Newcombe J et al. (2018), Diversity of Nicotinic Acetylcholine Receptor P...

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Mol Pharmacol 2018 Feb 01;932:128-140. doi: 10.1124/mol.117.110551.

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Diversity of Nicotinic Acetylcholine Receptor Positive Allosteric Modulators Revealed by Mutagenesis and a Revised Structural Model.

Newcombe J , Chatzidaki A , Sheppard TD , Topf M , Millar NS .

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By combining electrophysiological and computational approaches we have examined a series of positive allosteric modulators (PAMs) acting on the human α7 nicotinic acetylcholine receptor (nAChR). Electrophysiological studies have focused on three α7-selective PAMs (A-867744, TBS-516, and TQS) that display similar effects on wild-type α7 nAChRs. In addition to potentiating agonist-evoked responses, all three compounds reduce receptor desensitization and, consequently, are classed as type II PAMs. Despite having similar effects on wild-type receptors, A-867744 was found to have profoundly differing effects on mutated receptors compared with TBS-516 and TQS, a finding that is consistent with previous studies indicating that A-867744 may have a different mechanism of action compare with other α7-selective type II PAMs. Due to evidence that these PAMs bind within the α7 nAChR transmembrane region, we generated and validated new structural models of α7. Importantly, we have corrected a previously identified error in the transmembrane region of the original cryo-electron microscopy Torpedo model; the only pentameric ligand-gated ion channel imaged in a native lipid membrane. Real-space refinement was used to generate closed and open conformations on which the α7 models were based. Consensus docking with an extended series of PAMs with chemical similarity to A-867744, TBS-516, and TQS suggests that all bind to a broadly similar intersubunit transmembrane site. However, differences in the predicted binding of A-867744, compared with TBS-516 and TQS, may help to explain the distinct functional effects of A-867744. Thus, our revised structural models may provide a useful tool for interpreting functional effects of PAMs.

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Species referenced: Xenopus
Genes referenced: gabarap sall1

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	Fig. 1. Chemical structures of α7 nAChR PAMs and nAChR mutations. (A) The structure of the three compounds examined by electrophysiological techniques are shown (A-867744, TBS-516, and TQS). In addition, a larger group of PAMs with close chemical similarity to these three compounds (7 compounds similar to A-867744, 3 similar to TBS-516, and 24 similar to TQS) were selected for computer docking studies. Details of the chemical structure of all 37 compounds selected for docking studies is provided in Supplemental Table 1. (B) Location of α7 nAChR amino acids examined by site-directed mutagenesis. Mutated amino acids are indicated as spheres and correspond to W54 (coral), S222 (green), L247 (blue), M253 (yellow), and M260 (red).
	Fig. 2. Competition radioligand binding. Equilibrium radioligand binding was performed with [3H]-α-BTX (1 nM) in human kidney tsA201 cells expressing α7 nAChRs. A-867744 (3 nM to 30 μM) caused no significant displacement of [3H]-α-BTX binding, whereas the orthosteric antagonist MLA caused complete displacement of specific radioligand binding. Data points are means of triplicate samples (±S.E.M.) from a single experiment, and data are typical of three independent experiments.
	Fig. 3. Allosteric modulation of α7M260L by A-867744, TBS-516, and TQS. Mutated α7M260L nAChRs were expressed in Xenopus oocytes and examined by two-electrode voltage-clamp recording. (A) Concentration-response data illustrating agonist activation by TBS-516 and TQS but the absence of agonist activity with A-867744. Data are the mean ± S.E.M. of three independent experiments, each from different oocytes. Data are normalized to the maximum acetylcholine response. (B) Representative traces illustrating responses to acetylcholine (100 μM; left) together with acetylcholine responses from the same oocyte after preapplication (10 seconds) and coapplication of A-867744 (1 μM; right). (C) Representative traces illustrating responses to TQS (30 μM; left) together with TQS responses from the same oocyte after preapplication (10 seconds) and coapplication of A-867744 (1 μM; right). (D) Representative traces illustrating responses to TBS-516 (10 μM; left) together with TBS-516 responses from the same oocyte after preapplication (10 seconds) and coapplication of A-867744 (1 μM; right).
	Fig. 4. Allosteric modulation of α7L247T by A-867744, TBS-516, and TQS. Mutated α7L247T nAChRs were expressed in Xenopus oocytes and examined by two-electrode voltage-clamp recording. (A) Concentration-response data illustrating agonist activation by TBS-516 and TQS but the absence of agonist activity with A-867744. Data are the mean ± S.E.M. of three independent experiments, each from different oocytes. Data are normalized to the maximum acetylcholine response. (B) Representative traces illustrating responses to acetylcholine (10 μM; left) together with acetylcholine responses from the same oocyte after preapplication (10 seconds) and coapplication of A-867744 (1 μM; right). (C) Representative traces illustrating responses to TQS (10 μM; left) together with TQS responses from the same oocyte after preapplication (10 seconds) and coapplication of A-867744 (1 μM; right). (D) Representative traces illustrating responses to TBS-516 (10 μM; left) together with TBS-516 responses from the same oocyte after preapplication (10 seconds) and coapplication of A-867744 (1 μM; right).
	Fig. 5. Allosteric modulation of α7M253L by A-867744, TBS-516, and TQS. Mutated α7M253L nAChRs were expressed in Xenopus oocytes and examined by two-electrode voltage-clamp recording. (A) Representative traces illustrating responses to acetylcholine (100 μM; left), acetylcholine responses after preapplication (10 seconds) and coapplication of TQS (10 μM; middle), and response to acetylcholine after washing (right). (B) Representative traces illustrating responses to acetylcholine (100 μM; left), acetylcholine responses after preapplication (10 seconds) and coapplication of TBS-516 (10 μM; middle), and response to acetylcholine after washing (right). (C) Representative traces illustrating responses to acetylcholine (100 μM; left), and block of acetylcholine responses after preapplication (10 seconds) and coapplication of A-867744 (1 μM; middle). The acetylcholine response did not recover, even after a prolonged (10 minutes) wash (right). (D) Representative traces illustrating responses to acetylcholine (100 μM; left) and after preapplication of TQS (10 seconds), preapplication of A-867744 and TQS (5 seconds), and then coapplication of A-867744 (1 μM) and TQS (10 μM) (middle). TQS was preapplied for 10 seconds and A-867744 for 5 seconds. The acetylcholine response did not recover, even after a prolonged (10 minutes) wash (right). (E) Concentration-response data illustrating inhibition by A-867744 of responses to acetylcholine (100 μM). Data are the mean ± S.E.M. of four independent experiments, each from different oocytes.
	Fig. 6. Allosteric modulation of α7S222M by A-867744, TBS-516, and TQS. Mutated α7S222M nAChRs were expressed in Xenopus oocytes and examined by two-electrode voltage-clamp recording. (A) Representative traces illustrating responses to acetylcholine (100 μM; left), acetylcholine responses after preapplication (10 seconds) and coapplication of TQS (10 μM; middle), and response to acetylcholine after washing (right). (B) Representative traces illustrating responses to acetylcholine (100 μM; left), acetylcholine responses after preapplication (10 seconds) and coapplication of TBS-516 (10 μM; middle), and response to acetylcholine after washing (right). (C) Representative traces illustrating responses to acetylcholine (100 μM; left), block of acetylcholine responses after preapplication (10 seconds) and coapplication of A-867744 (1 μM; middle), and response to acetylcholine after washing (right). (D) Agonist concentration-response curve for acetylcholine in the absence and presence of A-867744 (1.5 μM; preapplied for 10 seconds and then coapplied with acetylcholine). Data are the mean ± S.E.M. of at least three independent experiments and are normalized to the maximum response obtained with ACh in the absence of A-867744. (E) When A-867744 was preapplied and coapplied with acetylcholine, positive modulatory effects (a reduction in agonist-evoked desensitization) was associated with a reduction in peak agonist responses.
	Fig. 7. Allosteric modulation of α7W54A by A-867744, TBS-516, and TQS. Mutated α7W54A nAChRs were expressed in Xenopus oocytes and examined by two-electrode voltage-clamp recording. (A) Concentration-response data illustrating agonist activation by TBS-516 and TQS but the absence of agonist activity with A-867744. Data are the mean ± S.E.M. of three independent experiments, each from different oocytes. Data are normalized to the maximum acetylcholine response. (B) Representative traces illustrating responses to acetylcholine (100 μM; left) together with acetylcholine responses from the same oocyte after preapplication (10 seconds) and coapplication of A-867744 (1 μM; right). (C) Representative traces illustrating responses to TQS (10 μM; left) together with TQS responses from the same oocyte after preapplication (10 seconds) and coapplication of A-867744 (1 μM; right). (D) Representative traces illustrating responses to TBS-516 (10 μM; left) together with TBS-516 responses from the same oocyte after preapplication (10 seconds) and coapplication of A-867744 (1 μM; right).
	Fig. 8. Refinement of the transmembrane domain of the Torpedo nAChR αγ subunit. (A) Sequence alignment and structural superposition of the TM1-TM2 loop region and TM2 helix of the Torpedo αγ subunit (PDB ID 2BG9; chain A) with that of the 5-HT3 receptor (PDB ID 4PIR; chain A) and GLIC (PDB ID 4HFI; chain A). As has been described previously (Supplemental Fig. 21 in Hibbs and Gouaux, 2011), amino acids from the TM1-TM2 loop of the Torpedo nAChR (e.g., Y234) superpose well with homologous amino acids from other pLGIC structures (C239 of 5-HT3R and W217 of GLIC; indicated by a straight line in the sequence alignment). In contrast, amino acids within the nAChR TM2 domain are out of register by ∼1 turn of the α-helix when compared with other pLGIC structures (e.g., compare E262 of the nAChR structure with D267 of 5-HT3R and T241 of GLIC; indicated by an angled line in the sequence alignment). (B) Alignment of amino acid sequence of the β10 strand and TM1 helix of Torpedo nAChR α subunit with that of related pLGICs (top panel). Also shown is the secondary structure before refinement (middle panel) and after refinement (bottom panel) of the Torpedo nAChR structure. Structural information is derived from the following PDB files: nAChR (2BG9; chain A), 5-HT3 receptor (4PIR; chain A), GABAA receptor (4COF; chain A), glycine receptor (3JAD; chain A), and GLIC (4HFI; chain A). Arrows denote β-strands, spirals denote α-helices, conserved residues are highlighted with white text on a red background, and residues with similar properties are highlighted with red text on a white background.
	Fig. 9. Docking of A-867744, TBS-516, and TQS and into α7 structural models. After docking studies with the closed and open models (top and bottom panels, respectively), representatives of binding mode clusters are illustrated for A-867744 (A and D; purple). TBS-516 (B and E; orange), and TQS (C and F; cyan). Amino acids that are discussed in the text are shown in stick representation. Predicted hydrogen bonds are shown with dashed lines. In each case, the principal subunit is shown in khaki (on the left in each panel) and the complimentary subunit is shown in olive (on the right in each panel).
	Fig. 10. Intersubunit transmembrane binding site for PAMs on the α7 nAChR. (A) Representation of the transmembrane domain viewed from above, looking down the axis of the channel pore. Black ellipses indicate the location of the intersubunit allosteric binding site identified in this study. (B) Predicted binding modes in the closed and open receptor models of A-867744 (purple), TBS-516 (orange), and TQS (cyan), shown in relation to transmembrane helices (gray rods) from the principal (+) and the complementary (−) subunit interface. The locations of predicted hydrogen bonds are shown with chain links to denote anchoring of the ligands within the binding site. The locations of amino acids M253, L247, and S222 are shown as yellow, blue, and green circles, respectively. An arrow at the top of TM2 from the complementary subunit denotes the motion required for the change in conformation from the open to the closed channel.

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