Transfection methods for high-throughput cellular assays of voltage-gated calcium and sodium channels involved in pain

Hasan, Md. Mahadhi; Ragnarsson, Lotten; Cardoso, Fernanda C.; Lewis, Richard J.

doi:10.1371/journal.pone.0243645

Cited by 12 publications

(14 citation statements)

References 45 publications

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“…The Na V 1.1/K V 2.1 chimera was subcloned into the mammalian vector pCDNA3.1 and new constructs confirmed by DNA sequencing. Plasmid constructs were used to transfect HEK293 cells using the FuGENE reagent as previously described ( Hasan et al, 2020 ). Briefly, HEK293 cells were transfected with 18 μg DNA using a ration of DNA:FuGENE at 1:3 and transfection media replaced with fresh media after 16 h incubation at 37°C 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Engineering of a Spider Peptide via Conserved Structure-Function Traits Optimizes Sodium Channel Inhibition In Vitro and Anti-Nociception In Vivo

Mawlawi

Zhao

et al. 2021

Front. Mol. Biosci.

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Venom peptides are potent and selective modulators of voltage-gated ion channels that regulate neuronal function both in health and in disease. We previously identified the spider venom peptide Tap1a from the Venezuelan tarantula Theraphosa apophysis that targeted multiple voltage-gated sodium and calcium channels in visceral pain pathways and inhibited visceral mechano-sensing neurons contributing to irritable bowel syndrome. In this work, alanine scanning and domain activity analysis revealed Tap1a inhibited sodium channels by binding with nanomolar affinity to the voltage-sensor domain II utilising conserved structure-function features characteristic of spider peptides belonging to family NaSpTx1. In order to speed up the development of optimized NaV-targeting peptides with greater inhibitory potency and enhanced in vivo activity, we tested the hypothesis that incorporating residues identified from other optimized NaSpTx1 peptides into Tap1a could also optimize its potency for NaVs. Applying this approach, we designed the peptides Tap1a-OPT1 and Tap1a-OPT2 exhibiting significant increased potency for NaV1.1, NaV1.2, NaV1.3, NaV1.6 and NaV1.7 involved in several neurological disorders including acute and chronic pain, motor neuron disease and epilepsy. Tap1a-OPT1 showed increased potency for the off-target NaV1.4, while this off-target activity was absent in Tap1a-OPT2. This enhanced potency arose through a slowed off-rate mechanism. Optimized inhibition of NaV channels observed in vitro translated in vivo, with reversal of nocifensive behaviours in a murine model of NaV-mediated pain also enhanced by Tap1a-OPT. Molecular docking studies suggested that improved interactions within loops 3 and 4, and C-terminal of Tap1a-OPT and the NaV channel voltage-sensor domain II were the main drivers of potency optimization. Overall, the rationally designed peptide Tap1a-OPT displayed new and refined structure-function features which are likely the major contributors to its enhanced bioactive properties observed in vivo. This work contributes to the rapid engineering and optimization of potent spider peptides multi-targeting NaV channels, and the research into novel drugs to treat neurological diseases.

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Section: Methodsmentioning

confidence: 99%

Engineering of a Spider Peptide via Conserved Structure-Function Traits Optimizes Sodium Channel Inhibition In Vitro and Anti-Nociception In Vivo

Mawlawi

Zhao

et al. 2021

Front. Mol. Biosci.

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“…Cells were transfected with ASOs using lipofectamine; the transfection mechanism was the formation of cationic liposomes that enabled the internalization of anionic ASOs. Then, in contact with the cell membrane, liposomes created endosomes to enter the cell [ 185 ]. The innovative in silico tool they proposed for ASO design succeeded in targeting different regions of the viral genome and reduced the viral yield and infection.…”

Section: The Landscape Of Nucleic Acid-based Therapies In Sars-cov-2mentioning

confidence: 99%

“…Cells were transfected using FuGENE, a cationic polymer that creates complexes with anionic ASOs. The complexes could create endosomes in the cell membrane to penetrate the cell [ 185 , 189 ]. Even though ASOs have been demonstrated to be efficient and specific in targeting SARS-CoV-2 in vitro and in vivo, further experiments are needed to confirm their potential use as therapeutic agents in humans against SARS-CoV-2.…”

Section: The Landscape Of Nucleic Acid-based Therapies In Sars-cov-2mentioning

confidence: 99%

Transcriptomics and RNA-Based Therapeutics as Potential Approaches to Manage SARS-CoV-2 Infection

Arriaga-Canon

Contreras-Espinosa

Rebollar-Vega

et al. 2022

IJMS

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SARS-CoV-2 is a coronavirus family member that appeared in China in December 2019 and caused the disease called COVID-19, which was declared a pandemic in 2020 by the World Health Organization. In recent months, great efforts have been made in the field of basic and clinical research to understand the biology and infection processes of SARS-CoV-2. In particular, transcriptome analysis has contributed to generating new knowledge of the viral sequences and intracellular signaling pathways that regulate the infection and pathogenesis of SARS-CoV-2, generating new information about its biology. Furthermore, transcriptomics approaches including spatial transcriptomics, single-cell transcriptomics and direct RNA sequencing have been used for clinical applications in monitoring, detection, diagnosis, and treatment to generate new clinical predictive models for SARS-CoV-2. Consequently, RNA-based therapeutics and their relationship with SARS-CoV-2 have emerged as promising strategies to battle the SARS-CoV-2 pandemic with the assistance of novel approaches such as CRISPR-CAS, ASOs, and siRNA systems. Lastly, we discuss the importance of precision public health in the management of patients infected with SARS-CoV-2 and establish that the fusion of transcriptomics, RNA-based therapeutics, and precision public health will allow a linkage for developing health systems that facilitate the acquisition of relevant clinical strategies for rapid decision making to assist in the management and treatment of the SARS-CoV-2-infected population to combat this global public health problem.

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“…Here, the opening ratio of the former was obtained from steady-state activation analysis, while that of the latter was obtained from both steady-state activation and inactivation analyses (see also Analysis of Whole-Cell Patch-Clamp Recordings subsection of the Experimental Procedures). Steady-state analysis of Na v in N2A cells was then performed to examine the effects of PUFAs, including AA (1), DGLA (5), EPA (6), and ETA (7) (Figure 4). It was found that the V a was −30.0 ± 0.7 mV, whereas the V h was −69.0 ± 0.2 mV.…”

Section: Steady-state Analysis Of Na V In N2a In the Presence Of Pufasmentioning

confidence: 99%

State-Dependent Inhibition of Voltage-Gated Sodium Channels in Neuroblastoma Neuro-2A Cells by Arachidonic Acid from Halichondria okadai

Chiba

Shimada

Yoshio

et al. 2022

Chem. Res. Toxicol.

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Voltage-gated sodium channels (Nav) are closely associated with epilepsy, cardiac and skeletal muscle diseases, and neuropathic pain. Several toxic compounds have been isolated from the marine sponge Halichondria okadai; however, toxic substances that modulate Nav are yet to be identified. This study aimed to identify Nav inhibitors from two snake venoms and H. okadai using mouse neuroblastoma Neuro-2A cells (N2A), which primarily express the specific Nav subtype Nav1.7, using whole-cell patch-clamp recordings. We successfully isolated arachidonic acid (AA, 1) from the hexane extract of H. okadai, and then the fatty acid-mediated modulation of Nav in N2A was investigated in detail for the first time. Octanoic acid (2), palmitic acid (3), and oleic acid (4) showed no inhibitory activity at 100 μM, whereas AA (1), dihomo-γ-linolenic acid (DGLA, 5), and eicosapentaenoic acid (EPA, 6) showed IC50 values of 6.1 ± 2.0, 58 ± 19, and 25 ± 4.0 μM, respectively (N = 4, mean ± SEM). Structure and activity relationships were investigated for the first time using two ω-3 polyunsaturated fatty acids (PUFAs), EPA (6) and eicosatetraenoic acid (ETA, 7), and two ω-6 PUFAs, AA (1) and DGLA (5), to determine their effects on a resting state, activated state, and inactivated state. Steady-state analysis showed that the half inactivation potential was largely hyperpolarized by 10 μM AA (1), while 50 μM DGLA (5), 50 μM EPA (6), and 10 μM ETA (7) led to a slight change. The percentages of the resting state block were 24 ± 1, 22 ± 1, 34 ± 4, and 38 ± 9% in the presence of AA (1), DGLA (5), EPA (6), and ETA (7), respectively, with EPA (6) and ETA (7) exhibiting a greater inhibition than both AA (1) and DGLA (5), and their inhibitions did not increase in the following depolarization pulses. None of the compounds exhibited the use-dependent block. The half recovery times from the inactivated state for the control, AA (1), DGLA (5), EPA (6), and ETA (7) were 7.67 ± 0.33, 34.3 ± 1.10, 15.5 ± 1.10, 10.7 ± 0.31, and 3.59 ± 0.18 ms, respectively, with AA (1) exhibiting a distinctively large effect. Overall, distributed binding to the resting and the inactivated states of Nav would be significant for the inhibition of Nav, which presumably depends on the active structure of each PUFA.

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Transfection methods for high-throughput cellular assays of voltage-gated calcium and sodium channels involved in pain

Cited by 12 publications

References 45 publications

Engineering of a Spider Peptide via Conserved Structure-Function Traits Optimizes Sodium Channel Inhibition In Vitro and Anti-Nociception In Vivo

Engineering of a Spider Peptide via Conserved Structure-Function Traits Optimizes Sodium Channel Inhibition In Vitro and Anti-Nociception In Vivo

Transcriptomics and RNA-Based Therapeutics as Potential Approaches to Manage SARS-CoV-2 Infection

State-Dependent Inhibition of Voltage-Gated Sodium Channels in Neuroblastoma Neuro-2A Cells by Arachidonic Acid from Halichondria okadai

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