Fluorescent Epibatidine Agonists for Neuronal and Muscle-Type Nicotinic Acetylcholine Receptors

2007 ◽  
Vol 119 (19) ◽  
pp. 3575-3578 ◽  
Author(s):  
Jörg Grandl ◽  
Elias Sakr ◽  
Florence Kotzyba-Hibert ◽  
Florian Krieger ◽  
Sonia Bertrand ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Muscle Type ◽  
Nicotinic Acetylcholine

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

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 603 ◽  
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.

scholarly journals “Weak Toxin” fromNaja kaouthiaIs a Nontoxic Antagonist of α7 and Muscle-type Nicotinic Acetylcholine Receptors

2001 ◽  
Vol 276 (19) ◽  
pp. 15810-15815 ◽  
Author(s):  
Yuri N. Utkin ◽  
Viktoriya V. Kukhtina ◽  
Elena V. Kryukova ◽  
Florence Chiodini ◽  
Daniel Bertrand ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Muscle Type ◽  
Nicotinic Acetylcholine

scholarly journals Interaction of Bupropion with Muscle-Type Nicotinic Acetylcholine Receptors in Different Conformational States

Biochemistry ◽  
2009 ◽  
Vol 48 (21) ◽  
pp. 4506-4518 ◽  
Author(s):  
Hugo R. Arias ◽  
Fernanda Gumilar ◽  
Avraham Rosenberg ◽  
Katarzyna M. Targowska-Duda ◽  
Dominik Feuerbach ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Muscle Type ◽  
Conformational States ◽  
Nicotinic Acetylcholine

Fluorescent Epibatidine Agonists for Neuronal and Muscle-Type Nicotinic Acetylcholine Receptors

2007 ◽  
Vol 46 (19) ◽  
pp. 3505-3508 ◽  
Author(s):  
Jörg Grandl ◽  
Elias Sakr ◽  
Florence Kotzyba-Hibert ◽  
Florian Krieger ◽  
Sonia Bertrand ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Muscle Type ◽  
Nicotinic Acetylcholine

scholarly journals Snake Toxins Labeled by Green Fluorescent Protein or Its Synthetic Chromophore are New Probes for Nicotinic acetylcholine Receptors

2021 ◽  
Vol 8 ◽  
Author(s):  
Igor E. Kasheverov ◽  
Alexey I. Kuzmenkov ◽  
Denis S. Kudryavtsev ◽  
Ivan S. Chudetskiy ◽  
Irina V. Shelukhina ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Fluorescent Protein ◽  
Full Length ◽  
Fluorescent Label ◽  
Muscle Type ◽  
Nicotinic Acetylcholine ◽  
Gfp Chromophore ◽  
Green Fluorescent

Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore (p-HOBDI-BF2) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p-HOBDI-BF2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures.

scholarly journals Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors

2019 ◽  
Vol 476 (8) ◽  
pp. 1285-1302 ◽  
Author(s):  
Yuri N. Utkin ◽  
Ulrich Kuch ◽  
Igor E. Kasheverov ◽  
Dmitry S. Lebedev ◽  
Ella Cederlund ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Dna Analysis ◽  
Binding Mode ◽  
Muscle Type ◽  
Nicotinic Acetylcholine ◽  
Mouse Muscle ◽  
Modeling And Analysis ◽  
Long Chain

Abstract αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α–δ subunits. Three isoforms (αδ-BgTx-1–3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17–0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve–muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α–δ site as compared with α–ε or α–γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs.

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