Tetrapeptide Ac-HAEE-NH2 Protects α4β2 nAChR from Inhibition by Aβ

The cholinergic deficit in Alzheimer’s disease (AD) may arise from selective loss of cholinergic neurons caused by the binding of Aβ peptide to nicotinic acetylcholine receptors (nAChRs). Thus, compounds preventing such an interaction are needed to address the cholinergic dysfunction. Recent findings suggest that the 11EVHH14 site in Aβ peptide mediates its interaction with α4β2 nAChR. This site contains several charged amino acid residues, hence we hypothesized that the formation of Aβ-α4β2 nAChR complex is based on the interaction of 11EVHH14 with its charge-complementary counterpart in α4β2 nAChR. Indeed, we discovered a 35HAEE38 site in α4β2 nAChR, which is charge-complementary to 11EVHH14, and molecular modeling showed that a stable Aβ42-α4β2 nAChR complex could be formed via the 11EVHH14:35HAEE38 interface. Using surface plasmon resonance and bioinformatics approaches, we further showed that a corresponding tetrapeptide Ac-HAEE-NH2 can bind to Aβ via 11EVHH14 site. Finally, using two-electrode voltage clamp in Xenopus laevis oocytes, we showed that Ac-HAEE-NH2 tetrapeptide completely abolishes the Aβ42-induced inhibition of α4β2 nAChR. Thus, we suggest that 35HAEE38 is a potential binding site for Aβ on α4β2 nAChR and Ac-HAEE-NH2 tetrapeptide corresponding to this site is a potential therapeutic for the treatment of α4β2 nAChR-dependent cholinergic dysfunction in AD.

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The Caenorhabditis elegans DEG-3/DES-2 Channel Is a Betaine-Gated Receptor Insensitive to Monepantel

Molecules ◽

10.3390/molecules27010312 ◽

2022 ◽

Vol 27 (1) ◽

pp. 312

Author(s):

Tina V. A. Hansen ◽

Heinz Sager ◽

Céline E. Toutain ◽

Elise Courtot ◽

Cédric Neveu ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Parasitic Nematode ◽

Nematode Species ◽

Xenopus Laevis Oocytes ◽

Allosteric Modulators ◽

C Elegans ◽

Electrode Voltage ◽

Insight Into

Natural plant compounds, such as betaine, are described to have nematocidal properties. Betaine also acts as a neurotransmitter in the free-living model nematode Caenorhabditis elegans, where it is required for normal motility. Worm motility is mediated by nicotinic acetylcholine receptors (nAChRs), including subunits from the nematode-specific DEG-3 group. Not all types of nAChRs in this group are associated with motility, and one of these is the DEG-3/DES-2 channel from C. elegans, which is involved in nociception and possibly chemotaxis. Interestingly, the activity of DEG-3/DES-2 channel from the parasitic nematode of ruminants, Haemonchus contortus, is modulated by monepantel and its sulfone metabolite, which belong to the amino-acetonitrile derivative anthelmintic drug class. Here, our aim was to advance the pharmacological knowledge of the DEG-3/DES-2 channel from C. elegans by functionally expressing the DEG-3/DES-2 channel in Xenopus laevis oocytes and using two-electrode voltage-clamp electrophysiology. We found that the DEG-3/DES-2 channel was more sensitive to betaine than ACh and choline, but insensitive to monepantel and monepantel sulfone when used as direct agonists and as allosteric modulators in co-application with betaine. These findings provide important insight into the pharmacology of DEG-3/DES-2 from C. elegans and highlight the pharmacological differences between non-parasitic and parasitic nematode species.

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Structural correlates of human muscle nicotinic acetylcholine receptor subunit assembly mediated by δ(+) interface residues

10.1101/2020.06.11.145466 ◽

2020 ◽

Author(s):

Max Epstein ◽

Susan Maxwell ◽

Thomas J. Piggot ◽

David Beeson ◽

Isabel Bermudez ◽

...

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Dynamical Behavior ◽

Human Muscle ◽

Amino Acid Residues ◽

Nicotinic Acetylcholine ◽

Specific Amino Acid ◽

Functional Differences ◽

Electrode Voltage ◽

Dynamics Simulations

AbstractMuscle nicotinic acetylcholine receptors are a class of heteropentameric ligand-gated cation channels with constituent subunits adopting a fixed stoichiometric arrangement. The specific amino acid residues that govern subunit ordering are however, only partially understood. By integrating all-atom molecular dynamics simulations, bioinformatics, two-electrode voltage clamp electrophysiology and 125I-α-bungarotoxin assays of chimeric nAChR subunits, we identify residues across the extracellular, transmembrane and extended M4 helix of the δ subunit that make structural signatures that contribute to intransigent assembly rules. Furthermore, functional differences observed in α2δ2β receptors can be rationalized by changes in dynamical behavior that manifest themselves at the agonist binding site.

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α-Conotoxin as Potential to α7-nAChR Recombinant Expressed in Escherichia coli

Marine Drugs ◽

10.3390/md18080422 ◽

2020 ◽

Vol 18 (8) ◽

pp. 422

Author(s):

Yanli Liu ◽

Yifeng Yin ◽

Yunyang Song ◽

Kang Wang ◽

Fanghui Wu ◽

...

Keyword(s):

Escherichia Coli ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

High Performance ◽

Cognitive Disorders ◽

Coli Strain ◽

Xenopus Laevis Oocytes ◽

Obvious Effect ◽

Nicotinic Acetylcholine ◽

Α7 Nachr

α7 nicotinic acetylcholine receptors (nAChR) is an important nicotinic acetylcholine receptors subtype and closely associated with cognitive disorders, such as Alzheimer’s and schizophrenia disease. The mutant ArIB (V11L, V16A) of α-conotoxin ArIB with 17-amino acid residues specifically targets α7 nAChR with no obvious effect on other nAChR subtypes. In the study, the synthetic gene encoding mature peptide of ArIB and mutant ArIB (V11L, V16A) carried a fusion protein Trx and 6 × His-tag was separately inserted in pET-32a (+) vector and transformed into Escherichia coli strain BL21(DE3) pLysS for expression. The expressions of Trx-ArIB-His6 and Trx-ArIB (V11L, V16A)-His6 were soluble in Escherichia coli, which were purified by Ni-NTA affinity chromatography column and cleaved by enterokinase to release rArIB and rArIB (V11L, V16A). Then, rArIB and rArIB (V11L, V16A) were purified by high-performance liquid chromatography (HPLC) and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bioactivity of rArIB and rArIB (V11L, V16A) was assessed by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing human nAChR subtypes. The results indicated that the yield of the fusion proteins was approximately 50 mg/L and rArIB (V11L, V16A) antagonized the α7 nAChR subtype selectively with 8-nM IC50. In summary, this study provides an efficient method to biosynthesize α-conotoxin ArIB and rArIB (V11L, V16A) in Escherichia coli, which could be economical to obtain massively bioactive disulfide-rich polypeptides at fast speed.

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Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

Invertebrate Neuroscience ◽

10.1007/s10158-003-0025-1 ◽

2003 ◽

Vol 5 (1) ◽

pp. 29-35 ◽

Cited By ~ 17

Author(s):

Val�rie Raymond Delpech ◽

Makoto Ihara ◽

Claudio Coddou ◽

Kazuhiko Matsuda ◽

David B. Sattelle

Keyword(s):

Xenopus Laevis ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Xenopus Laevis Oocytes ◽

Neuronal Nicotinic Acetylcholine Receptors ◽

Nicotinic Acetylcholine

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Identification of Crucial Residues in α-Conotoxin EI Inhibiting Muscle Nicotinic Acetylcholine Receptor

Toxins ◽

10.3390/toxins11100603 ◽

2019 ◽

Vol 11 (10) ◽

pp. 603 ◽

Cited By ~ 2

Author(s):

Jiong Ning ◽

Jie Ren ◽

Yang Xiong ◽

Yong Wu ◽

Manqi Zhangsun ◽

...

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Fold Increase ◽

Cd Spectroscopy ◽

Xenopus Laevis Oocytes ◽

Muscle Type ◽

Nicotinic Acetylcholine ◽

Activity Loss ◽

Spectroscopy Studies ◽

Key Residues

α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure–activity relationship (SAR) of EI revealed that the “Arg1–Asn2–Hyp3” residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1–3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1–3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.

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Distribution of Cholinergic Neurons in the Mammalian Brain with Special Reference to their Relationship with Neuronal Nicotinic Acetylcholine Receptors

Handbook of Experimental Pharmacology - Neuronal Nicotinic Receptors ◽

10.1007/978-3-642-57079-7_2 ◽

2000 ◽

pp. 13-30 ◽

Cited By ~ 12

Author(s):

M. Zoli

Keyword(s):

Special Reference ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Cholinergic Neurons ◽

Mammalian Brain ◽

Neuronal Nicotinic Acetylcholine Receptors ◽

Nicotinic Acetylcholine

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Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region.

The Journal of General Physiology ◽

10.1085/jgp.99.4.545 ◽

1992 ◽

Vol 99 (4) ◽

pp. 545-572 ◽

Cited By ~ 44

Author(s):

B N Cohen ◽

C Labarca ◽

L Czyzyk ◽

N Davidson ◽

H A Lester

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Ion Selectivity ◽

Point Mutations ◽

Reversal Potential ◽

Amino Acid Residues ◽

Wild Type ◽

Mouse Muscle ◽

Na Permeability ◽

Delta Subunit

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.

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