scholarly journals Structure-Function Elucidation of a New α-Conotoxin, MilIA, from Conus milneedwardsi

Marine Drugs ◽  
2019 ◽  
Vol 17 (9) ◽  
pp. 535 ◽  
Author(s):  
Steve Peigneur ◽  
Prabha Devi ◽  
Andrea Seldeslachts ◽  
Samuthirapandian Ravichandran ◽  
Loïc Quinton ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Xenopus Laevis Oocytes ◽  
Muscle Type ◽  
Isolation And Characterization ◽  
Neuronal Types ◽  
Peptide Toxins ◽  
Neuronal Subtype ◽  
Key Residues

The a-Conotoxins are peptide toxins that are found in the venom of marine cone snails and they are potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies. We describe the isolation and characterization of α-conotoxin MilIA, the first conopeptide from the venom of Conus milneedwardsi. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs that were expressed in Xenopus laevis oocytes. MilIA, which is a member of the α3/5 family, is an antagonist of muscle type nAChRs with a high selectivity for muscle versus neuronal subtype nAChRs. Several analogues were designed and investigated for their activity in order to determine the key epitopes of MilIA. Native MilIA and analogues both showed activity at the fetal muscle type nAChR. Two single mutations (Met9 and Asn10) allowed for MilIA to strongly discriminate between the two types of muscle nAChRs. Moreover, one analogue, MilIA [∆1,M2R, M9G, N10K, H11K], displayed a remarkable enhanced potency when compared to native peptide. The key residues that are responsible for switching between muscle and neuronal nAChRs preference were elucidated. Interestingly, the same analogue showed a preference for α9α10 nAChRs among the neuronal types.

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 The effective opening of nicotinic acetylcholine receptors with single agonist binding sites

2011 ◽  
Vol 137 (4) ◽  
pp. 369-384 ◽  
Author(s):  
Dustin K. Williams ◽  
Clare Stokes ◽  
Nicole A. Horenstein ◽  
Roger L. Papke
Keyword(s):  
Binding Site ◽  
Nicotinic Acetylcholine Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Muscle Type ◽  
Net Charge ◽  
Single Binding Site

We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of α7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed α7L119C subunits with pseudo wild-type α7C116S subunits, as well as ACh-insensitive α7Y188F subunits with wild-type α7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant α7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 µM ACh were retained by α7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be fewer than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and α1β1γδL121C receptors were equivalent until the α1β1γδL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (≤2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists.

Isolation and characterization of α-conotoxin LsIA with potent activity at nicotinic acetylcholine receptors

2013 ◽  
Vol 86 (6) ◽  
pp. 791-799 ◽  
Author(s):  
Marco C. Inserra ◽  
Shiva N. Kompella ◽  
Irina Vetter ◽  
Andreas Brust ◽  
Norelle L. Daly ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nicotinic Acetylcholine ◽  
Isolation And Characterization

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

Molecules ◽  
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.

Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells

2014 ◽  
Vol 1542 ◽  
pp. 1-11 ◽  
Author(s):  
Andreas H. Rasmussen ◽  
Dorte Strøbæk ◽  
Tino Dyhring ◽  
Marianne L. Jensen ◽  
Dan Peters ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Hek293 Cells ◽  
Pharmacological Characterization ◽  
Nicotinic Acetylcholine

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

Marine Drugs ◽  
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.

Molecular characterization of the specificity of interactions of various neurotoxins on two distinct nicotinic acetylcholine receptors

2000 ◽  
Vol 393 (1-3) ◽  
pp. 197-204 ◽  
Author(s):  
Denis Servent ◽  
Stéphanie Antil-Delbeke ◽  
Carole Gaillard ◽  
Pierre-Jean Corringer ◽  
Jean Pierre Changeux ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nicotinic Acetylcholine
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