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.

scholarly journals Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 164
Author(s):  
Lina Son ◽  
Elena Kryukova ◽  
Rustam Ziganshin ◽  
Tatyana Andreeva ◽  
Denis Kudryavtsev ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Gabaa Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Nicotinic Acetylcholine ◽  
High Affinity ◽  
Central Loop ◽  
Acetylcholine Binding Proteins ◽  
Α7 Nachrs

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

[3H]nicotine binding sites are associated with mammalian optic nerve terminals

1988 ◽  
Vol 1 (2) ◽  
pp. 245-248 ◽  
Author(s):  
Glen T. Prusky ◽  
Max S. Cynader
Keyword(s):  
Optic Nerve ◽  
Visual System ◽  
Nicotinic Acetylcholine Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Optic Tract ◽  
Nerve Fibers ◽  
Nerve Terminals ◽  
Nicotinic Acetylcholine ◽  
Retinotectal Transmission

AbstractThe autoradiographic distribution of [3H]nicotine binding sites was examined in the superior colliculus in normal rats and cats, and in animals in which one or both eyes were removed. [3H]Nicotine binding sites in normal animals were densely concentrated in the superficial layers of the colliculus corresponding to the zone of termination of optic nerve fibers. Following bilateral enucleation, [3H]nicotine binding in the superficial collicular layers was drastically reduced. Unilateral enucleation markedly reduced [3H]nicotine binding sites in the colliculus contralateral to the removed eye, with little effect on the ipsilateral colliculus. These results provide further evidence that nicotinic acetylcholine receptors have a presynaptic location on optic tract terminals and may therefore modulate retinotectal transmission in both the rat and cat visual system.

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 Roles of Amino Acids and Subunits in Determining the Inhibition of Nicotinic Acetylcholine Receptors by Competitive Antagonists

2007 ◽  
Vol 106 (6) ◽  
pp. 1186-1195 ◽  
Author(s):  
James P. Dilger ◽  
Ana Maria Vidal ◽  
Man Liu ◽  
Claire Mettewie ◽  
Takahiro Suzuki ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Hek293 Cells ◽  
Wild Type ◽  
Binding Studies ◽  
Response Curves ◽  
Nicotinic Acetylcholine ◽  
Mouse Muscle ◽  
Epsilon Subunit

Background Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the alpha-delta and alpha-epsilon subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the alpha-epsilon interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. Methods HEK293 cells were transfected with wild-type or mutant (alphaY198F, epsilonD59A, epsilonD59N, epsilonD173A, epsilonD173N, deltaD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. Results Effects of mutations were antagonist specific. alphaY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to epsilonD173 mutations. epsilonD59 mutations had large effects on metocurine and cisatracurium. deltaD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two delta subunits but no epsilon subunit. Differences in IC50 arose from differences in both dissociation and association rates. Conclusion Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the alpha-epsilon interface. Pancuronium, vecuronium, and cisatracurium were selective for the alpha-delta interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.

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