Macroscopic and Microscopic Activation of <i>α</i>7 Nicotinic Acetylcholine Receptors by the Structurally Unrelated Allosteric Agonist-Positive Allosteric Modulators (ago-PAMs) B-973B and GAT107

Quadri, Marta; Garai, Sumanta; Thakur, Ganesh A.; Stokes, Clare; Gulsevin, Alican; Horenstein, Nicole A.; Papke, Roger L.

doi:10.1124/mol.118.113340

Cited by 21 publications

(34 citation statements)

References 72 publications

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“…The Br-PBTC-binding site we located overlapped well with that of other known nAChR PAMs that have been docked within the transmembrane domains (31,40). Some PAMs bind at similar intrasubunit cavities as Br-PBTC and dFBr (40), and some bind at intersubunit cavities in the transmembrane domains (25,40,41). A common transmembrane-binding region suggests that perturbation of the transmembrane domains is a critical structural feature needed to potentiate nAChR opening.…”

Section: Discussionsupporting

confidence: 52%

Discovery of an intrasubunit nicotinic acetylcholine receptor–binding site for the positive allosteric modulator Br-PBTC

Norleans

Wang

Kuryatov

et al. 2019

Journal of Biological Chemistry

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Edited by Mike Shipston Nicotinic acetylcholine receptor (nAChR) ligands that lack agonist activity but enhance activation in the presence of an agonist are called positive allosteric modulators (PAMs). nAChR PAMs have therapeutic potential for the treatment of nicotine addiction and several neuropsychiatric disorders. PAMs need to be selectively targeted toward certain nAChR subtypes to tap this potential. We previously discovered a novel PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which selectively potentiates the opening of ␣4␤2*, ␣2␤2*, ␣2␤4*, and (␣4␤4) 2 ␣4 nAChRs and reactivates some of these subtypes when desensitized (* indicates the presence of other subunits). We located the Br-PBTC-binding site through mutagenesis and docking in ␣4. The amino acids Glu-282 and Phe-286 near the extracellular domain on the third transmembrane helix were found to be crucial for Br-PBTC's PAM effect. E282Q abolishes Br-PBTC potentiation. Using (␣4 E282Q ␤2) 2 ␣5 nAChRs, we discovered that the trifluoromethylated derivatives of Br-PBTC can potentiate channel opening of ␣5-containing nAChRs. Mutating Tyr-430 in the ␣5 M4 domain changed ␣5-selectivity among Br-PBTC derivatives. There are two kinds of ␣4 subunits in ␣4␤2 nAChRs. Primary ␣4 forms an agonist-binding site with another ␤2 subunit. Accessory ␣4 forms an agonist-binding site with another ␣4 subunit. The pharmacological effect of Br-PBTC depends both on its own and agonists' occupancy of primary and accessory ␣4 subunits. Br-PBTC reactivates desensitized (␣4␤2) 2 ␣4 nAChRs. Its full efficacy requires intact Br-PBTC sites in at least one accessory and one primary ␣4 subunit. PAM potency increases with higher occupancy of the agonist sites. Br-PBTC and its derivatives should prove useful as ␣ subunit-selective nAChR PAMs.

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Section: Discussionsupporting

confidence: 52%

Discovery of an intrasubunit nicotinic acetylcholine receptor–binding site for the positive allosteric modulator Br-PBTC

Norleans

Wang

Kuryatov

et al. 2019

Journal of Biological Chemistry

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“…The single most important residue for the a7-selective PAM activity of PNU-129596, which is unique to the a7 sequence, was the methionine residue at the 159 position (residue 254 in the human a7). Since the conversion of this residue to a leucine, which is the corresponding residue in most other nAChR subunits (all but a7 and a10), causes the essential loss of a7 PAM activity (Young et al, 2008;Quadri et al, 2019), we tested the effects of the reverse mutation (L159M) on the sensitivity of heteromeric nAChR to PAMs that are normally only effective on a7 receptors.…”

Section: Introductionmentioning

confidence: 99%

Heteromeric Neuronal Nicotinic Acetylcholine Receptors with Mutant β Subunits Acquire Sensitivity to α7-Selective Positive Allosteric Modulators

Stokes

Garai

Kulkarni

et al. 2019

J Pharmacol Exp Ther

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Homomeric a7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by a7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to a7 at the 159 position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L159M) in heteromeric nAChR receptors containing a4 and b2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the a7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c] quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3yl)-urea. The presence of the mutation in the b2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the a4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant b subunit, regardless of the position of the mutant b subunit within the pentameric complex. Similar results were obtained when b2L159M was coexpressed with a2 or a3 and when the L159M mutation was placed in b4 and coexpressed with a2, a3, or a4. Functional receptors were not observed when b1L159M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the a4b2L159M receptor compared with a7 and the availability of high-resolution a4b2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of a7 subunits but are insensitive to PAMs, which greatly increase the open probability of a7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in a7. Specifically, the mutation of the TM2 159 leucine to methionine in a subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal b subunits allows heteromeric receptors to respond to select a7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

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“…It has previously been suggested that the pores formed in PAM-potentiated a7 nAChR are biophysically different than the normal ACh-activated channels (Peng et al, 2013;Quadri et al, This article has not been copyedited and formatted. The final version may differ from this version.…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, there are differences that depend on the actual PAM used (Andersen et al, 2016). Also, PAM-potentiated currents do not show the inward rectification typical of a7 channels activated by ACh alone (Sitzia et al, 2011), and they have different sensitivity to channel blockers (Peng et al, 2013;Quadri et al, 2019). For example, the activation of a7 by the ago-PAM GAT107 is sensitive to mecamylamine, but currents evoked by the alternative ago-PAM B-973B are not (Quadri et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…Also, PAM-potentiated currents do not show the inward rectification typical of a7 channels activated by ACh alone (Sitzia et al, 2011), and they have different sensitivity to channel blockers (Peng et al, 2013;Quadri et al, 2019). For example, the activation of a7 by the ago-PAM GAT107 is sensitive to mecamylamine, but currents evoked by the alternative ago-PAM B-973B are not (Quadri et al, 2019). It has been previously reported that the properties of current rectification and calcium permeability are correlated in both glutamate receptors and nAChRs (Francis and Papke, 1996;Haghighi and Cooper, 2000).…”

Section: Introductionmentioning

confidence: 99%

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Allosterically Potentiated α7 Nicotinic Acetylcholine Receptors: Reduced Calcium Permeability and Current-Independent Control of Intracellular Calcium

et al. 2020

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The currents of a7 nicotinic acetylcholine receptors activated by acetylcholine (ACh) are brief. The channel has high permeability to calcium relative to monovalent cations and shows inward rectification. It has been previously noted that in the presence of positive allosteric modulators (PAMs), currents through the channels of a7 receptors differ from normal a7 currents both in sensitivity to specific channel blockers and their current-voltage (I-V) relationships, no longer showing inward rectification. Linear I-V functions are often associated with channels lacking calcium permeability, so we measured the I-V functions of a7 receptors activated by ACh when PAMs were bound to the allosteric binding site in the transmembrane domain. Currents were recorded in chloride-free Ringer's solution with low or high concentrations of extracellular calcium to determine the magnitude of the reversal potential shift in the two conditions as well as the I-V relationships. ACh-evoked currents potentiated by the ago-PAMs GAT107 and B-973B showed reduced inward rectification and calcium-dependent reversal potential shifts decreased by 80%, and 50%, respectively compared to currents activated by ACh alone, indicative of reduced calcium permeability. TQS-potentiated currents were also linear and showed no calciumdependent reversal potential shifts. The ago-PAMs GAT-107 and B-973B stimulated increases in intracellular calcium in stably transfected HEK293 cells. However, these calcium signals were delayed relative to channel activation produced by these agents and were insensitive to the channel blocker mecamylamine. Our results indicate that, although allosterically-activated a7 nAChR may affect intracellular calcium levels, such effects are not likely due to large channel-dependent calcium influx.

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Macroscopic and Microscopic Activation of α7 Nicotinic Acetylcholine Receptors by the Structurally Unrelated Allosteric Agonist-Positive Allosteric Modulators (ago-PAMs) B-973B and GAT107

Cited by 21 publications

References 72 publications

Discovery of an intrasubunit nicotinic acetylcholine receptor–binding site for the positive allosteric modulator Br-PBTC

Discovery of an intrasubunit nicotinic acetylcholine receptor–binding site for the positive allosteric modulator Br-PBTC

Heteromeric Neuronal Nicotinic Acetylcholine Receptors with Mutant β Subunits Acquire Sensitivity to α7-Selective Positive Allosteric Modulators

Allosterically Potentiated α7 Nicotinic Acetylcholine Receptors: Reduced Calcium Permeability and Current-Independent Control of Intracellular Calcium

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