Coexpression Of Nav1 Research Articles

Enhancement of EFL-inactivation gate interaction by truncation of Nav1.5 carboxy-terminal domain.

The voltage-gated sodium channel 1.5 (NaV1.5) participates in the initiation of the cardiac action potential by undergoing rapid activation which, within milliseconds, is followed by inactivation. Human channelopathic mutations in NaV1.5 are linked to various cardiac maladies, including cardiac arrythmia, atrial fibrillation, and dilated cardiomyopathies. The carboxy-terminal domain (CTD) of NaV1.5 is a well-recognized hotspot for channelopathic mutations. This domain harbors an EF-hand-like domain (EFL) and an IQ motif, which tune channel function via binding of auxiliary proteins, including the fibroblast growth factor homologous factors and calmodulin. In addition, the isolated NaV1.5 EFL has been reported to bind the channel inactivation gate (IG) via a partially buried hydrophobic pocket. However, whether this binding occurs in the full-length human channel and how it tunes NaV1.5 function are not well understood. Here, we have used high-resolution multi-channel electrophysiology recordings of disease-associated truncations and engineered NaV1.5 mutants to probe this interaction. We show that truncation of the NaV1.5 CTD increases late Na current, with a maximal increase observed for a truncation that includes helix V of the EFL (NaV1.5E[1864]∗), while further truncation resulted in reduced late Na current. Deletion of helix V increases the exposure of the EFL hydrophobic pocket, suggesting the large late Na current observed for NaV1.5E[1864]∗ may stem from a tighter interaction between the IG and the mutant EFL. Consistent with this, co-expression of NaV1.5E[1864]∗ with the WT NaV1.5 CTD or IG reduced late Na current, while expression with IG mutants designed to disrupt binding with either the pore-domain (IFM/QQQ) or EFL (K1504E/K1505E) showed only a partial reduction. These findings suggest that as NaV1.5 cycles between its functional states, the IG shuttles between the EFL and the pore domain shedding new light onto how these regions tune channel function.

Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice.

Trigeminal sensory neurons of transgenic knock-in (KI) mice expressing the R192Q missense mutation in the α1A subunit of neuronal voltage-gated CaV2.1 Ca2+ channels, which leads to familial hemiplegic migraine type 1 (FHM1) in patients, exhibit a hyperexcitability phenotype. Here, we show that the expression of NaV1.7 channels, linked to pain states, is upregulated in KI primary cultures of trigeminal ganglia (TG), as shown by increased expression of its α1 subunit. In the majority of TG neurons, NaV1.7 channels are co-expressed with ATP-gated P2X3 receptors (P2X3R), which are important nociceptive sensors. Reversing the trigeminal phenotype with selective CaV2.1 channel inhibitor ω-agatoxin IVA inhibited NaV1.7 overexpression. Functionally, KI neurons revealed a TTX-sensitive inward current of larger amplitude that was partially inhibited by selective NaV1.7 blocker Tp1a. Under current-clamp condition, Tp1a raised the spike threshold of both wild-type (WT) and KI neurons with decreased firing rate in KI cells. NaV1.7 activator OD1 accelerated firing in WT and KI neurons, a phenomenon blocked by Tp1a. Enhanced expression and function of NaV1.7 channels in KI TG neurons resulted in higher excitability and facilitated nociceptive signaling. Co-expression of NaV1.7 channels and P2X3Rs in TGs may explain how hypersensitivity to local stimuli can be relevant to migraine.

Small G-protein RhoA is a potential inhibitor of cardiac fast sodium current.

Small G-proteins of Rho family modulate the activity of several classes of ion channels, including K+ channels Kv1.2, Kir2.1, and ERG; Ca2+ channels; and epithelial Na+ channels. The present study was aimed to check the RhoA potential regulatory effects on Na+ current (INa) transferred by Na+ channel cardiac isoform NaV1.5 in heterologous expression system and in native rat cardiomyocytes. Whole-cell patch-clamp experiments showed that coexpression of NaV1.5 with the wild-type RhoA in CHO-K1 cell line caused 2.7-fold decrease of INa density with minimal influence on steady-state activation and inactivation. This effect was reproduced by the coexpression with a constitutively active RhoA, but not with a dominant negative RhoA. In isolated ventricular rat cardiomyocytes, a 5-h incubation with the RhoA activator narciclasine (5 × 10-6M) reduced the maximal INa density by 38.8%. The RhoA-selective inhibitor rhosin (10-5M) increased the maximal INa density by 25.3%. Experiments with sharp microelectrode recordings in isolated right ventricular wall preparations showed that 5 × 10-6M narciclasine induced a significant reduction of action potential upstroke velocity after 2h of incubation. Thus, RhoA might be considered as a potential negative regulator of sodium channels cardiac isoform NaV1.5.

Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells.

The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.

Effects of the β1 auxiliary subunit on modification of Rat Nav1.6 sodium channels expressed in HEK293 cells by the pyrethroid insecticides tefluthrin and deltamethrin

We expressed rat Nav1.6 sodium channels with or without the rat β1 subunit in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on whole-cell sodium currents. In assays with the Nav1.6 α subunit alone, both pyrethroids prolonged channel inactivation and deactivation and shifted the voltage dependence of channel activation and steady-state inactivation toward hyperpolarization. Maximal shifts in activation were ~18mV for tefluthrin and ~24mV for deltamethrin. These compounds also caused hyperpolarizing shifts of ~10–14mV in the voltage dependence of steady-state inactivation and increased in the fraction of sodium current that was resistant to inactivation. The effects of pyrethroids on the voltage-dependent gating greatly increased the size of sodium window currents compared to unmodified channels; modified channels exhibited increased probability of spontaneous opening at membrane potentials more negative than the normal threshold for channel activation and incomplete channel inactivation. Coexpression of Nav1.6 with the β1 subunit had no effect on the kinetic behavior of pyrethroid-modified channels but had divergent effects on the voltage-dependent gating of tefluthrin- or deltamethrin-modified channels, increasing the size of tefluthrin-induced window currents but decreasing the size of corresponding deltamethrin-induced currents. Unexpectedly, the β1 subunit did not confer sensitivity to use-dependent channel modification by either tefluthrin or deltamethrin. We conclude from these results that functional reconstitution of channels in vitro requires careful attention to the subunit composition of channel complexes to ensure that channels in vitro are faithful functional and pharmacological models of channels in neurons.

Reciprocal Changes in Phosphorylation and Methylation of Mammalian Brain Sodium Channels in Response to Seizures

Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.

Interaction of Voltage-gated Sodium Channel Nav1.6 (SCN8A) with Microtubule-associated Protein Map1b

The mechanism by which voltage-gated sodium channels are trafficked to the surface of neurons is not well understood. Our previous work implicated the cytoplasmic N terminus of the sodium channel Na(v)1.6 in this process. We report that the N terminus plus the first transmembrane segment (residues 1-153) is sufficient to direct a reporter to the cell surface. To identify proteins that interact with the 117-residue N-terminal domain, we carried out a yeast two-hybrid screen of a mouse brain cDNA library. Three clones containing overlapping portions of the light chain of microtubule-associated protein Map1b (Mtap1b) were recovered from the screen. Interaction between endogenous Na(v)1.6 channels and Map1b in mouse brain was confirmed by co-immunoprecipitation. Map1b did not interact with the N terminus of the related channel Na(v)1.1. Alanine-scanning mutagenesis of the Na(v)1.6 N terminus demonstrated that residues 77-80 (VAVP) contribute to interaction with Map1b. Co-expression of Na(v)1.6 with Map1b in neuronal cell line ND7/23 resulted in a 50% increase in current density, demonstrating a functional role for this interaction. Mutation of the Map1b binding site of Na(v)1.6 prevented generation of sodium current in transfected cells. The data indicate that Map1b facilitates trafficking of Na(v)1.6 to the neuronal cell surface.

Coexpression with auxiliary β subunits modulates the action of tefluthrin on rat Na v1.6 and Na v1.3 sodium channels

We expressed the rat Na v1.3 and Na v1.6 sodium channel α subunit isoforms in Xenopus oocytes either alone or with the rat β1 and β2 auxiliary subunits in various combinations and assessed the sensitivity of the expressed channels to resting and use-dependent modification by the pyrethroid insecticide tefluthrin using the two-electrode voltage clamp technique. Coexpression with the β1 and β2 subunits, either individually or in combination, did not affecting the resting sensitivity of Na v1.6 channels to tefluthrin. Modification by tefluthrin of Na v1.6 channels in the absence of β subunits was not altered by the application of trains of high-frequency depolarizing prepulses. By contrast, coexpression of the Na v1.6 channel with the β1 subunit enhanced the extent of channel modification 2-fold following repeated depolarization. Coexpression of Na v1.6 with the β2 subunit also slightly enhanced modification following repeated depolarization, but coexpression of Na v1.6 with both β subunits caused enhanced modification following repeated depolarization that was indistinguishable from that found with Na v1.6 + β1 channels. In contrast to Na v1.6, the resting modification by tefluthrin of Na v1.3 channels expressed in the absence of β subunits was reduced by repeated depolarization. However, tefluthrin modification of the Na v1.3 α subunit expressed with both β subunits was enhanced 1.7-fold by repeated depolarization, thereby confirming that β subunit modulation of use-dependent effects was not confined to the Na v1.6 isoform. These results show that the actions of pyrethroids on mammalian sodium channels in the Xenopus oocyte expression system are determined in part by the interactions of the sodium channel α subunit with the auxiliary β subunits that are part of the heteromultimeric sodium channel complexes found in neurons and other excitable cells.

A Novel Nonsense Variant in Nav1.5 Cofactor MOG1 Eliminates Its Sodium Current Increasing Effect and May Increase the Risk of Arrhythmias

Background The protein MOG1 is a cofactor of the cardiac sodium channel, Nav1.5. Overexpression of MOG1 in Nav1.5-expressing cells increases sodium current markedly. Mutations in the genes encoding Nav1.5 and its accessory proteins have been associated with cardiac arrhythmias of significant clinical impact. We sought to investigate whether MOG1 is implicated in cardiac arrhythmias. Methods We performed a genetic screening of the MOG1-encoding gene (gene symbol RANGRF, alias MOG1) in 220 Danish patients with cardiac arrhythmia. Of the 220, 197 were young patients with lone atrial fibrillation and 23 were patients with Brugada syndrome. The effect of one variant was investigated functionally by patch-clamping CHO-K1 cells coexpressing Nav1.5 with MOG1. Results We uncovered a novel heterozygous nonsense variant, c.181G>T (p.E61X), that, however, was also present in control subjects, albeit at a lower frequency (1.8% vs 0.4%, P = 0.078). Electrophysiological investigation showed that the p.E61X variant completely eliminates the sodium current–increasing effect of MOG1 and thereby causes loss of function in the sodium current. When mimicking heterozygosity by coexpression of Nav1.5 with wild-type MOG1 and p.E61X-MOG1, no current decrease was seen. Conclusions Our screening of Nav1.5 cofactor MOG1 uncovered a novel nonsense variant that appeared to be present at a higher frequency among patients than control subjects. This variant causes MOG1 loss of function and therefore might be disease causing or modifying under certain conditions.

Loss-of-function mutation of the SCN3B-encoded sodium channel 3 subunit associated with a case of idiopathic ventricular fibrillation

Loss-of-function mutations in the SCN5A-encoded sodium channel SCN5A or Nav1.5 have been identified in idiopathic ventricular fibrillation (IVF) in the absence of Brugada syndrome phenotype. Nav1.5 is regulated by four sodium channel auxiliary beta subunits. Here, we report a case with IVF and a novel mutation in the SCN3B-encoded sodium channel beta subunit Navbeta3 that causes a loss of function of Nav1.5 channels in vitro. Comprehensive open reading frame mutational analysis of KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, GPD1L, four sodium channel beta subunit genes (SCN1-4B), and targeted scan of RYR2 was performed. A novel missense mutation, Navbeta3-V54G, was identified in a 20-year-old male following witnessed collapse and defibrillation from VF. The ECG exhibited epsilon waves, and imaging studies demonstrated a structurally normal heart. The mutated residue was highly conserved across species, localized to the Navbeta3 extracellular domain, and absent in 800 reference alleles. We found that HEK-293 cells had endogenous Navbeta3, but COS cells did not. Co-expression of Nav1.5 with Navbeta3-V54G (with or without co-expression of the Navbeta1 subunit) in both HEK-293 cells and COS cells revealed a significant decrease in peak sodium current and a positive shift of inactivation compared with WT. Co-immunoprecipitation experiments showed association of Navbeta3 with Nav1.5, and immunocytochemistry demonstrated a dramatic decrease in trafficking to the plasma membrane when co-expressed with mutant Navbeta3-V54G. This study provides molecular and cellular evidence implicating mutations in Navbeta3 as a cause of IVF.

Molecular changes in neurons in multiple sclerosis: altered axonal expression of Nav1.2 and Nav1.6 sodium channels and Na+/Ca2+ exchanger.

Although voltage-gated sodium channels are known to be deployed along experimentally demyelinated axons, the molecular identities of the sodium channels expressed along axons in human demyelinating diseases such as multiple sclerosis (MS) have not been determined. Here we demonstrate changes in the expression of sodium channels in demyelinated axons in MS, with Nav1.6 confined to nodes of Ranvier in controls but with diffuse distribution of Nav1.2 and Nav1.6 along extensive regions of demyelinated axons within acute MS plaques. Using triple-labeled fluorescent immunocytochemistry, we also show that Nav1.6, which is known to produce a persistent sodium current, and the Na+/Ca2+ exchanger, which can be driven by persistent sodium current to import damaging levels of calcium into axons, are colocalized with beta-amyloid precursor protein, a marker of axonal injury, in acute MS lesions. Our results demonstrate the molecular identities of the sodium channels expressed along demyelinated and degenerating axons in MS and suggest that coexpression of Nav1.6 and Na+/Ca2+ exchanger is associated with axonal degeneration in MS.

Expression of auxiliary β subunits of sodium channels in primary afferent neurons and the effect of nerve injury

Multiple voltage-gated sodium channels are the primary mediators of cell excitability. They are multimers that consist of the pore-forming α subunit and auxiliary β subunits. Although ion permeability and voltage sensing are primarily determined by the α subunit, β subunits are important modulators of sodium channel function. The purpose of this study was to assess the effect of axotomy on the expression of β subunits (β 1, β 2 and β 3) and coexpression of Na v1.3 and β 3 subunits in the dorsal root ganglion (DRG). We used sciatic nerve transection models or spared nerve injury (SNI) models in the rat. In reverse transcriptase–polymerase chain reaction analysis, there were no significant differences between contralateral and ipsilateral DRGs of β 1 and β 2 mRNA 3 days after axotomy. β 3 mRNA expression in ipsilateral DRGs increased significantly compared with contralateral DRGs 3 days after axotomy. In in situ hybridization histochemistry, β 1 mRNA was predominantly expressed in medium- to large-size neurons, whereas β 2 mRNA was expressed in small- to large-size neurons. There were no significant differences in β 1 and β 2 mRNA between contralateral and ipsilateral DRGs 3 days after axotomy. In contrast, β 3 mRNA was mainly expressed in small neurons and occasionally in medium- to large-size neurons, and β 3 mRNA expression in small c-type neurons in ipsilateral DRGs was increased significantly compared with contralateral DRGs. We examined β 3 mRNA expression with one of α subunits, Na v1.3-ir, in DRG neurons after axotomy using the double labeling method. We found a high percentage of coexpression in injured DRG neurons: 83.6±2.8% of neurons expressing β 3 mRNA were labeled for Na v1.3-ir; 70.1±3.1% of Na v1.3-ir neurons expressed β 3 mRNA. We also examined the expression of β 3 mRNA in DRG neurons in the SNI model, a neuropathic pain model. We used activating transcription factor 3 to identify axotomized neurons, and found that β 3 mRNA up-regulation occurred mainly in axotomized neurons in the neuropathic pain model. These data strongly suggest that β 3 expression in injured DRG neurons following axotomy might be an important pathomechanism of post-nerve injury pain in primary sensory neurons.

Neurotrophin Depolarization

Neurotrophins are best known for their role in neuron survival and differentiation, effects that are mediated by kinase cascades activated by receptor tyrosine kinases. Brain-derived neurotrophic factor (BDNF) is also able to stimulate rapid depolarization through the activation of a Na+ current (INa). Blum et al. identified a saxitoxin (STX)-sensitive, tetrodotoxin (TTX)-insensitive INa that was stimulated by BDNF in both cultured hippocampal neurons and differentiated SH-SY5Y cells. They used antisense RNA to inhibit the expression of the TrkB receptor and the voltage-gated sodium channel Nav1.9 (predicted to be STX-sensitive, TTX-insensitive on the basis of its amino acid sequence) in SH-SY5Y neuroblastoma cells. Inhibition of TrkB or Nav1.9 resulted in the complete loss of BDNF-stimulated INa. Antisense treatment for TrkC or a TTX-sensitive Na+ channel had no effect, even though these two genes were expressed in the SH-SY5Y cells. BDNF-stimulated INa was reconstituted in human embryonic kidney (HEK) 293 cells by coexpression of Nav1.9 and TrkB, These results suggest that a voltage-gated ion channel may be also ligand activated. Presumably, TrkB can associate directly or indirectly with the sodium channel to transmit the growth factor signal to the channel. Barde discusses the implications of these results for pain perception and the possibility that similar interactions may occur between other neurotrophins, their receptors, and voltage-gated ion channels.R. Blum, K. W. Kafitz, A. Konnerth, Neurotrophin-evoked depolarization requires the sodium channel Nav1.9. Nature 419, 687-693 (2002). [Online Journal]Y.A. Barde, Neurotrophin channels excitement. Nature 419, 683-684 (2002). [Online Journal]

Neurotrophin Depolarization

Neurotrophins are best known for their role in neuron survival and differentiation, effects that are mediated by kinase cascades activated by receptor tyrosine kinases. Brain-derived neurotrophic factor (BDNF) is also able to stimulate rapid depolarization through the activation of a Na + current ( I Na ). Blum et al. identified a saxitoxin (STX)-sensitive, tetrodotoxin (TTX)-insensitive I Na that was stimulated by BDNF in both cultured hippocampal neurons and differentiated SH-SY5Y cells. They used antisense RNA to inhibit the expression of the TrkB receptor and the voltage-gated sodium channel Na v 1.9 (predicted to be STX-sensitive, TTX-insensitive on the basis of its amino acid sequence) in SH-SY5Y neuroblastoma cells. Inhibition of TrkB or Na v 1.9 resulted in the complete loss of BDNF-stimulated I Na . Antisense treatment for TrkC or a TTX-sensitive Na + channel had no effect, even though these two genes were expressed in the SH-SY5Y cells. BDNF-stimulated I Na was reconstituted in human embryonic kidney (HEK) 293 cells by coexpression of Na v 1.9 and TrkB, These results suggest that a voltage-gated ion channel may be also ligand activated. Presumably, TrkB can associate directly or indirectly with the sodium channel to transmit the growth factor signal to the channel. Barde discusses the implications of these results for pain perception and the possibility that similar interactions may occur between other neurotrophins, their receptors, and voltage-gated ion channels. R. Blum, K. W. Kafitz, A. Konnerth, Neurotrophin-evoked depolarization requires the sodium channel Nav1.9. Nature 419 , 687-693 (2002). [Online Journal] Y.A. Barde, Neurotrophin channels excitement. Nature 419 , 683-684 (2002). [Online Journal]

Characterization of sodium channel alpha- and beta-subunits in rat and mouse cardiac myocytes.

Sodium channels isolated from mammalian brain are composed of alpha-, beta(1)-, and beta(2)-subunits. The composition of sodium channels in cardiac muscle, however, has not been defined, and disagreement exists over which beta-subunits are expressed in the myocytes. Some investigators have demonstrated beta(1) expression in heart. Others have not detected any auxiliary subunits. On the basis of Northern blot analysis of total RNA, beta(2) expression has been thought to be exclusive to neurons and absent from cardiac muscle. The goal of this study was to define the subunit composition of cardiac sodium channels in myocytes. We show that cardiac sodium channels are composed of alpha-, beta(1)-, and beta(2)-subunits. Nav1.5 and Nav1.1 are expressed in myocytes and are associated with beta(1)- and beta(2)-subunits. Immunocytochemical localization of Nav1.1, beta(1), and beta(2) in adult heart sections showed that these subunits are expressed at the Z lines, as shown previously for Nav1.5. Coexpression of Nav1.5 with beta(2) in transfected cells resulted in no detectable changes in sodium current. Cardiac sodium channels are composed of alpha- (Nav1.1 or Nav1.5), beta(1)-, and beta(2)-subunits. Although beta(1)-subunits modulate cardiac sodium channel current, beta(2)-subunit function in heart may be limited to cell adhesion.