Veratridine produces distinct calcium response profiles in mouse Dorsal Root Ganglia neurons

Mohammed, Zainab A.; Doran, Ciara; Grundy, David; Nassar, Mohammed A.

doi:10.1038/srep45221

Cited by 23 publications

(25 citation statements)

References 40 publications

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“…2b), which is in agreement with the expression of Nav1.8 in 80-90% of nociceptors [5; 21]. Our identification of nociceptors is based on responsiveness to one or more of three nociceptive agonists [16]. This definition will encompass six of the putative eleven molecular subtypes (the NP1-3, PEP1-2 and TH types) of sensory neurons as defined in one single-cell RNA sequencing study [23].…”

Section: Discussionmentioning

confidence: 58%

“…The knowledge gap includes information on the effect of a drug on nociceptors as a whole (rather than just the molecular target of interest) and the concentrations needed for a potent effect on nociceptors with a minimal effect on non-nociceptors (as a proxy for unwanted side effects on CNS and motor neurons). We discovered that nociceptors and nonnociceptors respond to veratridine with distinct response profiles [16]. Here we show that a veratridine-based assay allows for a simultaneous assessment of drugs' action on both populations and therefore provides an efficient and more informative assay for screening for analgesics on sensory neurons.…”

Section: Discussionmentioning

confidence: 75%

“…Given that Nav1.7 is expressed in all DRG neurons, why was there no significant reduction in the SD population in Nav1.7KO? Veratridine primarily activates TTX-sensitive VGSCs and indeed, we previously showed that TTX silences all OS and SD neurons [16]. However, while Nav1.7 is the main TTX-sensitive channel in nociceptors, non-nociceptors rely on other TTX-sensitive subtypes, namely Nav1.1, Nav1.2 and Nav1.6.…”

Section: A C C E P T E Dmentioning

confidence: 78%

“…Voltage gated sodium channels (VGSCs) are critical for the excitability in DRG neurons. We discovered that the VGSC opener, veratridine, produces distinct calcium responses in cultured sensory neurons [16]. We found that most nociceptors show an oscillatory response (OS), while most nonnociceptors show a slowly decaying response (SD), Fig 1. Here we provide evidence that the OS and SD veratridine response profiles can be used as readouts in an assay to evaluate the effect of drugs on nociceptors and non-nociceptors.…”

Section: Introductionmentioning

confidence: 91%

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An unbiased and efficient assessment of excitability of sensory neurons for analgesic drug discovery

Mohammed

Kaloyanova²,

Nassar³

2020

Pain

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Alleviating chronic pain is challenging, due to lack of drugs that effectively inhibit nociceptors without off-target effects on motor or central neurons. Dorsal root ganglia (DRG) contain nociceptive and non-nociceptive neurons. Drug screening on cultured DRG neurons, rather than cell lines, allows for the identification of drugs most potent on nociceptors with no effects on non-nociceptors (as a proxy for unwanted side effects on central nervous system and motor neurons). However, screening using DRG neurons is currently a low-throughput process, and there is a need for assays to speed this process for analgesic drug discovery. We previously showed that veratridine elicits distinct response profiles in sensory neurons. Here, we show evidence that a veratridine-based calcium assay allows for an unbiased and efficient assessment of a drug effect on nociceptors (targeted neurons) and non-nociceptors (nontargeted neurons). We confirmed the link between the oscillatory profile and nociceptors, and the slow-decay profile and non-nociceptors using 3 transgenic mouse lines of known pain phenotypes. We used the assay to show that blockers for Nav1.7 and Nav1.8 channels, which are validated targets for analgesics, affect non-nociceptors at concentrations needed to effectively inhibit nociceptors. However, a combination of low doses of both blockers had an additive effect on nociceptors without a significant effect on non-nociceptors, indicating that the assay can also be used to screen for combinations of existing or novel drugs for the greatest selective inhibition of nociceptors.

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Section: Discussionmentioning

confidence: 58%

Section: Discussionmentioning

confidence: 75%

Section: A C C E P T E Dmentioning

confidence: 78%

Section: Introductionmentioning

confidence: 91%

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An unbiased and efficient assessment of excitability of sensory neurons for analgesic drug discovery

Mohammed

Kaloyanova²,

Nassar³

2020

Pain

Self Cite

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“…Nav1.7 plays important roles in sensory and sense-driven functions such as pain, cough, olfactory reflexes, and acid sensation, which make it an important drug target [25,26], and this channel is expressed abundantly in peripheral sensory neurons such as dorsal root ganglion (DRG) neurons, where it regulates cell excitability by modulating the neuronal action potential threshold [19,22,25]. In DRG neurons, veratridine inhibits sodium channel conductance in a dose-dependent manner, exerts different modulatory effects on the voltage gating of TTX-sensitive and TTX-resistant channels [27], and induces different Ca 2+ responses [28]. The modulatory effect of veratridine on the characteristics of sodium channels has been reported mostly for skeletal muscle Nav1.4 [12,29] and murine vas deferens myocyte Nav1.6 [11,30], while little is known about its effect on neuronal Nav1.7 [11]; in particular, its effect on the gating properties of Nav1.7 is largely unknown.…”

Section: Introductionmentioning

confidence: 99%

Veratridine modifies the gating of human voltage-gated sodium channel Nav1.7

Zhang

et al. 2018

Acta Pharmacol Sin

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Veratridine is a lipid-soluble neurotoxin derived from plants in the family Liliaceae. It has been broadly investigated for its action as a sodium channel agonist. However, the effects of veratridine on subtypes of sodium channels, especially Nav1.7, remain to be studied. Here, we investigated the effects of veratridine on human Nav1.7 ectopically expressed in HEK293A cells and recorded Nav1.7 currents from the cells using whole-cell patch clamp technique. We found that veratridine exerted a dose-dependent inhibitory effect on the peak current of Nav1.7, with the half-maximal inhibition concentration (IC) of 18.39 µM. Meanwhile, veratridine also elicited tail current (linearly) and sustained current [half-maximal concentration (EC): 9.53 µM], also in a dose-dependent manner. Veratridine (75 µM) shifted the half-maximal activation voltage of the Nav1.7 activation curve in the hyperpolarized direction, from -21.64 ± 0.75 mV to -28.14 ± 0.66 mV, and shifted the half-inactivation voltage of the steady-state inactivation curve from -59.39 ± 0.39 mV to -73.78 ± 0.5 mV. An increased frequency of stimulation decreased the peak and tail currents of Nav1.7 for each pulse along with pulse number, and increased the accumulated tail current at the end of train stimulation. These findings reveal the different modulatory effects of veratridine on the Nav1.7 peak current and tail current.

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Human neuronal signaling and communication assays to assess functional neurotoxicity

et al. 2020

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Prediction of drug toxicity on the human nervous system still relies mainly on animal experiments. Here, we developed an alternative system allowing assessment of complex signaling in both individual human neurons and on the network level. The LUHMES cultures used for our approach can be cultured in 384-well plates with high reproducibility. We established here high-throughput quantification of free intracellular Ca2+ concentrations [Ca2+]i as broadly applicable surrogate of neuronal activity and verified the main processes by patch clamp recordings. Initially, we characterized the expression pattern of many neuronal signaling components and selected the purinergic receptors to demonstrate the applicability of the [Ca2+]i signals for quantitative characterization of agonist and antagonist responses on classical ionotropic neurotransmitter receptors. This included receptor sub-typing and the characterization of the anti-parasitic drug suramin as modulator of the cellular response to ATP. To exemplify potential studies on ion channels, we characterized voltage-gated sodium channels and their inhibition by tetrodotoxin, saxitoxin and lidocaine, as well as their opening by the plant alkaloid veratridine and the food-relevant marine biotoxin ciguatoxin. Even broader applicability of [Ca2+]i quantification as an end point was demonstrated by measurements of dopamine transporter activity based on the membrane potential-changing activity of this neurotransmitter carrier. The substrates dopamine or amphetamine triggered [Ca2+]i oscillations that were synchronized over the entire culture dish. We identified compounds that modified these oscillations by interfering with various ion channels. Thus, this new test system allows multiple types of neuronal signaling, within and between cells, to be assessed, quantified and characterized for their potential disturbance.

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Veratridine produces distinct calcium response profiles in mouse Dorsal Root Ganglia neurons

Cited by 23 publications

References 40 publications

An unbiased and efficient assessment of excitability of sensory neurons for analgesic drug discovery

An unbiased and efficient assessment of excitability of sensory neurons for analgesic drug discovery

Veratridine modifies the gating of human voltage-gated sodium channel Nav1.7

Human neuronal signaling and communication assays to assess functional neurotoxicity

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