Voltage-dependence Of Steady-state Fast Inactivation Research Articles

Beyond CBD: Inhibitory effects of lesser studied phytocannabinoids on human voltage-gated sodium channels.

Introduction: Cannabis contains cannabidiol (CBD), the main non-psychoactive phytocannabinoid, but also many other phytocannabinoids that have therapeutic potential in the treatment of epilepsy. Indeed, the phytocannabinoids cannabigerolic acid (CBGA), cannabidivarinic acid (CBDVA), cannabichromenic acid (CBCA) and cannabichromene (CBC) have recently been shown to have anti-convulsant effects in a mouse model of Dravet syndrome (DS), an intractable form of epilepsy. Recent studies demonstrate that CBD inhibits voltage-gated sodium channel function, however, whether these other anti-convulsant phytocannabinoids affect these classic epilepsy drug-targets is unknown. Voltage-gated sodium (NaV) channels play a pivotal role in initiation and propagation of the neuronal action potential and NaV1.1, NaV1.2, NaV1.6 and NaV1.7 are associated with the intractable epilepsies and pain conditions. Methods: In this study, using automated-planar patch-clamp technology, we assessed the profile of the phytocannabinoids CBGA, CBDVA, cannabigerol (CBG), CBCA and CBC against these human voltage-gated sodium channels subtypes expressed in mammalian cells and compared the effects to CBD. Results: CBD and CBGA inhibited peak current amplitude in the low micromolar range in a concentration-dependent manner, while CBG, CBCA and CBC revealed only modest inhibition for this subset of sodium channels. CBDVA inhibited NaV1.6 peak currents in the low micromolar range in a concentration-dependent fashion, while only exhibiting modest inhibitory effects on NaV1.1, NaV1.2, and NaV1.7 channels. CBD and CBGA non-selectively inhibited all channel subtypes examined, whereas CBDVA was selective for NaV1.6. In addition, to better understand the mechanism of this inhibition, we examined the biophysical properties of these channels in the presence of each cannabinoid. CBD reduced NaV1.1 and NaV1.7 channel availability by modulating the voltage-dependence of steady-state fast inactivation (SSFI, V0.5 inact), and for NaV1.7 channel conductance was reduced. CBGA also reduced NaV1.1 and NaV1.7 channel availability by shifting the voltage-dependence of activation (V0.5 act) to a more depolarized potential, and for NaV1.7 SSFI was shifted to a more hyperpolarized potential. CBDVA reduced channel availability by modifying conductance, SSFI and recovery from SSFI for all four channels, except for NaV1.2, where V0.5 inact was unaffected. Discussion: Collectively, these data advance our understanding of the molecular actions of lesser studied phytocannabinoids on voltage-gated sodium channel proteins.

Biochemical and Functional Evidence for Homodimerization of Voltage-Gated Sodium Channels (Navs)

Sodium channels in the peripheral nervous system play a major role in the initiation and transmission of action potentials, which can lead to the perception of pain. Mutations in different channel subtypes can induce pain syndromes, which are associated with increased pain or total loss of pain. Interestingly, most pain-linked mutations are heterozygous, resulting in the simultaneous presence of WT and mutated channels in the expressing cells. In 2017, Clatot et al. (Nat. comm. https://doi.org/10.1038/s41467-017-02262-0) provided evidence that Nav1.5 channels operate as dimers, which can be uncoupled by difopein, an antagonist of 14-3-3. Here, we performed patch-clamp experiments on Nav1.7 stably expressed in HEK cells. We found that the voltage-dependence of steady-state fast inactivation is shifted by co-transfection with difopein, suggesting that dimerziation can have an impact on Nav1.7 channel gating. To biochemically confirm Nav dimerization, we expressed C-terminally GFP-tagged versions of Nav1.2, Nav1.5. Nav1.7 and Nav1.8 in Xenopus laevis oocytes. We extracted the Navs by a dodecyl maltoside-based non-denaturing buffer from these cells and resolved the proteins by colorless SDS-PAGE and high resolution clear native PAGE (hrCN-PAGE; Wittig et al. Mol Cell Proteomics 2007. https://doi.org/10.1074/mcp.M700076-MCP200). As a mass marker, we co-analyzed TRPV1-GFP (calculated protomer mass 123 kDa). We visualized the GFP-tagged proteins by Typhoon fluorescence scanning. All the Nav proteins migrated in hrCN-PAGE gels as defined bands of molecular masses of about 560 kDa compared to the TRPV1-GFP tetramer (492 kDa). Following denaturing with SDS, the Navs migrated in hrCN-PAGE gels at about 270 kDa, i.e. close to the masses of the Nav protomers. We conclude from these findings that Nav1.2, Nav1.5. Nav1.7 and Nav1.8 exist in cells as stable non-covalently associated homodimers, and that at least the gating of Nav1.7 is affected by dimerization.

Effects of acidosis on neuronal voltage-gated sodium channels: Nav1.1 and Nav1.3

ABSTRACTVoltage-gated sodium channels are key contributors to membrane excitability. These channels are expressed in a tissue-specific manner. Mutations and modulation of these channels underlie various physiological and pathophysiological manifestations. The effects of changes in extracellular pH on channel gating have been studied on several sodium channel subtypes. Among these, Nav1.5 is the most pH-sensitive channel, with Nav1.2 and Nav1.4 being mostly pH-resistant channels. However, pH effects have not been characterized on other sodium channel subtypes. In this study, we sought to determine whether Nav1.1 and Nav1.3 display resistance or sensitivity to changes in extracellular pH. These two sodium channel subtypes are predominantly found in inhibitory neurons. The expression of these channels highly depends on age and the developmental stage of neurons, with Nav1.3 being found mostly in neonatal neurons, and Nav1.1 being found in adult neurons. Our present results indicate that, during extracellular acidosis, both channels show a depolarization in the voltage-dependence of activation and moderate reduction in current density. Voltage-dependence of steady-state fast inactivation and recovery from fast inactivation were unchanged. We conclude that Nav1.1 and Nav1.3 have similar pH-sensitivities.

Molecular and functional characterization of a novel sodium channel TipE-like auxiliary subunit from the American cockroach Periplaneta americana

In Drosophila melanogaster, the functions of voltage-gated sodium (Nav) channels are modulated by TipE and its orthologs. Here, we describe a novel TipE homolog of the American cockroach, Periplaneta americana, called PaTipE. Like DmTipE, PaTipE mRNAs are ubiquitously expressed. Surprisingly, PaTipE mRNA was undetectable in neurosecretory cells identified as dorsal unpaired median neurons. Phylogenetic analysis placed this new sequence in TipE clade, indicating an independent evolution from a common ancestor. Contrary to previous reports, our data indicate that the auxiliary subunits of insect Nav channels are very distant from the mammalian BKCa auxiliary subunits. To decipher the functional roles of PaTipE, we characterized the gating properties of DmNav1-1 channels co-expressed with DmTipE or PaTipE, in Xenopus oocytes. Compared to DmTipE, PaTipE increased Na+ currents by a 4.2-fold. The voltage-dependence of steady-state fast inactivation of DmNav1-1/PaTipE channels was shifted by 5.8 mV to more negative potentials than that of DmNav1-1/DmTipE channels. DmNav1-1/PaTipE channels recovered 3.2-fold slower from the fast-inactivated state than DmNav1-1/DmTipE channels. In conclusion, this study supports that the insect Nav auxiliary subunits share functional features with their mammalian counterparts, although structurally and phylogenetically distant.

Carvacrol modulates voltage-gated sodium channels kinetics in dorsal root ganglia

Recent studies have shown that many of plant-derived compounds interact with specific ion channels and thereby modulate many sensing mechanisms, such as nociception. The monoterpenoid carvacrol (5-isopropyl-2-methylphenol) has an anti-nociceptive effect related to a reduction in neuronal excitability and voltage-gated Na+ channels (NaV) inhibition in peripheral neurons. However, the detailed mechanisms of carvacrol-induced inhibition of neuronal NaV remain elusive. This study explores the interaction between carvacrol and NaV in isolated dorsal root ganglia neurons. Carvacrol reduced the total voltage-gated Na+ current and tetrodotoxin-resistant (TTX-R) Na+ current component in a concentration-dependent manner. Carvacrol accelerates current inactivation and induced a negative-shift in voltage-dependence of steady-state fast inactivation in total and TTX-R Na+ current. Furthermore, carvacrol slowed the recovery from inactivation. Carvacrol provoked a leftward shift in both the voltage-dependence of steady-state inactivation and activation of the TTX-R Na+ current component. In addition, carvacrol-induced inhibition of TTX-R Na+ current was enhanced by an increase in stimulation frequency and when neurons were pre-conditioned with long depolarization pulse (5s at −50mV). Taken all results together, we herein demonstrated that carvacrol affects NaV gating properties. The present findings would help to explain the mechanisms underlying the analgesic activity of carvacrol.

Nav1.5 C-Terminal Domains Influence Calcium Regulation of Fast Inactivation Separately from Calmodulin Interaction

The cardiac sodium channel (Nav1.5) has a complex ‘intracellular Ca2+ sensing apparatus’ within its C-terminal domain (CTD) consisting of a partial EF-hand domain (CTD-EF) and a calmodulin (CaM) binding IQ motif (CTD-IQ). There are additional CaM binding motifs within the DIII-DIV linker. Variation in intracellular Ca2+ concentration influences the voltage-dependence of steady-state fast inactivation (SSI) by an unclear mechanism. Here, we mutated 16 key residues implicated in the interactions between CTD-EF and CTD-IQ, CTD-IQ and CaM, or DIII-DIV and CaM and explored their impact on NaV1.5 function and biochemistry. We used quantitative yeast-two-hybrid assays to measure effects of mutations on the interaction between the full length CTD on CaM, and evaluated SSI in high (1 µM free Ca2+) and low [Ca2+]i conditions. Using either BAPTA or HEDTA as chelator, 1 µM free [Ca2+]i was sufficient to shift SSI (elicited by 50 ms prepulses) towards depolarized potentials. We observed that 3 of 5 CTD-IQ mutations (F1912A, A1924T and IQ/AA) strongly reduced the interaction between the CTD and CaM. However, these mutations did not affect the [Ca2+]i effect on SSI suggesting that SSI [Ca2+]i sensitivity does not depend on CaM binding to the CTD-IQ. Unexpectedly, single and combination mutations of the CTD-EF (L1786A, F1791A, Q1807A, L1862A, E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) diminished the CTD-CaM interaction, and some of these mutations (L1786A, E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) also suppressed the [Ca2+]i effect on SSI. Indeed, the only mutations we studied that blunted the [Ca2+]i effect on SSI were within the CTD-EF. These results suggest that the CTD-EF influences NaV1.5 [Ca2+]i sensitivity and mutations in this domain can also alter the interaction of the CTD with CaM, but CaM interaction with the CTD-IQ is not required for the effect of intracellular Ca2+ on inactivation.

Mexiletine as a treatment for primary erythromelalgia: normalization of biophysical properties of mutant L858F NaV 1.7 sodium channels.

Background and PurposeThe non-selective sodium channel inhibitor mexiletine has been found to be effective in several animal models of chronic pain and has become popular in the clinical setting as an orally available alternative to lidocaine. It remains unclear why patients with monogenic pain disorders secondary to gain-of-function SCN9a mutations benefit from a low systemic concentration of mexiletine, which does not usually induce adverse neurological side effects. The aim of this study was, therefore, to investigate the biophysical effects of mexiletine on the L858F primary erythromelalgia NaV1.7 mutation in vitro.Experimental ApproachHuman wild-type and L858F-mutated NaV1.7 channels were expressed in HEK293A cells. Whole-cell currents were recorded by voltage-clamp techniques to characterize the effect of mexiletine on channel gating properties.Key ResultsWhile the concentration-dependent tonic block of peak currents by mexiletine was similar in wild-type and L858F channels, phasic block was more pronounced in cells transfected with the L858F mutation. Moreover, mexiletine substantially shifted the pathologically-hyperpolarized voltage-dependence of steady-state activation in L858F-mutated channels towards wild-type values and the voltage-dependence of steady-state fast inactivation was shifted to more hyperpolarized potentials, leading to an overall reduction in window currents.Conclusion and ImplicationsMexiletine has a normalizing effect on the pathological gating properties of the L858F gain-of-function mutation in NaV1.7, which, in part, might explain the beneficial effects of systemic treatment with mexiletine in patients with gain-of-function sodium channel disorders.

Enhancement of Slow Inactivation of Voltage-Gated Na + Channels by Ranolazine

Ranolazine is an anti-anginal drug that blocks cardiac (Nav1.5) late Na+ current (INa) at therapeutic concentrations (2-10 μM). Recent electrophysiological studies have shown that ranolazine also blocks skeletal muscle (Nav1.4) and neuronal (Nav1.1, Nav1.7, Nav1.8) INa. We investigated the effects of ranolazine on peak INa and on both sustained repetitive firing (SRF) and 0 mM Mg2+- induced (0 [Mg2+]O) continuous high frequency firing of action potentials in cultured rat hippocampal neurons. Ranolazine caused a voltage (−60 mV) and frequency (10 Hz)-dependent inhibition of INa with IC50 values of 0.48 and 61.8 μM (n=4-6), respectively. Ranolazine (10 μM, n=4) did not shift the voltage-dependence of steady-state fast inactivation; however, it caused a 15-mV hyperpolarizing shift (p<0.05, compared to control, n=4) in the slow inactivation. Consistently, ranolazine (10 μM) reduced SRF only during a 4-sec burst but not with a 1-sec depolarization step (Fig.1). Furthermore, ranolazine (10 μM) reduced 0 [Mg2+]O -induced high frequency firing of spontaneous action potentials from 0.78 to 0.45 Hz (p<0.05, n=5). Taken together, these data suggest that ranolazine suppresses the propagation and conduction of action potentials by preferentially interacting with Na+ channels in the slow inactivated state.View Large Image | View Hi-Res Image | Download PowerPoint Slide