Sooyeon Jo

Nav1.7 voltage-gated sodium channels are strongly expressed in human primary pain-sensing neurons (nociceptors), and selective Nav1.7 inhibitors have been developed as possible therapeutic agents for treating pain, so far with disappointing clinical results. In contrast, a selective Nav1.8 channel inhibitor (suzetrigine) has had successful clinical trials. Because nociceptors express both Nav1.7 and Nav1.8 channels, it is of interest to compare effects of Nav1.7 and Nav1.8 inhibitors on the excitability of human nociceptors. To compare with previous results with suzetrigine, we characterized the effects of a selective Nav1.7 inhibitor, AM-2099, on action potential generation and repetitive firing of dissociated human dorsal root ganglion (DRG) neurons, studied at 37°C. Inhibition of Nav1.7 channels by 600 nM AM-2099 generally produced a substantial depolarizing shift of action potential threshold, an increase in rheobase, a decrease in action potential upstroke velocity, decrease in action potential peak, and prolongation of the refractory period. Compared to inhibition of Nav1.8 channels, inhibition of Nav1.7 channels had larger effects on threshold and maximal upstroke velocity, while action potential peak was reduced similarly by both. Nav1.8 inhibition produced much more dramatic reduction of repetitive firing than Nav1.7 inhibition. The results show that although the excitability of human DRG neurons is affected by inhibition of Nav1.7 channels, most notably by an increase in threshold and increase in refractory period, repetitive firing of the neurons in response to strong stimuli is little affected.

CACNA1A encodes the α1A subunit of the Ca _V 2.1 voltage–gated calcium channel essential for neuronal excitability, neurotransmitter release, and calcium signaling in neurons. Pathogenic CACNA1A variants are linked to diverse neurological and developmental disorders, including hemiplegic migraine, epilepsy, developmental delay, and ataxia. However, the contribution of CACNA1A variants to neurodevelopmental disorders has been less well established. Clinical heterogeneity and the complex impact of missense variants on channel function have made genotype-phenotype relationships difficult to resolve. Here, we characterized the biophysical properties of 42 de novo CACNA1A missense variants identified in a neurodevelopmental disorder cohort of over 31,000 individuals, alongside eight common variants from the general population. All but one de novo variant altered biophysical properties of the Ca _V 2.1 channel, with 50% abolishing measurable currents. Among variants with detectable currents, nearly half disrupted voltage-dependent gating, whereas common variants had no measurable effect. Similar results were obtained using representative variants in the presence of β3 or β4 auxiliary subunits. The expression of two selected variants in CACNA1A -knockdown hiPSC-derived neurons confirmed similar functional alterations in human neurons. Simulations using the NEURON model of a Purkinje neuron demonstrated that these biophysical changes profoundly affected excitability. Coupling functional data with AlphaMissense predictions, we found that Ca _V 2.1 missense variants contribute to the risk for developmental epileptic encephalopathy. Finally, correlating clinical features with molecular consequences of each variant revealed meaningful associations between specific channel dysfunctions and distinct clinical outcomes. These findings provide insights into CACNA1A molecular pathology and highlight the potential for precision medicine treatment of CACNA1A -associated disorders.

Nav1.8 sodium channels are expressed in pain-sensing neurons, and some Nav1.8 inhibitors significantly reduce pain in clinical trials. Several Nav1.8 inhibitors have an unusual state dependence whereby inhibition is relieved by depolarization. We compared the state-dependent action of several Nav1.8 channel inhibitors to test whether inhibition is relieved during action potential (AP) firing under physiological conditions to produce “reverse use dependence.” A-887826 inhibition was substantially relieved by AP waveforms applied at 20 Hz at 37°C. In contrast, there was no relief during AP trains with suzetrigine (VX-548) or LTGO-33, even though inhibition could be effectively removed by long, strong depolarizations. These differences were explained by differences in the voltage dependence and kinetics with which the compounds dissociate from depolarized channels and rebind to resting state channels. Suzetrigine required the strongest depolarizations for relief (midpoint +33 mV) and relief was slow (tau >300 ms at +20 mV), so almost no relief occurred during an AP waveform. Relief from A-887826 required weaker depolarizations (midpoint +13 mV) and was much faster, so some relief occurred during each AP waveform and accumulated during 20-Hz trains. LTGO-33 required the weakest depolarizations for relief (midpoint −11 mV) and relief was even faster than for A-887826, but reinhibition between AP waveforms was far faster than for A-887826, so that relief did not accumulate during AP trains at 20 Hz. The results show that, unlike A-887826, there is no use-dependent relief of inhibition by suzetrigine or LTGO-33 with physiological voltage waveforms at physiological temperatures, but each for different reasons.

Nav1.8 channels are an attractive therapeutic target for pain because they are prominent in primary pain-sensing neurons with little expression in most other kinds of neurons. Recently, two Nav1.8-targeted compounds, VX-150 and VX-548, have shown efficacy in clinical trials for reducing pain. We examined the characteristics of Nav1.8 inhibition by these compounds. The active metabolite form of VX-150 (VX-150m) inhibited human Nav1.8 channels with an IC₅₀ of 15 nM. VX-548 (suzetrigine) was even more potent (IC₅₀ 0.27 nM). Both VX-150m and VX-548 had the unusual property of "reverse use-dependence", whereby inhibition could be relieved by repetitive depolarizations, a property seen before with another Nav1.8 inhibitor, A-887826. The relief of VX-548 inhibition by large depolarizations occurred with a time constant of ~40 ms that was not concentration-dependent. Re-inhibition at negative voltages occurred with a rate that was nearly proportional to drug concentration, consistent with the idea that relief of inhibition reflects dissociation of drug from the channel and re-inhibition reflects re-binding. The relief of inhibition by depolarization suggests a remarkably strong and unusual state-dependence for both VX-150m and VX-548, with very weak binding to channels with fully activated voltage sensors despite very tight binding to channels with voltage sensors in the resting state. Significance Statement The Nav1.8 sodium channel is a current target for new drugs for pain. This work describes the potency, selectivity, and state-dependent characteristics of inhibition of Nav1.8 channels by VX-150 and VX-548, compounds that have recently shown efficacy for relief of pain in clinical trials but whose mechanism of interaction with channels has not been described. The results show that the compounds share an unusual property whereby inhibition is relieved by depolarization, demonstrating a state-dependence different than most sodium channel inhibitors.

Sodium channel inhibitors used as local anesthetics, antiarrhythmics, or antiepileptics typically have the property of use-dependent inhibition, whereby inhibition is enhanced by repetitive channel activation. For targeting pain, Nav1.8 channels are an attractive target because they are prominent in primary pain-sensing neurons, with little or no expression in most other kinds of neurons, and a number of Nav1.8-targeted compounds have been developed. We examined the characteristics of Nav1.8 inhibition by one of the most potent Nav1.8 inhibitors so far described, A-887826, and found that when studied with physiologic resting potentials and physiologic temperatures, inhibition had strong “reverse use dependence”, whereby inhibition was relieved by repetitive short depolarizations. This effect was much stronger with A-887826 than with A-803467, another Nav1.8 inhibitor. The use-dependent relief from inhibition was seen in both human Nav1.8 channels studied in a cell line and in native Nav1.8 channels in mouse dorsal root ganglion (DRG) neurons. In native Nav1.8 channels, substantial relief of inhibition occurred during repetitive stimulation by action potential waveforms at 5 Hz, suggesting that the phenomenon is likely important under physiologic conditions. SIGNIFICANCE STATEMENT Nav1.8 sodium channels are expressed in primary pain-sensing neurons and are a prime current target for new drugs for pain. This work shows that one of the most potent Nav1.8 inhibitors, A-887826, has the unusual property that inhibition is relieved by repeated short depolarizations. This “reverse use dependence” may reduce inhibition during physiological firing and should be selected against in drug development.

Phenotypic screens using human induced pluripotent stem cell–derived nociceptors hold promise for the development of better analgesics.