NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE) or sodium channels (EKEE or EEKE). NALCN channels with alternative calcium, (EEEE) and sodium, (EKEE or EEKE) -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K)-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG(+) impermeant and blockable with 10 µM Gd(3+) ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2):371-83.

Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. β and γENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.

The aim of the present study was to determine whether the TRPV1 channel is involved in the onset of sodium appetite. For this purpose, we used TRPV1-knockout mice to investigate sodium depletion-induced drinking at different times (2/24 h) after furosemide administration combined with a low sodium diet (FURO-LSD). In sodium depleted wild type and TRPV1 KO (SD-WT/SD-TPRV1-KO) mice, we also evaluated the participation of other sodium sensors, such as TPRV4, NaX and angiotensin AT1-receptors (by RT-PCR), as well as investigating the pattern of neural activation shown by Fos immunoreactivity, in different nuclei involved in hydromineral regulation. TPRV1 SD-KO mice revealed an increased sodium preference, ingesting a higher hypertonic cocktail in comparison with SD-WT mice. Our results also showed in SD-WT animals that SFO-Trpv4 expression increased 2 h after FURO-LSD, compared to other groups, thus supporting a role of SFO-Trpv4 channels during the hyponatremic state. However, the SD-TPRV1-KO animals did not show this early increase, and maybe as a consequence drank more hypertonic cocktail. Regarding the SFO-NaX channel expression, in both genotypes our findings revealed a reduction 24 h after FURO-LSD. In addition, there was an increase in the OVLT-NaX expression of SD-WT 24 h after FURO-LSD, suggesting the participation of OVLT-NaX channels in the appearance of sodium appetite, possibly as an anticipatory response in order to limit sodium intake and to induce thirst. Our work demonstrates changes in the expression of different osmo‑sodium-sensitive channels at specific nuclei, related to the body sodium status in order to stimulate an adequate drinking.

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.

Voltage-gated sodium channels are targets for a range of pharmaceutical drugs developed for the treatment of neurological diseases. Cannabidiol (CBD), the non-psychoactive compound isolated from cannabis plants, was recently approved for treatment of two types of epilepsy associated with sodium channel mutations. This study used high-resolution X-ray crystallography to demonstrate the detailed nature of the interactions between CBD and the NavMs voltage-gated sodium channel, and electrophysiology to show the functional effects of binding CBD to these channels. CBD binds at a novel site at the interface of the fenestrations and the central hydrophobic cavity of the channel. Binding at this site blocks the transmembrane-spanning sodium ion translocation pathway, providing a molecular mechanism for channel inhibition. Modelling studies suggest why the closely-related psychoactive compound tetrahydrocannabinol may not have the same effects on these channels. Finally, comparisons are made with the TRPV2 channel, also recently proposed as a target site for CBD. In summary, this study provides novel insight into a possible mechanism for CBD interactions with sodium channels.

Voltage-gated sodium (NaV) channels are a family of transmembrane ion channel proteins. They function by forming a gated, water-filled pore to help establish and control cell membrane potential via control of the flow of ions between the intracellular and the extracellular environments. Blockade of NaVs has been successfully accomplished in the clinic to enable control of pathological firing patterns that occur in a diverse range of conditions such as chronic pain, epilepsy, and cardiac arrhythmias. First generation sodium channel modulator drugs, despite low inherent subtype selectivity, preferentially act on over-excited cells which reduces undesirable side effects in the clinic. However, the limited therapeutic indices observed with the first generation demanded a new generation of sodium channel inhibitors. The structure, function and the state of the art in sodium channel modulator drug discovery are discussed in this chapter.

Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs), this AP back-propagation is supported by dendritic voltage-gated Na+ (Nav) channels, whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we revealed the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here, the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability.

Bacterial sodium channels are important models for understanding ion permeation and selectivity. However, their homotetrameric structure limits their use as models for understanding the more complex eukaryotic voltage-gated sodium channels (which have a pseudo-heterotetrameric structure formed from an oligomer composed of four domains). To bridge this gap we attempted to synthesise oligomers made from four covalently linked bacterial sodium channel monomers and thus resembling their eukaryotic counterparts.

Voltage-gated sodium channels (NaV channels) are essential for the initiation and propagation of action potentials that critically influence our ability to respond to a diverse range of stimuli. Physiological and pharmacological studies have linked abnormal function of NaV channels to many human disorders, including chronic neuropathic pain. These findings, along with the description of the functional properties and expression pattern of NaV channel subtypes, are helping to uncover subtype specific roles in acute and chronic pain and revealing potential opportunities to target these with selective inhibitors. High-throughput screens and automated electrophysiology platforms have identified natural toxins as a promising group of molecules for the development of target-specific analgesics. In this review, the role of toxins in defining the contribution of NaV channels in acute and chronic pain states and their potential to be used as analgesic therapies are discussed.

Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown.

We examined the contribution of the sodium channel isoform Nav1.8 to retinal function using the specific blocker A803467. We found that A803467 has little influence on the electroretinogram (ERG) a- and b-waves, but significantly reduces the oscillatory potentials (OPs) to 40-60% of their original amplitude, with significant changes in implicit time in the rod-driven range. To date, only two cell types were found in mouse to express Nav1.8; the starburst amacrine cells (SBACs), and a subtype of retinal ganglion cells (RGCs). When we recorded light responses from ganglion cells using a multielectrode array we found significant and opposing changes in two physiological groups of RGCs. ON-sustained cells showed significant decreases while transient ON-OFF cells showed significant increases. The effects on ON-OFF transient cells but not ON-sustained cells disappeared in the presence of an inhibitory cocktail. We have previously shown that RGCs have only a minor contribution to the OPs (Smith et al., 2014), therefore suggesting that SBACs might be a significant contributor to this ERG component. Targeting SBACs with the cholinergic neurotoxin ethylcholine mustard aziridinium (AF64A) caused a reduction in the amplitude of the OPs similar to A803467. Our results, both using the ERG and MEA recordings from RGCs, suggest that Nav1.8 plays a role in modulating specific aspects of the retinal physiology and that SBACs are a fundamental cellular contributor to the OPs in mice, a clear demonstration of the dichotomy between ERG b-wave and OPs.

Voltage-gated sodium channels (VGSCs) are strategically positioned to mediate neuronal plasticity because of their influence on action potential waveform. VGSC function may be strongly inhibited by local anesthetic and antiepileptic drugs and modestly modulated via second messenger pathways. Here, we report that the allosteric modulators of the calcium-sensing receptor (CaSR) cinacalcet, calindol, calhex, and NPS 2143 completely inhibit VGSC current in the vast majority of cultured mouse neocortical neurons. This form of VGSC current block persisted in CaSR-deficient neurons, indicating a CaSR-independent mechanism. Cinacalcet-mediated blockade of VGSCs was prevented by the guanosine diphosphate (GDP) analog GDPβs, indicating that G-proteins mediated this effect. Cinacalcet inhibited VGSCs by increasing channel inactivation, and block was reversed by prolonged hyperpolarization. Strong cinacalcet inhibition of VGSC currents was also present in acutely isolated mouse cortical neurons. These data identify a dynamic signaling pathway by which G-proteins regulate VGSC current to indirectly modulate central neuronal excitability.

Cementum, which is excreted by cementoblasts, provides an attachment site for collagen fibers that connect to the alveolar bone and fix the teeth into the alveolar sockets. Transmembrane ionic signaling, associated with ionic transporters, regulate various physiological processes in a wide variety of cells. However, the properties of the signals generated by plasma membrane ionic channels in cementoblasts have not yet been described in detail. We investigated the biophysical and pharmacological properties of ion channels expressed in human cementoblast (HCEM) cell lines by measuring ionic currents using conventional whole-cell patch-clamp recording. The application of depolarizing voltage steps in 10 mV increments from a holding potential (Vh) of -70 mV evoked outwardly rectifying currents at positive potentials. When intracellular K+ was substituted with an equimolar concentration of Cs+, the outward currents almost disappeared. Using tail current analysis, the contributions of both K+ and background Na+ permeabilities were estimated for the outward currents. Extracellular application of tetraethylammonium chloride (TEA) and iberiotoxin (IbTX) reduced the densities of the outward currents significantly and reversibly, whereas apamin and TRAM-34 had no effect. When the Vh was changed to -100 mV, we observed voltage-dependent inward currents in 30% of the recorded cells. These results suggest that HCEM express TEA- and IbTX-sensitive large-conductance Ca2+-activated K+ channels and voltage-dependent Na+ channels.

The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene variants associated with hypertension, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC activity and SSI. We evaluated a commercially available automated two-electrode voltage-clamp (TEVC) system that records transmembrane currents of ENaC-expressing Xenopus oocytes in 96-well microtiter plates. We employed guinea pig, human and Xenopus laevis ENaC orthologs that display specific magnitudes of SSI. While demonstrating some limitations over traditional TEVC systems with customized perfusion chambers, the automated TEVC system was able to detect the established SSI characteristics of the employed ENaC orthologs. We were able to confirm a reduced SSI in a gene variant, leading to C479R substitution in the human α-ENaC subunit that has been reported in Liddle syndrome. In conclusion, automated TEVC in Xenopus oocytes can detect SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended.

Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogs, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors.

ND7/23 cells are gaining traction as a host model to express peripheral sodium channels such as NaV1.8 and NaV1.9 that have been difficult to express in widely utilized heterologous cells, like CHO and HEK293. Use of ND7/23 as a model cell to characterize the properties of sodium channels requires clear understanding of the endogenous ion channels. To define the nature of the background sodium currents in ND7/23 cells, we aimed to comprehensively profile the voltage-gated sodium channel subunits by endpoint and quantitative reverse transcription-PCR and by whole-cell patch clamp electrophysiology. We found that untransfected ND7/23 cells express endogenous peak sodium currents that average -2.12nA (n = 15) and with kinetics typical of fast sodium currents having activation and inactivation completed within few milliseconds. Furthermore, sodium currents were reduced to virtually nil upon exposure to 100nM tetrodotoxin, indicating that ND7/23 cells have essentially null background for tetrodotoxin-resistant (TTX-R) currents. qRT-PCR profiling indicated a major expression of TTX-sensitive (TTX-S) NaV1.6 and NaV1.7 at similar levels and very low expression of TTX-R NaV1.9 transcripts. There was no expression of TTX-R NaV1.8 in ND7/23 cells. There was low expression of NaV1.1, NaV1.2, NaV1.3 and no expression of cardiac or skeletal muscle sodium channels. As for the sodium channel auxiliary subunits, β1 and β3 subunits were expressed, but not the β2 and β4 subunits that covalently associate with the α-subunits. In addition, our results also showed that only the mouse forms of NaV1.6, NaV1.7 and NaV1.9 sodium channels were expressed in ND7/23 cells that was originally generated as a hybridoma of rat embryonic DRG and mouse neuroblastoma cell-line. By molecular profiling of auxiliary β- and principal α-subunits of the voltage gated sodium channel complex, our results define the background sodium channels expressed in ND7/23 cells, and confirm their utility for detailed functional studies of emerging pain channelopathies ascribed to mutations of the TTX-R sodium channels of sensory neurons.

Fast opening and closing of voltage-gated sodium channels are crucial for proper propagation of the action potential through excitable tissues. Unlike potassium channels, sodium channel α-subunits are believed to form functional monomers. Yet, an increasing body of literature shows inconsistency with the traditional idea of a single α-subunit functioning as a monomer. Here we demonstrate that sodium channel α-subunits not only physically interact with each other but they actually assemble, function and gate as a dimer. We identify the region involved in the dimerization and demonstrate that 14-3-3 protein mediates the coupled gating. Importantly we show conservation of this mechanism among mammalian sodium channels. Our study not only shifts conventional paradigms in regard to sodium channel assembly, structure, and function but importantly this discovery of the mechanism involved in channel dimerization and biophysical coupling could open the door to new approaches and targets to treat and/or prevent sodium channelopathies.

Propranolol is a widely used, non-selective β-adrenergic receptor antagonist with proven efficacy in treating cardiovascular disorders and in the prevention of migraine headaches. At plasma concentrations exceeding those required for β-adrenergic receptor inhibition, propranolol also exhibits anti-arrhythmic ("membrane stabilizing") effects that are not fully explained by β-blockade. Previous in vitro studies suggested that propranolol may have local anesthetic effects. We directly tested the effects of propranolol on heterologously expressed recombinant human cardiac (NaV1.5) and brain (NaV1.1, NaV1.2, NaV1.3) sodium channels using whole-cell patch-clamp recording. We found that block was not stereospecific as we observed approximately equal IC50 values for tonic and use-dependent block by R-(+) and S-(-) propranolol (tonic block: R: 21.4 μM vs S: 23.6 μM; use-dependent block: R: 2.7 μM vs S: 2.6 μM). Metoprolol and nadolol did not block NaV1.5 indicating that sodium channel block is not a class effect of β-blockers. The biophysical effects of R-(+)-propranolol on NaV1.5 and NaV1.1 resembled that of the prototypical local anesthetic lidocaine including the requirement for a critical phenylalanine residue (F1760 in NaV1.5) in the domain 4 S6 segment. Finally, we observed that brain sodium channels exhibited less sensitivity to R-(+)-propranolol than NaV1.5 channels. Our findings establish sodium channels as targets for propranolol and may help explain some beneficial effects of the drug in treating cardiac arrhythmias, and may explain certain adverse central nervous system effects.

Action potential generation is governed by the opening, inactivation, and recovery of voltage-gated sodium channels. A channel's voltage-sensing and pore-forming α subunit bears an intrinsic fast inactivation particle that mediates both onset of inactivation upon membrane depolarization and rapid recovery upon repolarization. We describe here a novel inactivation particle housed within an accessory channel subunit (A-type FHF protein) that mediates rapid-onset, long-term inactivation of several sodium channels. The channel-intrinsic and tethered FHF-derived particles, both situated at the cytoplasmic face of the plasma membrane, compete for induction of inactivation, causing channels to progressively accumulate into the long-term refractory state during multiple cycles of membrane depolarization. Intracellular injection of a short peptide corresponding to the FHF particle can reproduce channel long-term inactivation in a dose-dependent manner and can inhibit repetitive firing of cerebellar granule neurons. We discuss potential structural mechanisms of long-term inactivation and potential roles of A-type FHFs in the modulation of action potential generation and conduction.

Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.