Functional and structural insights into activation of TRPV2 by weak acids.

Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.

Local Anesthetic Like Inhibition of the Cardiac Na+ Channel Nav1.5 by Chloroquine and Hydroxychloroquine.

Introduction: Chloroquine (CQ) and its derivate hydroxychloroquine (HCQ) are successfully deployed for different diseases beyond the prophylaxis and treatment of malaria. Both substances exhibit antiviral properties and have been proposed for prophylaxis and treatment of COVID-19 caused by SARS-CoV-2. CQ and HCQ cause similar adverse events including life-threatening cardiac arrhythmia generally based on QT-prolongation, which is one of the most reported adverse events for both agents associated with the treatment of COVID-19. Various drugs known to induce QT-prolongation have been proven to exert local anesthetic (LA)-like properties regarding their impact on the cardiac Na+ channel Nav1.5. Inhibition of Nav1.5 is considered as the primary mechanism of cardiotoxicity caused by LAs. However, the mechanism of the arrhythmogenic effects of CQ and HCQ related to Nav1.5 has not yet been fully investigated. Therefore, the exact mechanism of how CQ and HCQ affect the sodium currents generated by Nav1.5 need to be further elucidated. Objective: This in vitro study aims to investigate the effects of CQ and HCQ on Nav1.5-generated sodium currents to identify possible LA-like mechanisms that might contribute to their arrhythmogenic properties. Methods: The effects of CQ and HCQ on Nav1.5-generated sodium currents by HEK-293 cells expressing either wild-type human Nav1.5 or mutant Nav1.5 F1760A are measured using the whole-cell patch-clamp technique. Results: Both agents induce a state-dependent inhibition of Nav1.5. Furthermore, CQ and HCQ produce a use-dependent block of Nav1.5 and a shift of fast and slow inactivation. Results of experiments investigating the effect on the LA-insensitive mutant Nav1.5-F1760A indicate that both agents at least in part employ the proposed LA-binding site of Nav1.5 to induce inhibition. Conclusion: This study demonstrated that CQ and HCQ exert LA-typical effects on Nav1.5 involving the proposed LA binding site, thus contributing to their arrhythmogenic properties.

Sodium Channel Nav1.3 Is Expressed by Polymorphonuclear Neutrophils during Mouse Heart and Kidney Ischemia In Vivo and Regulates Adhesion, Transmigration, and Chemotaxis of Human and Mouse Neutrophils In Vitro.

BACKGROUND: Voltage-gated sodium channels generate action potentials in excitable cells, but they have also been attributed noncanonical roles in nonexcitable cells. We hypothesize that voltage-gated sodium channels play a functional role during extravasation of neutrophils. METHODS: Expression of voltage-gated sodium channels was analyzed by polymerase chain reaction. Distribution of Nav1.3 was determined by immunofluorescence and flow cytometry in mouse models of ischemic heart and kidney injury. Adhesion, transmigration, and chemotaxis of neutrophils to endothelial cells and collagen were investigated with voltage-gated sodium channel inhibitors and lidocaine in vitro. Sodium currents were examined with a whole cell patch clamp. RESULTS: Mouse and human neutrophils express multiple voltage-gated sodium channels. Only Nav1.3 was detected in neutrophils recruited to ischemic mouse heart (25 ± 7%, n = 14) and kidney (19 ± 2%, n = 6) in vivo. Endothelial adhesion of mouse neutrophils was reduced by tetrodotoxin (56 ± 9%, unselective Nav-inhibitor), ICA121431 (53 ± 10%), and Pterinotoxin-2 (55 ± 9%; preferential inhibitors of Nav1.3, n = 10). Tetrodotoxin (56 ± 19%), ICA121431 (62 ± 22%), and Pterinotoxin-2 (59 ± 22%) reduced transmigration of human neutrophils through endothelial cells, and also prevented chemotactic migration (n = 60, 3 × 20 cells). Lidocaine reduced neutrophil adhesion to 60 ± 9% (n = 10) and transmigration to 54 ± 8% (n = 9). The effect of lidocaine was not increased by ICA121431 or Pterinotoxin-2. CONCLUSIONS: Nav1.3 is expressed in neutrophils in vivo; regulates attachment, transmigration, and chemotaxis in vitro; and may serve as a relevant target for antiinflammatory effects of lidocaine.

Activation of the capsaicin-receptor TRPV1 by the acetaminophen metabolite N-arachidonoylaminophenol results in cytotoxicity.

AIMS: The anandamide reuptake inhibitor N-arachidonoylaminophenol (AM404) and the reactive substance N-acetyl-p-benzoquinone imine (NAPQI) are both metabolites of acetaminophen and may contribute to acetaminophen-induced analgesia by acting at TRPV1 expressed in the peripheral or central nervous system. While NAPQI slowly sensitizes and activates TRPV1 by interacting with distinct intracellular cysteine residues, detailed properties of AM404 as an agonist of TRPV1 have not yet been reported on. We explored the effects of AM404 on recombinant human TRPV1 and in rodent dorsal root ganglion (DRG) neurons. MATERIALS AND METHODS: HEK 293 cells expressing different isoforms of recombinant TRPV1 and rodent DRG neurons were employed for patch clamp and calcium imaging experiments. Cytotoxicity was assessed by propidium iodide and Annexin V staining on TRPV1-HEK 293 cells and with trypan blue staining on DRG neurons. KEY FINDINGS: AM404 activates hTRPV1 at concentrations >1µM and in a concentration-dependent manner. AM404 also potentiates TRPV1-mediated currents evoked by heat and anandamide. Moreover, AM404-evoked currents are potentiated by NAPQI. While the partly capsaicin-insensitive rabbit (o) TRPV1 fails to respond to AM404, AM404-sensitivity is restored by insertion of the capsaicin binding-domain of rat TRPV1 into oTRPV1. In DRG neurons, AM404-evoked calcium influx as well as cell death is mediated by TRPV1. SIGNIFICANCE: AM404 gates TRPV1 by interacting with the vanilloid-binding site, and TRPV1 is the main receptor for AM404 in DRG neurons. While direct activation of TRPV1 requires high concentrations of AM404, it is possible that synergistic effects of AM404 with further TRPV1-agonists may occur at clinically relevant concentrations.

Lactate is a potent inhibitor of the capsaicin receptor TRPV1.

Tissue ischemia results in an accumulation of lactate and local or systemic lactic acidosis. In nociceptive sensory neurons, lactate was reported to sensitize or activate the transient receptor potential ion channel TRPA1 and acid-sensing ion channels (ASICs). However, it is unclear how lactate modulates the TRPV1 regarded as the main sensor for acidosis in sensory neurons. In this study we investigated the effects of lactate (LA) on recombinant and native TRPV1 channels and on TRPV1-mediated release of neuropeptides from mouse nerves. TRPV1-mediated membrane currents evoked by protons, capsaicin or heat are inhibited by LA at concentrations ranging from 3 µM to 100 mM. LA inhibits TRPV1-mediated proton-induced Ca2+-influx in dorsal root ganglion neurons as well as proton-evoked neuropeptide release from mouse nerves. Inhibition of TRPV1 by LA is significantly stronger on inward currents as compared to outward currents since LA affects channel gating, shifting the activation curve towards more positive potentials. The mutation I680A in the pore lower gate displays no LA inhibition. Cell-attached as well as excised inside- and outside-out patches suggest an interaction through an extracellular binding site. In conclusion, our data demonstrate that lactate at physiologically relevant concentrations is a potent endogenous inhibitor of TRPV1.

Local-anesthetic like inhibition of the cardiac sodium channel Nav1.5 α-subunit by 5-HT3 receptor antagonists.

5-hydroxytryptamine 3 receptor (5-HT3 receptor) antagonists are administered for prevention and therapy of nausea and vomiting. Although regarded as safe therapeutics, they can also provoke arrhythmias by prolonging the QRS interval. However, the mechanisms mediating this cardiotoxicity are poorly understood. Here we investigated effects of 5-HT3 receptor antagonists on the cardiac Na(+) channel Nav1.5. We explored the interaction of dolasetron, tropisetron, granisetron and ondansetron on the human α-subunit Nav1.5 heterologously expressed in HEK293 cells. Sodium currents were explored by means of whole-cell patch clamp recordings. All four substances inhibited the Nav1.5 in a concentration and state-dependent manner. Dolasetron displayed the lowest blocking efficacy, and tropisetron was the most potent blocker with a half maximum blocking concentration of 18µM for tonic block of inactivated channels. Tropisetron was also the most potent use-dependent inhibitor, and it also induced a strong open -channel block. Both tonic and use-dependent block by tropisetron were abbreviated on the local-anesthetic insensitive mutant Nav1.5-F1760A. Co-administration of tropisetron and the local anesthetic bupivacaine or the hypnotic propofol augmented inhibition of Nav1.5. Our data demonstrate that 5-HT3 receptor antagonists induce a local-anesthetic like inhibition of Nav1.5, and that they display different blocking efficacies. Reports on a relevant cardiotoxicity of dolasetron as opposed to other 5-HT3 receptor antagonists do not seem to correlate with a block of Nav1.5. As inhibition of Nav1.5 was enhanced by propofol and bupivacaine however, it is possible that a combined administration of Na(+) channel blockers and 5-HT3 receptor antagonists can provoke arrhythmias.