An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na⁺ currents by removing the inactivation of voltage-gated Na⁺ channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na⁺ currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na⁺ currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na⁺ currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na⁺ currents significantly stronger as compared to peak Na⁺ currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na⁺ channels expressed in cardiomyocytes may be relevant for the generation of late Na⁺ currents following oxidative stress.

Background: A large number of patients suffer from neuropathic pain, and systemic therapy often remains ineffective while inducing severe side effects. Topical therapy with the TRPV1-agonist capsaicin is an established alternative, and the identification of co-therapeutics that modulate TRPV1 may be a promising approach to reduce the dose of capsaicin while maintaining efficacy. Here, we aimed to determine if the antidepressant amitriptyline displays properties rendering it a potential co-therapeutic agent. Methods: We performed patch clamp and calcium imaging experiments on HEK293T cells expressing human (h) TRPV1 as well as on dorsal root ganglion (DRG) neurons from adult mice. Results: Amitriptyline induced an increase in intracellular calcium in both HEK293T and mouse DRG neurons expressing TRPV1. Patch clamp experiments revealed a concentration-dependent activation of hTRPV1 by amitriptyline that was also evident in cell-free inside-out patches. When hTRPV1 was fully activated by capsaicin, amitriptyline induced concentration-dependent and partly reversible current inhibition. In contrast, amitriptyline potentiated small responses to capsaicin, heat and protons. We also found that amitriptyline desensitized hTRPV1 to capsaicin. This effect was reduced by the intracellular application of the strong calcium chelator BAPTA. Furthermore, the non-desensitizing mutant hTRPV1-Y672K displayed a reduced amitriptyline-induced desensitization. Conclusions: Our data showed that amitriptyline can activate, sensitize, desensitize and even inhibit TRPV1. Together with its property as a strong local anesthetic, our data suggest that amitriptyline may be a promising adjunct to topical capsaicin.

The recently identified proton-activated chloride (PAC) channel is ubiquitously expressed, and it regulates several proton-sensitive physiological and pathophysiological processes. While the PAC channel is activated by strong acids due to the binding of protons to extracellular binding sites, here, we describe the way in which weak acids inhibit the PAC channel by a mechanism involving a distinct extracellular binding site. Whole-cell patch clamp was performed on wildtype HEK293T cells, PAC-knockout HEK293 cells expressing human (h)PAC mutant constructs, and on hiPSC-derived cardiomyocytes. Proton-induced cytotoxicity was examined in HEK293T cells. Acetic acid inhibited endogenous PAC channels in HEK 293T cells in a reversible, concentration-dependent, and pH-dependent manner. The inhibition of PAC channels was also induced by lactic acid, propionic acid, itaconic acid, and β-hydroxybutyrate. Weak acids also inhibited recombinant wildtype hPAC channels and PAC-like currents in hiPSC-derived cardiomyocytes. Replacement of the extracellular arginine 93 by an alanine (hPAC–Arg93Ala) strongly reduced the inhibition by some weak acids, including arachidonic acid. Although lactic acid inhibited PAC, it did not reduce the proton-induced cytotoxicity examined in wildtype HEK 293 cells. To conclude, weak acids inhibit PAC via an extracellular mechanism involving Arg93. These data warrant further investigations into the regulation of the PAC channel by endogenous weak acids.

Several transient receptor potential vanilloid (TRPV) ion channels are proton-sensitive, and recent structural studies have identified poorly conserved mechanisms for the proton sensitivity of TRPV1, TRPV2 and TRPV5. While such detailed studies are lacking for TRPV3, three distinct intracellular motifs were suggested to be required for a direct channel activation by cytosolic acidification. In this study, we investigated if these mechanisms are also relevant for the activation of TRPV3 by weak acids. Wildtype (WT) and several mutants of human TRPV3 transiently expressed in HEK 293T cells were investigated by whole-cell patch clamp electrophysiology. Cells expressing TRPV3-WT generated membrane currents induced by acetic acid (HOAc), formic acid and carbonic acid at pH 5.0. Activation induced by HOAc was concentration-dependent and increased with decreasing pH values. HOAc also strongly potentiated TRPV3-mediated responses to carvacrol and heat. Among the three suggested motifs for the binding of intracellular protons, only the mutant TRPV3-Asp512Ala exhibited an almost complete loss of HOAc sensitivity. The mutation of two C-terminal charged residues (Gln689/Asp727) even resulted in a clear gain of function for both HOAc and heat, and the mutation of the 2-APB-binding site His426 did not significantly abrogate HOAc sensitivity. Finally, insertion of the recently identified binding site in TRPV2 for the weak acid probenecid into TRPV3 (Glu216His) resulted in an increased HOAc sensitivity. To conclude, our data confirm that TRPV3 is sensitized and activated by several weak acids. While Asp512 appears to be a critical intracellular proton-modulating site, a more profound understanding of the mechanisms dictating the proton sensitivity of TRPV3 may require structural studies.

Summary: Peripheral nociceptive sensory neurons integrate various noxious inputs, resulting in local depolarization that triggers the firing of action potentials and thus the sensation of pain. We recently reported that nociceptor depolarization itself initiates signaling by the calcium channel Cav1.2 causing acute hyperalgesia in vivo . However, whether this mechanism initiates excitation-transcription (E-T) coupling and thereby leads to long-lasting modulation of nociceptor activity remains poorly understood. Using high content imaging of dorsal root ganglion (DRG) neurons, we here found that depolarization of nociceptors induces phosphorylation of the transcription factor (TF) cAMP-response element binding protein (CREB), which was affected by inhibition of protein kinase A (PKA) and calcineurin, but not Ca ²⁺ /calmodulin-dependent protein kinases. Genetic deletion or pharmacological inhibition of Cav1.2 confirmed its role in calcium-dependent kinase signaling and CREB phosphorylation after depolarization. In line with this, pharmacological modulation of Cav1 channels affected the expression of a subset of depolarization-regulated immediate early genes known to orchestrate a broader transcriptional response. Indeed, RNA-Seq analysis of DRG neurons from mice with a tissue-specific deletion of Cav1.2 in nociceptive sensory neurons (SNS-Cacna1c ^-/- mice) revealed downregulation of multiple calcium and potassium channel subunits as well as proteins involved in synaptic vesicle release and cell adhesion. Furthermore, repetitive firing of action potentials and release of the neuropeptide CGRP was impaired in Cav1.2-deficient sensory neurons. SNS-Cacna1c ^-/- mice showed increased sensitivity to noxious heat and exacerbated inflammatory but not neuropathic pain. In conclusion, our data suggest a Cav1.2-dependent E-T coupling mechanism in nociceptors that counteracts nociception in vivo . Highlights: Depolarization induces Cav1.2- and PKA-dependent phosphorylation of the transcriptional regulator CREB in nociceptors Cav1.2 regulates the expression of depolarization-induced immediate early genes and induces reorganization of the signaling network in nociceptors Cav1.2-deficient nociceptors downregulate multiple ion channel subunits as well as proteins involved in synaptic vesicle release and cell adhesion Cav1.2-deficient nociceptors show impaired depolarization-induced PKA-II signaling, CREB phosphorylation, repetitive firing of action potentials, and release of the neuropeptide CGRP Nociceptor-specific Cav1.2-deficiency in mice increases the sensitivity to noxious heat and exacerbates inflammatory pain In brief: Isensee et al. report that depolarization of nociceptors induces calcium influx through Cav1.2 channels, thereby initiating a signaling cascade that links neuronal excitation to transcriptional regulation of immediate early genes (E-T coupling). Nociceptor-specific knockout of Cav1.2 led to transcriptional changes in nociceptor-specific genes and was associated with impaired depolarization-induced signaling, action potential firing, and neuropeptide release. As the Cav1.2-deficient mice show increased noxious heat sensitivity and exacerbated inflammatory pain, these findings suggest that Cav1.2-dependent E-T coupling mechanism counteracts nociception in vivo .

BACKGROUND: Cannabidiol (CBD) inhibits voltage-gated sodium channels (Na _v ). CBD is highly lipophilic and exhibits slow binding kinetics on Na _v s, a property that may enable CBD to enhance and prolong regional anesthesia. Here we explored the effects of CBD on lidocaine-induced Na _v -inhibition and neurotoxicity. METHODS: Patch clamp recordings were performed on tetrodotoxin (TTX)-sensitive Na _v s and the TTX-resistant Na _v 1.8 in ND7/23 cells or murine dorsal root ganglion neurons (DRG). Cytotoxicity was analyzed using flow cytometry. RESULTS: CBD induced a potent tonic inhibition of sodium currents with slow onset and offset kinetics. Na _v 1.8 exhibited a higher sensitivity to tonic block (IC ₅₀ 1.4 µM) as compared to TTX-sensitive sodium channels (IC ₅₀ 2.9 µM). Similar to local anesthetics, CBD also induced a shift of the steady-state inactivation and a modest use-dependent block at 10 Hz. In contrast to local anesthetics, inhibition of Na _v s by CBD is pH-independent. The co-application of CBD and lidocaine resulted in an additive tonic block of sodium currents in ND7/23 cells, but not in DRG neurons. While only high concentrations of CBD induced cytotoxicity, it potentiated lidocaine-induced cytotoxicity. CONCLUSIONS: CBD demonstrates promising characteristics as a long-lasting local anesthetic, or as an adjunct to local anesthetics used for regional or topical anesthesia.