Non-Equivalent Ligand Selectivity of Agonist Sites in (α4β2)2α4 Nicotinic Acetylcholine Receptors: A Key Determinant of Agonist Efficacy

Mazzaferro, Simone; Gasparri, Federica; New, Karina; Alcaino, Constanza; Bermúdez, Isabel

doi:10.1016/j.bpj.2014.11.2344

Cited by 13 publications

(27 citation statements)

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“…The additional agonist site on the (␣4␤2) 2 ␣4 receptor receptors plays a dominant role in determining the signature properties of this receptor type, as compared with the (␣4␤2) 2 ␤2 receptor (29 -31). When the additional agonist site is ablated, (␣4␤2) 2 ␣4 receptor displays (␣4␤2) 2 ␤2-like pharmacology (30,31), including sensitivity to agonists and allosteric modulators (11,20,21). Significantly, introducing ␣4F312A in one of the two agonist sites of the (␣4␤2) 2 ␤2 receptor reduced dFBr efficacy by ϳ50% (Fig.…”

Section: Resultsmentioning

confidence: 99%

Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors

Alcaino

Musgaard

Minguez

et al. 2017

Journal of Biological Chemistry

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Edited by F. Anne StephensonAllosteric modulators of pentameric ligand-gated ion channels are thought to act on elements of the pathways that couple agonist binding to channel gating. Using ␣4␤2 nicotinic acetylcholine receptors and the ␣4␤2-selective positive modulators 17␤-estradiol (␤EST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pentameric ligand-gated ion channels. Previous studies have shown that the binding site for potentiating ␤EST is in the C-terminal (post-M4) region of the ␣4 subunit. Here, using homology modeling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top half of a cavity between the third (M3) and fourth transmembrane (M4) ␣-helices of the ␣4 subunit. We found that the binding sites for ␤EST and dFBr communicate with the Cys loop, through interactions between the last residue of post-M4 and Phe 170 of the conserved FPF sequence of the Cys loop, and that these interactions affect potentiating efficacy. In addition, interactions between a residue in M3 (Tyr 309 ) and Phe 167 , a residue adjacent to the Cys loop FPF motif, also affect dFBr potentiating efficacy. Thus, the Cys loop acts as a key control element in the allosteric transduction pathway for potentiating ␤EST and dFBr. Overall, we propose that positive allosteric modulators that bind the M3-M4 cavity or post-M4 region increase the efficacy of channel gating through interactions with the Cys loop.The ␣4␤2 nicotinic acetylcholine receptor (nAChR) 2 belongs to the superfamily of pentameric ligand-gated ion channels (pLGICs). In humans, this superfamily comprises the Cys loop receptors (including muscle and neuronal nAChRs, 5-HT 3 , GABA A , and glycine receptors), which mediate all fast central nervous system synaptic inhibition and much of fast peripheral excitation. Cys loop receptor subunits assemble as a pentamer of identical (homomeric channels) or different (heteromeric channels) subunits around a central ion channel. The individual subunits have a large extracellular N-terminal domain (ECD) that consists of 10 ␤-strands (␤1-␤10) folded into a ␤-sandwich and a transmembrane domain (TMD) with four transmembrane ␣ helices (M1-M4) connected by linkers (M1-M2, M2-M3, and M3-M4), as well as intracellular domains and a highly variable extracellular C-terminal (post-M4) region (1). There are 2-5 neurotransmitter binding sites at subunit interfaces within the ECD, and these sites are functionally coupled to the transmembrane ion channel located ϳ50 Å away. In the nAChR, the subunit contributing the principal face of the agonist binding site (an ␣ subunit) couples agonist-triggered agonist binding site movements to channel gating (2, 3). The coupling is achieved at the ECD-TMD interface by a principal pathway that couples the pre-M1 region in the ECD to the M2-M3 linker through the ␤1-␤2 loop (4) and the canonical FPF motif of the ␤6-...

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Section: Resultsmentioning

confidence: 99%

Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors

Alcaino

Musgaard

Minguez

et al. 2017

Journal of Biological Chemistry

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“…A recent study has suggested that sazetidine-A is excluded from binding at the a-a interface because of steric clashes with a4H142 (Mazzaferro et al, 2014). However, if this were the case, it could be expected that NS3573-which, like sazetidine-A, contains a large substituent in the pyridine 5-position-would be affected in a similar way.…”

Section: Discussionmentioning

confidence: 99%

Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

Shahsavar¹,

Ahring²,

Olsen³

et al. 2015

Mol Pharmacol

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Neuronal a4b2 nicotinic acetylcholine receptors are attractive drug targets for psychiatric and neurodegenerative disorders and smoking cessation aids. Recently, a third agonist binding site between two a4 subunits in the (a4) 3 (b2) 2 receptor subpopulation was discovered. In particular, three residues, H142, Q150, and T152, were demonstrated to be involved in the distinct pharmacology of the a4-a4 versus a4-b2 binding sites. To obtain insight into the three-dimensional structure of the a4-a4 binding site, a surrogate protein reproducing a4-a4 binding characteristics was constructed by introduction of three point mutations, R104H, L112Q, and M114T, into the binding pocket of Lymnaea stagnalis acetylcholine-binding protein (Ls-AChBP). Cocrystallization with two agonists possessing distinct pharmacologic profiles, NS3920 [1-(6-bromopyridin-3-yl)-1,4-diazepane] and NS3573 [1-(5-ethoxypyridin-3-yl)-1,4-diazepane], highlights the roles of the three residues in determining binding affinities and functional properties of ligands at the a4-a4 interface. Confirmed by mutational studies, our structures suggest a unique ligandspecific role of residue H142 on the a4 subunit. In the cocrystal structure of the mutated Ls-AChBP with the high-efficacy ligand NS3920, the corresponding histidine forms an intersubunit bridge that reinforces the ligand-mediated interactions between subunits. The structures further reveal that the binding site residues gain different and ligand-dependent interactions that could not be predicted based on wild-type Ls-AChBP structures in complex with the same agonists. The results show that an unprecedented correlation between binding in engineered AChBPs and functional receptors can be obtained and provide new opportunities for structure-based design of drugs targeting specific nicotinic acetylcholine receptor interfaces.

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“…Partial agonism of sazetidine arises from its exclusive and high affinity action at the ␣4/␤2 agonist sites; i.e. sazetidine is a primary SSAg (21,22). Cytisine is an intrinsic partial agonist that non-selectively binds to both the ␣4/␤2 and ␣4/␣4 agonist sites (20).…”

Section: Ns9283 Increases Activation Bymentioning

confidence: 99%

“…The drug sazetidine is a full agonist at ␣4/␤2 primary agonist sites but does not bind to the ␣4/␣4 accessory ACh binding site (20 -22). Cytisine acts at both types of ACh binding sites as a partial agonist (16,20,22). Dihydro-␤-erythroidine is an antagonist at both ␣4/␤2 and ␣4/␣4 ACh binding sites (3).…”

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confidence: 99%

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors

Wang

Kuryatov

Sriram

et al. 2015

Journal of Biological Chemistry

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Background: In (␣4␤2) 2 ␣4 nicotinic acetylcholine receptors, there is an agonist binding site at the ␣4/␣4 subunit interface. Results: ␣2, ␣3, and ␣6 accessory subunits can form an agonist site with ␣4. These promote activation upon agonist binding at ␣4/␤2 agonist sites. Conclusion: Accessory subunit agonist sites greatly influence receptor function. Significance: These sites are promising drug targets.

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Non-Equivalent Ligand Selectivity of Agonist Sites in (α4β2)2α4 Nicotinic Acetylcholine Receptors: A Key Determinant of Agonist Efficacy

Cited by 13 publications

References 0 publications

Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors

Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors

Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors

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