Native Nav1 Research Articles

Use-Dependent Relief of Inhibition of Nav1.8 Channels by A-887826.

Sodium channel inhibitors used as local anesthetics, antiarrhythmics, or antiepileptics typically have the property of use-dependent inhibition, whereby inhibition is enhanced by repetitive channel activation. For targeting pain, Nav1.8 channels are an attractive target because they are prominent in primary pain-sensing neurons, with little or no expression in most other kinds of neurons, and a number of Nav1.8-targeted compounds have been developed. We examined the characteristics of Nav1.8 inhibition by one of the most potent Nav1.8 inhibitors so far described, A-887826, and found that when studied with physiologic resting potentials and physiologic temperatures, inhibition had strong "reverse use dependence", whereby inhibition was relieved by repetitive short depolarizations. This effect was much stronger with A-887826 than with A-803467, another Nav1.8 inhibitor. The use-dependent relief from inhibition was seen in both human Nav1.8 channels studied in a cell line and in native Nav1.8 channels in mouse dorsal root ganglion (DRG) neurons. In native Nav1.8 channels, substantial relief of inhibition occurred during repetitive stimulation by action potential waveforms at 5 Hz, suggesting that the phenomenon is likely important under physiologic conditions. SIGNIFICANCE STATEMENT: Nav1.8 sodium channels are expressed in primary pain-sensing neurons and are a prime current target for new drugs for pain. This work shows that one of the most potent Nav1.8 inhibitors, A-887826, has the unusual property that inhibition is relieved by repeated short depolarizations. This "reverse use dependence" may reduce inhibition during physiological firing and should be selected against in drug development.

Differential roles of NaV1.2 and NaV1.6 in regulating neuronal excitability at febrile temperature and distinct contributions to febrile seizures

Dysregulation of voltage-gated sodium channels (VGSCs) is associated with multiple clinical disorders, including febrile seizures (FS). The contribution of different sodium channel subtypes to environmentally triggered seizures is not well understood. Here we demonstrate that somatic and axonal sodium channels primarily mediated through NaV1.2 and NaV1.6 subtypes, respectively, behave differentially at FT, and might play distinct roles in FS generation. In contrast to sodium channels on the main axonal trunk, somatic ones are more resistant to inactivation and display significantly augmented currents, faster gating rates and kinetics of recovery from inactivation at FT, features that promote neuronal excitabilities. Pharmacological inhibition of NaV1.2 by Phrixotoxin-3 (PTx3) suppressed FT-induced neuronal hyperexcitability in brain slice, while up-regulation of NaV1.2 as in NaV1.6 knockout mice showed an opposite effect. Consistently, NaV1.6 knockout mice were more susceptible to FS, exhibiting much lower temperature threshold and shorter onset latency than wildtype mice. Neuron modeling further suggests that NaV1.2 is the major subtype mediating FT-induced neuronal hyperexcitability, and predicts potential outcomes of alterations in sodium channel subtype composition. Together, these data reveal a role of native NaV1.2 on neuronal excitability at FT and its important contribution to FS pathogenesis.

Dual Action of Mexiletine and Its Pyrroline Derivatives as Skeletal Muscle Sodium Channel Blockers and Anti-oxidant Compounds: Toward Novel Therapeutic Potential.

Mexiletine (Mex) has been recently appointed as an orphan-drug in myotonic-syndromes, being a potent use-dependent blocker of skeletal-muscle sodium channels (NaV1.4). Available evidences about a potential anti-oxidant effect of Mex and its tetramethyl-pyrroline-derivatives in vivo, suggest the possibility to further enlarge the therapeutic potential of Mex-like compounds in myopathies in which alteration of excitation-contraction coupling is paralleled by oxidative stress. In line with this and based on our previous structure-activity-relationship studies, we synthesized new compounds with a tetramethyl-pyrroline-ring on the amino-group of both Mex (VM11) and of its potent use-dependent isopropyl-derivative (CI16). The compounds were tested for their ability to block native NaV1.4 and to exert cyto-protective effects against oxidative-stress injury in myoblasts. Voltage-clamp-recordings on adult myofibers were performed to assess the tonic and use-dependent block of peak sodium-currents (INa) by VM11 and CI16, as well as Mex, VM11 and CI16 were 3 and 6-fold more potent than Mex in producing a tonic-block of peak sodium-currents (INa), respectively. Interestingly, CI16 showed a 40-fold increase of potency with respect to Mex during high-frequency stimulation (10-Hz), resulting the strongest use-dependent Mex-like compound so far. The derivatives also behaved as inactivated channel blockers, however the voltage dependent block was modest. The experimental data fitted with the molecular-modeling simulation based on previously proposed interaction of main pharmacophores with NaV1.4 binding-site. CI16 and VM11 were then compared to Mex and its isopropyl derivative (Me5) for the ability to protect C2C12-cells from H2O2-cytotoxicity in the concentration range effective on Nav1.4. Mex and Me5 showed a moderate cyto-protective effect in the presence of H2O2, Importantly, CI16 and VM11 showed a remarkable cyto-protection at concentrations effective for use-dependent block of NaV1.4. This effect was comparable to that of selected anti-oxidant drugs proved to exert protective effect in preclinical models of progressive myopathies such as muscular dystrophies. Then, the tetramethyl-pyrroline compounds have increased therapeutic profile as sodium channel blockers and an interesting cyto-protective activity. The overall profile enlarges therapeutic potential from channelopathies to myopathies in which alteration of excitation-contraction coupling is paralleled by oxidative-stress, i.e., muscular dystrophies.

C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

Loperamide inhibits sodium channels to alleviate inflammatory hyperalgesia

Previous studies demonstrated that Loperamide, originally known as an anti-diarrheal drug, is a promising analgesic agent primarily targeting mu-opioid receptors. However some evidences suggested that non-opioid mechanisms may be contributing to its analgesic effect. In the present study, Loperamide was identified as a Nav1.7 blocker in a pilot screen. In HEK293 cells expressing Nav1.7 sodium channels, Loperamide blocked the resting state of Nav1.7 channels (IC50 = 1.86 ± 0.11 μM) dose-dependently and reversibly. Loperamide produced a 10.4 mV of hyperpolarizing shift for the steady-state inactivation of Nav1.7 channels without apparent effect on the voltage-dependent activation. The drug displayed a mild use- and state-dependent inhibition on Nav1.7 channels, which was removed by the local anesthetic-insensitive construct Nav1.7-F1737A. Inhibition of Nav1.7 at resting state was not altered significantly by the F1737A mutation. Compared to its effects on Nav1.7, Loperamide exhibited higher potency on recombinant Nav1.8 channels in ND7/23 cells (IC50 = 0.60 ± 0.10 μM) and weaker potency on Nav1.9 channels (3.48 ± 0.33 μM). Notably more pronounced inhibition was observed in the native Nav1.8 channels (0.11 ± 0.08 μM) in DRG neurons. Once mu-opioid receptor was antagonized by Naloxone in DRG neurons, potency of Loperamide on Nav1.8 was identical to that of recombinant Nav1.8 channels. The inhibition on Nav channels may be the main mechanism of Loperamide for pain relief beyond mu-opioid receptor. In the meanwhile, the opioid receptor pathway may also influence the blocking effect of Loperamide on sodium channels, implying a cross-talk between sodium channels and opioid receptors in pain processing.

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.

Nav1.5 channels can reach the plasma membrane through distinct N-glycosylation states

BackgroundLike many voltage-gated sodium channels, the cardiac isoform Nav1.5 is well known as a glycoprotein which necessarily undergoes N-glycosylation processing during its transit to the plasma membrane. In some cardiac disorders, especially the Brugada syndrome (BrS), mutations in Nav1.5 encoding gene lead to intracellular retention and consequently trafficking defect of these proteins. We used two BrS mutants as tools to clarify both Nav1.5 glycosylation states and associated secretory behaviors. MethodsPatch-clamp recordings and surface biotinylation assays of HEK293T cells expressing wild-type (WT) and/or mutant Nav1.5 proteins were performed to assess the impact of mutant co-expression on the membrane activity and localization of WT channels. Enzymatic deglycosylation assays and brefeldin A (BFA) treatments were also employed to further characterize recombinant and native Nav1.5 maturation. ResultsThe present data demonstrate that Nav1.5 channels mainly exist as two differentially glycosylated forms. We reveal that dominant negative effects induced by BrS mutants upon WT channel current result from the abnormal surface expression of the fully-glycosylated forms exclusively. Furthermore, we show that core-glycosylated channels can be found at the surface membrane of BFA-treated or untreated cells, but obviously without generating any sodium current. ConclusionsOur findings provide evidence that native and recombinant Nav1.5 subunits are expressed as two distinct matured forms. Fully-glycosylated state of Nav1.5 seems to determine its functionality whereas core-glycosylated forms might be transported to the plasma membrane through an unconventional Golgi-independent secretory route. General significanceThis work highlights that N-linked glycosylation processing would be critical for Nav1.5 membrane trafficking and function.

Selective blocking effects of 4,9-anhydrotetrodotoxin, purified from a crude mixture of tetrodotoxin analogues, on NaV1.6 channels and its chemical aspects.

Tetrodotoxin (TTX) is a potent neurotoxin found in a number of marine creatures including the pufferfish, where it is synthesized by bacteria and accumulated through the food chain. It is a potent and selective blocker of some types of voltage-gated Na+ channel (NaV channel). 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX) was purified from a crude mixture of TTX analogues (such as TTX, 4-epiTTX, 6-epiTTX, 11-oxoTTX and 11-deoxyTTX) by the use of liquid chromatography-fluorescence detection (LC-FLD) techniques. Recently, it has been reported that 4,9-anhydroTTX selectively blocks the activity of NaV1.6 channels with a blocking efficacy 40–160 times higher than that for other TTX-sensitive NaV1.x channel isoforms. However, little attention has been paid to the molecular properties of the α-subunit in NaV1.6 channels and the characteristics of binding of 4,9-anhydroTTX. From a functional point of view, it is important to determine the relative expression of NaV1.6 channels in a wide variety of tissues. The aim of this review is to discuss briefly current knowledge about the pharmacology of 4,9-anhydroTTX, and provide an analysis of the molecular structure of native NaV1.6 channels. In addition, chemical aspects of 4,9-anhydroTTX are briefly covered.

Mass Spectrometry-Based Identification of Native Cardiac Nav1.5 Channel α Subunit Phosphorylation Sites

Cardiac voltage-gated Na+ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified in situ phosphorylation sites. Taken together, the results presented provide the first in situ map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit.

Biophysical and Pharmacological Characterisation of Native Human NaV1.8 Channels from Isolated Dorsal Root Ganglia (DRG)

NaV1.8 is a tetrodotoxin resistant (TTX-r) sodium channel expressed in sensory neurones that has a depolarised activation threshold, slow inactivation kinetics and a recovers rapidly from inactivation compared to tetrodotoxin sensitive (TTX-s) channels (Cummins & Waxman 1997; Akopian et al., 1996). These biophysical properties mean that NaV1.8 contributes to both electrogenesis and the maintenance of repetitive firing of action potentials (Blair & Bean 2002; Renganathan et al., 2001; Waxman et al., 2001). The expression and biophysical properties of NaV1.8 can be modulated by ongoing nociceptive input and findings in the literature strongly support a key role for NaV1.8 in pain signalling (England et al., 1996; Roza et al., 2003; Kerr et al., 2001; Coward et al., 2000; Akopian et al., 1999). The selective NaV1.8 antagonist A803467 has provided further evidence for a role of NaV1.8 in nociceptive sensory input (Jarvis et al., 2007) however characterisation and pharmacology of native NaV1.8 currents has been shown using cells isolated from non human species. We have characterised NaV1.8 currents recorded from TTX-r channels in human DRG and shown that they can be inhibited by a selective modulator of TTX-r current, A803467. Isolated currents had a V1/2 inactivation of −35 mV and a V1/2 of activation of −10 mV. Action potentials evoked by increasing current injections were inhibited by TTX and by the selective NaV1.8 modulator A803467 which abolished repetitive firing. Furthermore, we have compared the biophysical properties of the native channel with those of recombinantly expressed human NaV1.8 channel. This work not only provides a means by which we can assess the biophysics and pharmacological modulation of native human NaV1.8 currents, but will also help us to understand the role of the channel in human pain signalling.

Trauma-Induced Changes in Recombinant and Native Nav1.2 Channel Kinetics

Nav1.2, the Nav isoform of neuronal cell bodies and proximal axon initial segments (AIS), is a “high threshold” channel compared to Nav1.6, the distal AIS and node of Ranvier isoform. For recombinant Nav1.6, we showed that bleb formation (mechanically-induced by aspiration of cell-attached oocyte patches) irreversibly left-shifts availability and activation (Wang et al 2009 Am J Physiol 297:C823); in situ, the resultant window current shift would constitute a pathological Na-leak. Here we show that, like human Nav1.6, recombinant rat Nav1.2 expressed in oocytes (without/with rat β1 subunit) and monitored in cell-attached patches, current availability and activation left-shifted progressively and irreversibly with progressively stronger pipette aspiration. Sometimes, just the act of patch formation was mechanically traumatic enough to left-shift channel operation (as we found previously with Nav1.6). In computations, left-shifting even a fraction of an axon's Nav channel population yields multiple deleterious effects for excitability and ion homeostasis (Boucher, Joós, Morris; BPSoc2011 abstracts) and this makes it important to determine if native Nav channels misbehave like recombinants when subjected to membrane trauma. To test native Nav1.2, we did cell-attached patching of entorhinal cortex pyramidal neurons in rat brain slices. Well-clamped whole-cell recordings from these neurons (Milescu et al 2010 J Neurosci 30:7740) show the threshold for Nav1.2 activation to be −45 mV. Seals were difficult to maintain (spontaneous break-in was frequent within a minute of sealing), but assuming Vrest=0 or =-65 mV (i.e. hiK bath; normal bath, respectively), we observed Nav current starting at Vm= −60 mV or −55 mV, i.e. close to threshold activation values for maximally traumatized recombinant Nav1.2 current in oocyte patches. Establishing the cell-attached configuration in these neurons, we conclude, unavoidably subjected patches to mechanical trauma that put the native Nav1.2 channels in a permanently left-shifted condition.

A sodium channel mutation linked to epilepsy increases ramp and persistent current of Nav1.3 and induces hyperexcitability in hippocampal neurons

Voltage-gated sodium channelopathies underlie many excitability disorders. Genes SCN1A, SCN2A and SCN9A, which encode pore-forming α-subunits NaV1.1, NaV1.2 and NaV1.7, are clustered on human chromosome 2, and mutations in these genes have been shown to underlie epilepsy, migraine, and somatic pain disorders. SCN3A, the gene which encodes NaV1.3, is part of this cluster, but until recently was not associated with any mutation. A charge-neutralizing mutation, K345Q, in the NaV1.3 DI/S5-6 linker has recently been identified in a patient with cryptogenic partial epilepsy. Pathogenicity of the NaV1.3/K354Q mutation has been inferred from the conservation of this residue in all sodium channels and its absence from control alleles, but functional analysis has been limited to the corresponding substitution in the cardiac muscle sodium channel NaV1.5. Since identical mutations may produce different effects within different sodium channel isoforms, we assessed the K354Q mutation within its native NaV1.3 channel and studied the effect of the mutant NaV1.3/K354Q channels on hippocampal neuron excitability. We show here that the K354Q mutation enhances the persistent and ramp currents of NaV1.3, reduces current threshold and produces spontaneous firing and paroxysmal depolarizing shift-like complexes in hippocampal neurons. Our data provide a pathophysiological basis for the pathogenicity of the first epilepsy-linked mutation within NaV1.3 channels and hippocampal neurons.

Sodium Channel β1 Subunit-mediated Modulation of Nav1.2 Currents and Cell Surface Density Is Dependent on Interactions with Contactin and Ankyrin

Voltage-gated sodium channels are composed of a pore-forming alpha subunit and at least one auxiliary beta subunit. Both beta1 and beta2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, beta1, but not beta2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of beta1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for beta1 association with contactin, ankyrin, and Nav1.2. beta1/beta2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the beta1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. We show that native Nav1.2 associates with ankyrinG in cells in the absence of beta subunits and that this interaction is enhanced in the presence of beta1 but not beta1Y181E, a mutant that does not interact with ankyrinG. beta1Y181E does not modulate Nav1.2 channel function despite efficient association with Nav1.2 and contactin. beta1Y181E increases Nav1.2 cell surface expression, but not as efficiently as wild type beta1. beta1/beta2 chimeras exchanging various regions of the beta1 Ig loop were all ineffective in increasing Nav1.2 cell surface density. Our results demonstrate that full-length beta1 is required for channel modulation and enhancement of sodium channel cell surface expression.

Regulation of Neuronal Traits by a Novel Transcriptional Complex

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.