scholarly journals Cell-Free Expression of Sodium Channel Domains for Pharmacology Studies. Noncanonical Spider Toxin Binding Site in the Second Voltage-Sensing Domain of Human Nav1.4 Channel

2019 ◽  
Vol 10 ◽  
Author(s):  
Mikhail Yu. Myshkin ◽  
Roope Männikkö ◽  
Olesya A. Krumkacheva ◽  
Dmitrii S. Kulbatskii ◽  
Anton O. Chugunov ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Binding Site ◽  
Free Expression ◽  
Voltage Sensing ◽  
Toxin Binding ◽  
Spider Toxin ◽  
Voltage Sensing Domain

scholarly journals Voltage-Sensing Domain of the Third Repeat of Human Skeletal Muscle NaV1.4 Channel As a New Target for Spider Gating Modifier Toxins

Acta Naturae ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 134-139
Author(s):  
M. Yu. Myshkin ◽  
A. S. Paramonov ◽  
D. S. Kulbatsky ◽  
E. A. Surkova ◽  
A. A. Berkut ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
New Drugs ◽  
Modular Architecture ◽  
Voltage Sensing ◽  
Toxin Binding ◽  
Voltage Gated Sodium Channels ◽  
Nav Channels ◽  
Gating Modifier ◽  
Voltage Sensing Domain

Voltage-gated sodium channels (NaV) have a modular architecture and contain five membrane domains. The central pore domain is responsible for ion conduction and contains a selectivity filter, while the four peripheral voltage-sensing domains (VSD-I/IV) are responsible for activation and rapid inactivation of the channel. Gating modifier toxins from arthropod venoms interact with VSDs, influencing the activation and/or inactivation of the channel, and may serve as prototypes of new drugs for the treatment of various channelopathies and pain syndromes. The toxin-binding sites located on VSD-I, II and IV of mammalian NaV channels have been previously described. In this work, using the example of the Hm-3 toxin from the crab spider Heriaeus melloteei, we showed the presence of a toxin-binding site on VSD-III of the human skeletal muscle NaV1.4 channel. A developed cell-free protein synthesis system provided milligram quantities of isolated (separated from the channel) VSD-III and its 15N-labeled analogue. The interactions between VSD-III and Hm-3 were studied by NMR spectroscopy in the membrane-like environment of DPC/LDAO (1 : 1) micelles. Hm-3 has a relatively high affinity to VSD-III (dissociation constant of the complex Kd ~6 M), comparable to the affinity to VSD-I and exceeding the affinity to VSD-II. Within the complex, the positively charged Lys25 and Lys28 residues of the toxin probably interact with the S1S2 extracellular loop of VSD-III. The Hm-3 molecule also contacts the lipid bilayer surrounding the channel.

scholarly journals Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-Like Toxin Binding Properties

Toxins ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 722
Author(s):  
Ryan Schroder ◽  
Leah Cohen ◽  
Ping Wang ◽  
Joekeem Arizala ◽  
Sébastien Poget
Keyword(s):  
Pain Perception ◽  
Dissociation Constants ◽  
Recombinant Expression ◽  
Single Amino Acid ◽  
Binding Studies ◽  
Voltage Sensing ◽  
Toxin Binding ◽  
Channel Inhibition ◽  
Gating Modifier ◽  
Voltage Sensing Domain

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.

scholarly journals The Guanidinium Toxin Binding Site on the Sodium Channel.

1996 ◽  
Vol 37 (5) ◽  
pp. 683-692 ◽  
Author(s):  
Harry A. FOZZARD ◽  
Gregory LIPKIND
Keyword(s):  
Sodium Channel ◽  
Binding Site ◽  
Toxin Binding

scholarly journals Voltage-Sensing Domain of the Third Repeat of Human Skeletal Muscle NaV1.4 Channel As a New Target for Spider Gating Modifier Toxins

Acta Naturae ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 134-139
Author(s):  
Mikhail Yu. Myshkin ◽  
Alexander S. Paramonov ◽  
Dmitrii S. Kulbatskii ◽  
Yelizaveta A. Surkova ◽  
Antonina A. Berkut ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
New Drugs ◽  
Modular Architecture ◽  
Voltage Sensing ◽  
Toxin Binding ◽  
Voltage Gated Sodium Channels ◽  
Nav Channels ◽  
Gating Modifier ◽  
Voltage Sensing Domain

Voltage-gated sodium channels (NaV) have a modular architecture and contain five membrane domains. The central pore domain is responsible for ion conduction and contains a selectivity filter, while the four peripheral voltage-sensing domains (VSD-I/IV) are responsible for activation and rapid inactivation of the channel. Gating modifier toxins from arthropod venoms interact with VSDs, influencing the activation and/or inactivation of the channel, and may serve as prototypes of new drugs for the treatment of various channelopathies and pain syndromes. The toxin-binding sites located on VSD-I, II and IV of mammalian NaV channels have been previously described. In this work, using the example of the Hm-3 toxin from the crab spider Heriaeus melloteei, we showed the presence of a toxin-binding site on VSD-III of the human skeletal muscle NaV1.4 channel. A developed cell-free protein synthesis system provided milligram quantities of isolated (separated from the channel) VSD-III and its 15N-labeled analogue. The interactions between VSD-III and Hm-3 were studied by NMR spectroscopy in the membrane-like environment of DPC/LDAO (1 : 1) micelles. Hm-3 has a relatively high affinity to VSD-III (dissociation constant of the complex Kd ~6 M), comparable to the affinity to VSD-I and exceeding the affinity to VSD-II. Within the complex, the positively charged Lys25 and Lys28 residues of the toxin probably interact with the S1S2 extracellular loop of VSD-III. The Hm-3 molecule also contacts the lipid bilayer surrounding the channel.

New Molecular Scaffolds for Fluorescent Voltage Indicators

2018 ◽  
Author(s):  
Steven Boggess ◽  
Shivaani Gandhi ◽  
Brian Siemons ◽  
Nathaniel Huebsch ◽  
Kevin Healy ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Action Potentials ◽  
Induced Pluripotent Stem Cell ◽  
Molecular Wire ◽  
Voltage Sensing ◽  
Design Synthesis ◽  
Cardiac Action ◽  
Action Potential Waveform ◽  
Induced Pluripotent ◽  
Voltage Sensing Domain

<div> <p>The ability to non-invasively monitor membrane potential dynamics in excitable cells like neurons and cardiomyocytes promises to revolutionize our understanding of the physiology and pathology of the brain and heart. Here, we report the design, synthesis, and application of a new class of fluorescent voltage indicator that makes use of a fluorene-based molecular wire as a voltage sensing domain to provide fast and sensitive measurements of membrane potential in both mammalian neurons and human-derived cardiomyocytes. We show that the best of the new probes, fluorene VoltageFluor 2 (fVF 2) readily reports on action potentials in mammalian neurons, detects perturbations to cardiac action potential waveform in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, shows a substantial decrease in phototoxicity compared to existing molecular wire-based indicators, and can monitor cardiac action potentials for extended periods of time. Together, our results demonstrate the generalizability of a molecular wire approach to voltage sensing and highlights the utility of fVF 2 for interrogating membrane potential dynamics.</p> </div>

Quantum Calculation of Proton and Other Charge Transfer Steps in Voltage Sensing in the Kv1.2 Channel

2019 ◽  
Author(s):  
Alisher M Kariev ◽  
Michael Green
Keyword(s):  
Charge Transfer ◽  
Energy Charge ◽  
Quantum Calculation ◽  
Quantum Calculations ◽  
Gating Current ◽  
Transmembrane Segment ◽  
Voltage Sensing ◽  
Standard Models ◽  
Charge Displacement ◽  
Voltage Sensing Domain

Quantum calculations on 976 atoms of the voltage sensing domain of the K<sub>v</sub>1.2 channel, with protons in several positions, give energy, charge transfer, and other properties. Motion of the S4 transmembrane segment that accounts for gating current in standard models is shown not to occur; there is H<sup>+ </sup>transfer instead. The potential at which two proton positions cross in energy approximately corresponds to the gating potential for the channel. The charge displacement seems approximately correct for the gating current. Two mutations are accounted for (Y266F, R300cit, cit =citrulline). The primary conclusion is that voltage sensing depends on H<sup>+</sup> transfer, not motion of arginine charges.

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