Cannabidiol Inhibition of Murine Primary Nociceptors: Tight Binding to Slow Inactivated States of Nav1.8 Channels.

The nonpsychoactive phytocannabinoid cannabidiol (CBD) has been shown to have analgesic effects in animal studies but little is known about its mechanism of action. We examined the effects of CBD on intrinsic excitability of primary pain-sensing neurons. Studying acutely dissociated capsaicin-sensitive mouse DRG neurons at 37°C, we found that CBD effectively inhibited repetitive action potential firing, from 15-20 action potentials evoked by 1 s current injections in control to 1-3 action potentials with 2 µm CBD. Reduction of repetitive firing was accompanied by a reduction of action potential height, widening of action potentials, reduction of the afterhyperpolarization, and increased propensity to enter depolarization block. Voltage-clamp experiments showed that CBD inhibited both TTX-sensitive and TTX-resistant (TTX-R) sodium currents in a use-dependent manner. CBD showed strong state-dependent inhibition of TTX-R channels, with fast binding to inactivated channels during depolarizations and slow unbinding on repolarization. CBD alteration of channel availability at various voltages suggested that CBD binds especially tightly [Kd (dissociation constant), ∼150 nm] to the slow inactivated state of TTX-R channels, which can be substantially occupied at voltages as negative as -40 mV. Remarkably, CBD was more potent in inhibiting TTX-R channels and inhibiting action potential firing than the local anesthetic bupivacaine. We conclude that CBD might produce some of its analgesic effects by direct effects on neuronal excitability, with tight binding to the slow inactivated state of Nav1.8 channels contributing to effective inhibition of repetitive firing by modest depolarizations.SIGNIFICANCE STATEMENT Cannabidiol (CBD) has been shown to inhibit pain in various rodent models, but the mechanism of this effect is unknown. We describe the ability of CBD to inhibit repetitive action potential firing in primary nociceptive neurons from mouse dorsal root ganglia and analyze the effects on voltage-dependent sodium channels. We find that CBD interacts with TTX-resistant sodium channels in a state-dependent manner suggesting particularly tight binding to slow inactivated states of Nav1.8 channels, which dominate the overall inactivation of Nav1.8 channels for small maintained depolarizations from the resting potential. The results suggest that CBD can exert analgesic effects in part by directly inhibiting repetitive firing of primary nociceptors and suggest a strategy of identifying compounds that bind selectively to slow inactivated states of Nav1.8 channels for developing effective analgesics.

Prefrontal cortex pyramidal neurons express functional Nav1.8 tetrodotoxin-resistant sodium currents.

It has been repeatedly proved that Nav1.8 tetrodotoxin (TTX)-resistant sodium currents are expressed in peripheral sensory neurons where they play important role in nociception. There are very few publications that show the presence of TTX-resistant sodium currents in central neurons. The aim of this study was to assess if functional Nav1.8 TTX-resistant sodium currents are expressed in prefrontal cortex pyramidal neurons. All recordings were performed in the presence of TTX in the extracellular solution to block TTX-sensitive sodium currents. The TTX-resistant sodium current recorded in this study was mainly carried by the Nav1.8 sodium channel isoform because the Nav1.9 current was inhibited by the -65 mV holding potential that we used throughout the study. Moreover, the sodium current that we recorded was inhibited by treatment with the selective Nav1.8 inhibitor A-803467. Confocal microscopy experiments confirmed the presence of the Nav1.8 α subunit in prefrontal cortex pyramidal neurons. Activation and steady state inactivation properties of TTX-resistant sodium currents were also assessed in this study and they were similar to activation and inactivation properties of TTX-resistant sodium currents expressed in dorsal root ganglia (DRG) neurons. Moreover, this study showed that carbamazepine (60 µM) inhibited the maximal amplitude of the TTX-resistant sodium current. Furthermore, we found that carbamazepine shifts steady state inactivation curve of TTX-resistant sodium currents toward hyperpolarization. This study suggests that the Nav1.8 TTX-resistant sodium channel is expressed not only in DRG neurons, but also in cortical neurons and may be molecular target for antiepileptic drugs such as carbamazepine.

SCN11A gene deletion causes sensorineural hearing loss by impairing the ribbon synapses and auditory nerves.

BACKGROUND: The SCN11A gene, encoded Nav1.9 TTX resistant sodium channels, is a main effector in peripheral inflammation related pain in nociceptive neurons. The role of SCN11A gene in the auditory system has not been well characterized. We therefore examined the expression of SCN11A in the murine cochlea, the morphological and physiological features of Nav1.9 knockout (KO) ICR mice. RESULTS: Nav1.9 expression was found in the primary afferent endings beneath the inner hair cells (IHCs). The relative quantitative expression of Nav1.9 mRNA in modiolus of wild-type (WT) mice remains unchanged from P0 to P60. The number of presynaptic CtBP2 puncta in Nav1.9 KO mice was significantly lower than WT. In addition, the number of SGNs in Nav1.9 KO mice was also less than WT in the basal turn, but not in the apical and middle turns. There was no lesion in the somas and stereocilia of hair cells in Nav1.9 KO mice. Furthermore, Nav1.9 KO mice showed higher and progressive elevated ABR threshold at 16 kHz, and a significant increase in CAP thresholds. CONCLUSIONS: These data suggest a role of Nav1.9 in regulating the function of ribbon synapses and the auditory nerves. The impairment induced by Nav1.9 gene deletion mimics the characters of cochlear synaptopathy.

Valproic acid inhibits TTX-resistant sodium currents in prefrontal cortex pyramidal neurons.

Valproic acid is frequently prescribed and used to treat epilepsy, bipolar disorder and other conditions. However, the mechanism of action of valproic acid has not been fully elucidated. The aim of this study was to assess the influence of valproic acid (200 µM) on TTX-resistant sodium currents in mPFC pyramidal neurons. Valproic acid inhibited the maximal amplitude and did not change the activation parameters of TTX-resistant sodium currents. Moreover, valproic acid (2 µM and 200 µM) shifted the TTX-resistant sodium channel inactivation curve towards hyperpolarisation. In the presence of valproic acid, TTX-resistant sodium currents recovered from inactivation more slowly. Valproic acid did not influence the use-dependent blockade of TTX-resistant sodium currents. This study suggests that a potential new mechanism of the antiepileptic action of valproic acid is, among others, inhibition of TTX-resistant sodium currents.

MrgprX1 Mediates Neuronal Excitability and Itch Through Tetrodotoxin-Resistant Sodium Channels.

In this study, we sought to elucidate the molecular mechanism underlying human Mas-related G protein-coupled receptor X1 (MrgprX1) mediated itch sensation. We found that activation of MrgprX1 by BAM8-22 triggered robust action potential discharges in dorsal root ganglion (DRG) neurons. This neuronal excitability is not mediated by Transient receptor potential (TRP) cation channels, M-type potassium channels, or chloride channels. Instead, activation of MrgprX1 lowers the activation threshold of TTX-resistant sodium channels and induces inward sodium currents. These MrgprX1-elicited action potential discharges can be blocked by Pertussis toxin (PTX) and a Gßγ inhibitor - Gallein. Behavioral results showed that Nav1.9 knockout but not Trpa1 knockout significantly reduced BAM8-22 evoked scratching behavior. Collectively, these data suggest that activation of MrgprX1 triggers itch sensation by increasing the activity of TTX-resistant voltage-gated sodium channels.

Acute inflammation reveals GABAA receptor-mediated nociception in mouse dorsal root ganglion neurons via PGE2 receptor 4 signaling.

Gamma-aminobutyric acid (GABA) depolarizes dorsal root ganglia (DRG) primary afferent neurons through activation of Cl- permeable GABAA receptors but the physiologic role of GABAA receptors in the peripheral terminals of DRG neurons remains unclear. In this study, we investigated the role of peripheral GABAA receptors in nociception using a mouse model of acute inflammation. In vivo, peripheral administration of the selective GABAA receptor agonist muscimol evoked spontaneous licking behavior, as well as spinal wide dynamic range (WDR) neuron firing, after pre-conditioning with formalin but had no effect in saline-treated mice. GABAA receptor-mediated pain behavior after acute formalin treatment was abolished by the GABAA receptor blocker picrotoxin and cyclooxygenase inhibitor indomethacin. In addition, treatment with prostaglandin E2 (PGE2) was sufficient to reveal muscimol-induced licking behavior. In vitro, GABA induced sub-threshold depolarization in DRG neurons through GABAA receptor activation. Both formalin and PGE2 potentiated GABA-induced Ca2+ transients and membrane depolarization in capsaicin-sensitive nociceptive DRG neurons; these effects were blocked by the prostaglandin E2 receptor 4 (EP4) antagonist AH23848 (10 µmol/L). Furthermore, potentiation of GABA responses by PGE2 was prevented by the selective Nav1.8 antagonist A887826 (100 nmol/L). Although the function of the Na+-K+-2Cl- co-transporter NKCC1 was required to maintain the Cl- ion gradient in isolated DRG neurons, NKCC1 was not required for GABAA receptor-mediated nociceptive behavior after acute inflammation. Taken together, these results demonstrate that GABAA receptors may contribute to the excitation of peripheral sensory neurons in inflammation through a combined effect involving PGE2-EP4 signaling and Na+ channel sensitization.

Control of sensory neuron excitability by serotonin involves 5HT2C receptors and Ca(2+)-activated chloride channels.

Serotonin (5HT) is a constituent of the so-called "inflammatory soup" that sensitizes nociceptors during inflammation. Nevertheless, receptors and signaling mechanisms that mediate an excitation of dorsal root ganglion (DRG) neurons by 5HT remained controversial. Therefore, capsaicin-sensitive nociceptive neurons dissociated from rat DRGs were used to investigate effects of 5HT on membrane excitability and currents through ligand- as well as voltage-gated ion channels. In 58% of the neurons tested, 5HT increased action potential firing, an effect that was abolished by the 5HT2 receptor antagonist ritanserin, but not by the 5HT3 antagonist tropisetron. Unlike other algogenic mediators, such as PGE2 and bradykinin, 5HT did not affect currents through TTX-resistant Na(+) channels or Kv7 K(+) channels. In all neurons investigated, 5HT potentiated capsaicin-evoked currents through TRPV1 channels, an effect that was attenuated by antagonists at 5HT2A (4 F 4 PP), 5HT2B (SB 204741), as well as 5HT2C (RS 102221) receptors. 5HT triggered slowly arising inward Cl(-) currents in 53% of the neurons. This effect was antagonized by the 5HT2C receptor blocker only, and the current was prevented by an inhibitor of Ca(2+)-activated chloride channels (CaCC). The 5HT-induced increase in action potential firing was also abolished by this CaCC blocker and by the TRPV1 inhibitor capsazepine. Amongst the subtype selective 5HT2 antagonists, only RS 102221 (5HT2C-selectively) counteracted the rise in action potential firing elicited by 5HT. These results show that 5HT excites DRG neurons mainly via 5HT2C receptors which concomitantly mediate a sensitization of TRPV1 channels and an opening of CaCCs.

Synthesis and biological characterization of synthetic analogs of Huwentoxin-IV (Mu-theraphotoxin-Hh2a), a neuronal tetrodotoxin-sensitive sodium channel inhibitor.

Huwentoxin-IV (HWTX-IV, also named Mu-theraphotoxin-Hh2a) is a typical inhibitor cystine knot peptide isolated from the venom of Chinese tarantula Ornithoctonus huwena and is found to inhibit tetrodotoxin-sensitive (TTX-S) sodium channels from mammalian sensory neurons. This peptide binds to neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II in neuronal sodium channels. However, the molecular surface of HWTX-IV interaction with sodium channels remains unknown. In this study, we synthesized HWTX-IV and three mutants (T28D, R29A and Q34D) and characterized their functions on TTX-S sodium channels from adult rat dorsal root ganglion (DRG) neurons. Analysis of liquid chromatography, mass spectrometry and circular dichroism spectrum indicated that all four synthetic peptides are properly folded. Synthetic HWTX-IV exhibited the same activity as native HWTX-IV, while three mutations reduced toxin binding affinities by 10-200 fold, indicating that the basic or vicinal polar residues Thr²8, Arg²9, and Gln³4 in C-terminus might play critical roles in the interaction of HWTX-IV with TTX-S sodium channels.

Neuroplastic alteration of TTX-resistant sodium channel with visceral pain and morphine-induced hyperalgesia.

The discovery of the tetrodotoxin-resistant (TTX-R) Na(+) channel in nociceptive neurons has provided a special target for analgesic intervention. In a previous study we found that both morphine tolerance and persistent visceral inflammation resulted in visceral hyperalgesia. It has also been suggested that hyperexcitability of sensory neurons due to altered TTX-R Na(+) channel properties and expression contributes to hyperalgesia; however, we do not know if some TTX-R Na(+) channel property changes can be triggered by visceral hyperalgesia and morphine tolerance, or whether there are similar molecular or channel mechanisms in both situations. To evaluate the effects of morphine tolerance and visceral inflammation on the channel, we investigated the dorsal root ganglia (DRG) neuronal change following these chronic treatments. Using whole-cell patch clamp recording, we recorded TTX-R Na(+) currents in isolated adult rat lumbar and sacral (L6-S2) DRG neurons from normal and pathologic rats with colon inflammatory pain or chronic morphine treatment. We found that the amplitudes of TTX-R Na(+) currents were significantly increased in small-diameter DRG neurons with either morphine tolerance or visceral inflammatory pain. Meanwhile, the result also showed that those treatments altered the kinetics properties of the electrical current (ie, the activating and inactivating speed of the channel was accelerated). Our current results suggested that in both models, visceral chronic inflammatory pain and morphine tolerance causes electrophysiological changes in voltage-gated Na channels due to the chronic administration of these medications. For the first time, the present investigation explored the adaptations of this channel, which may contribute to the hyperexcitability of primary afferent nerves and hyperalgesia during these pathologic conditions. The results also suggest that neurophysiologic mechanisms of morphine tolerance and visceral hyperalgesia are related at the TTX-R Na(+) channel.