responses with α7 nAChRs expressed in Xenopus oocytes. Application of acetylcholine (1 mM) resulted in rapidly desensitizing responses that are typical of α7 nAChRs (left hand traces). Pre-application (20 s) followed by co-application of either NS1738 (10 μM) or PNU-120596 (3 μM) resulted in potentiation of responses evoked by acetylcholine (right panels). In contrast, no agonist activity was observed when NS1738 (10 μM) and PNU-120596 (3 μM) were applied in the absence of acetylcholine (middle traces). Applications of acetylcholine and PAMs are indicated by horizontal lines.
	Fig. 3. Influence of α7 nAChR transmembrane point mutations upon potentiation by NS1738 and PNU-120596. Histograms illustrating the influence of α7 point mutations on the level of potentiation caused by maximal concentrations of NS1738 (10 μM; open bars) and PNU-120596 (3 μM; filled bars). Potentiation of agonist-evoked responses were determined with an EC20 concentration of acetylcholine (see Table 1). Data are means ± SEM of 3–10 independent experiments (***P < 0.001; Student’s t-test). Data for PNU-120596 are taken from (Young et al., 2008).
	Fig. 4. Altered receptor desensitization in α7 nAChRs containing the M260L mutation. A) Model of the TM2 and TM3 transmembrane region derived from the α7 nAChR homology model (Cheng et al., 2007). The location of a 10 amino acid region ‘M2-M3 segment’ (AEIMPATSDS) described by Bertrand et al. (2008) is shaded in black. Also illustrated (as black spheres) is the side chain of M260, the amino acid that when mutated has a selective effect on NS1738. B) Differences in desensitization of wild type α7 and α7M260L nAChRs are illustrated by responses to 5 s applications of an EC20 concentration of acetylcholine. Solid lines represent normalized means of 9 (wild type α7) or 10 (α7M260L) independent recordings. Dotted lines represent SEMs.
	Fig. 5. PNU-120596 (a type II PAM) facilitates recovery of α7 nAChRs from desensitization. Exposure of α7 nAChR to a high concentration of acetylcholine (1 mM) resulted in rapid desensitization. The subsequent co-application of NS1738 (10 μM) to desensitized α7 nAChRs had little effect (left hand trace). In contrast, the co-application of PNU-120596 after desensitization by acetylcholine (3 μM) causes rapid opening, indicating a recovery from the receptor’s desensitized state (right hand trace).
	Fig. 6. Block of recovery from desensitization by NS1738 and MLA. The ability of PNU-120596 to cause recovery from desensitization of α7 nAChRs is blocked by NS1738 and MLA. A) Representative trace showing activation of α7 nAChRs followed by rapid desensitization in response to acetylcholine (1 mM; lower horizontal line). Subsequent application of PNU-120596 (1 μM; middle horizontal line) allows recovery from desensitization. Recovery can be blocked by subsequent application of NS1738 (upper horizontal line). B) Dose-response curves showing the ability of NS1738 (filled circles) and MLA (filled squares) to block recovery from desensitization caused by co-application of acetylcholine (1 mM) and PNU-120596 (1 μM). Also shown (open circles) is the dose-response curve for NS1738 obtained when the experiment was performed in the presence of the same concentration of acetylcholine but with a higher concentration of PNU-120596 (3 μM). Data are means ± SEM of 3–7 independent experiments.
	Fig. 7. Computer docking simulation with a homology model of the α7 transmembrane domain. Computer docking simulations were performed with AutoDock 4 using a homology model of the α7 nAChR transmembrane domain (Cheng et al., 2007; Young et al., 2008). The backbone of the four transmembrane α helices (TM1-TM4) is shown in grey and the side chains of amino acids which, when mutated, had a significant effect on potentiation by both NS1738 and PNU-120596 (S222, A225, M253, F455 and C459) are shaded in red. The side chain of M260 (an amino acid that, when mutated, reduced receptor desensitization and had a selective effect on potentiation by NS1738) is shown in blue. The backbone region corresponding to the 10 amino acid ‘M2-M3 segment’, that has been described previously as having a selective effect on potentiation by NS1738 (Bertrand et al., 2008), is also shown in blue. The lowest energy (highest predicted binding affinity) docked positions of NS1738 and PNU-120596 are shown in yellow and green, respectively. Initially, a ‘blind docking’ approach was used in which no assumptions were made as to where within the transmembrane region the compounds might bind (left panel). Subsequently, more energetically favourable docked conformations were identified by conducting further simulations focussed upon the central region of the model (where the of the lowest energy docked conformations are located) using a modified homology model in which flexibility was permitted within the side chains of the five amino acid that have been identified as influencing potentiation of NS1738 and PNU-120596 (right panel). For clarity, the lower part of the TM3 α-helical domain has been omitted from the figure.

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