Recombinant Nav1 Research Articles

NaV1.5 autoantibodies in Brugada syndrome: pathogenetic implications.

Patients suffering from Brugada syndrome (BrS) are predisposed to life-threatening cardiac arrhythmias. Diagnosis is challenging due to the elusive electrocardiographic (ECG) signature that often requires unconventional ECG lead placement and drug challenges to be detected. Although NaV1.5 sodium channel dysfunction is a recognized pathophysiological mechanism in BrS, only 25% of patients have detectable SCN5A variants. Given the emerging role of autoimmunity in cardiac ion channel function, this study explores the presence and potential impact of anti-NaV1.5 autoantibodies in BrS patients. Using engineered HEK293A cells expressing recombinant NaV1.5 protein, plasma from 50 BrS patients and 50 controls was screened for anti-NaV1.5 autoantibodies via western blot, with specificity confirmed by immunoprecipitation and immunofluorescence. The impact of these autoantibodies on sodium current density and their pathophysiological effects were assessed in cellular models and through plasma injection in wild-type mice. Anti-NaV1.5 autoantibodies were detected in 90% of BrS patients vs. 6% of controls, yielding a diagnostic area under the curve of .92, with 94% specificity and 90% sensitivity. These findings were consistent across varying patient demographics and independent of SCN5A mutation status. Electrophysiological studies demonstrated a significant reduction specifically in sodium current density. Notably, mice injected with BrS plasma showed Brugada-like ECG abnormalities, supporting the pathogenic role of these autoantibodies. The study demonstrates the presence of anti-NaV1.5 autoantibodies in the majority of BrS patients, suggesting an immunopathogenic component of the syndrome beyond genetic predispositions. These autoantibodies, which could serve as additional diagnostic markers, also prompt reconsideration of the underlying mechanisms of BrS, as evidenced by their role in inducing the ECG signature of the syndrome in wild-type mice. These findings encourage a more comprehensive diagnostic approach and point to new avenues for therapeutic research.

Isolation and transfection of myenteric neurons from mice for patch-clamp applications.

The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses that extend along the entire length of the gastrointestinal tract. Largely independent of the central nervous system, the ENS coordinates motility and peristalsis of the digestive tract, regulates secretion and absorption, and is involved in immunological processes. Electrophysiological methods such as the patch-clamp technique are particularly suitable to study the function of neurons as well as the biophysical parameters of the underlying ion channels under both physiological and pathophysiological conditions. However, application of the patch-clamp method to ENS neurons remained difficult because they are embedded in substantial tissue layers that limit access to and targeted manipulation of these cells. Here, we present a robust step-by-step protocol that involves isolation of ENS neurons from adult mice, culturing of the cells, their transfection with plasmid DNA, and subsequent electrophysiological characterization of individual neurons in current-clamp and voltage-clamp recordings. With this protocol, ENS neurons can be prepared, transfected, and electrophysiologically characterized within 72 h. Using isolated ENS neurons, we demonstrate the feasibility of the approach by functional overexpression of recombinant voltage-gated NaV1.9 mutant channels associated with hereditary sensory and autonomic neuropathy type 7 (HSAN-7), a disorder characterized by congenital analgesia and severe constipation that can require parenteral nutrition. Although our focus is on the electrophysiological evaluation of isolated ENS neurons, the presented methodology is also useful to analyze molecules other than sodium channels or to apply alternative downstream assays including calcium imaging, proteomic and nucleic acid approaches, or immunochemistry.

Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling.

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure–activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a “CXCE” motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

Potent Inactivation-Dependent Inhibition of Adult and Neonatal NaV1.5 Channels by Lidocaine and Levobupivacaine

Cardiotoxic effects of local anesthetics (LAs) involve inhibition of NaV1.5 voltage-gated Na channels. Metastatic breast and colon cancer cells also express NaV1.5, predominantly the neonatal splice variant (nNaV1.5) and their inhibition by LAs reduces invasion and migration. It may be advantageous to target cancer cells while sparing cardiac function through selective blockade of nNaV1.5 and/or by preferentially affecting inactivated NaV1.5, which predominate in cancer cells. We tested the hypotheses that lidocaine and levobupivacaine differentially affect (1) adult (aNaV1.5) and nNaV1.5 and (2) the resting and inactivated states of NaV1.5. The whole-cell voltage-clamp technique was used to evaluate the actions of lidocaine and levobupivacaine on recombinant NaV1.5 channels expressed in HEK-293 cells. Cells were transiently transfected with cDNAs encoding either aNaV1.5 or nNaV1.5. Voltage protocols were applied to determine depolarizing potentials that either activated or inactivated 50% of maximum conductance (V½ activation and V½ inactivation, respectively). Lidocaine and levobupivacaine potently inhibited aNaV1.5 (IC50 mean [SD]: 20 [22] and 1 [0.6] μM, respectively) and nNaV1.5 (IC50 mean [SD]: 17 [10] and 3 [1.6] μM, respectively) at a holding potential of -80 mV. IC50s differed significantly between lidocaine and levobupivacaine with no influence of splice variant. Levobupivacaine induced a statistically significant depolarizing shift in the V½ activation for aNaV1.5 (mean [SD] from -32 [4.6] mV to -26 [8.1] mV) but had no effect on the voltage dependence of activation of nNaV1.5. Lidocaine had no effect on V½ activation of either variant but caused a significantly greater depression of maximum current mediated by nNaV1.5 compared to aNaV1.5. Similar statistically significant shifts in the V½ inactivation (approximately -10 mV) occurred for both LAs and NaV1.5 variants. Levobupivacaine (1 μM) caused a significantly greater slowing of recovery from inactivation of both variants than did lidocaine (10 μM). Both LAs caused approximately 50% tonic inhibition of aNaV1.5 or nNaV1.5 when holding at -80 mV. Neither LA caused tonic block at a holding potential of either -90 or -120 mV, voltages at which there was little steady-state inactivation. Higher concentrations of either lidocaine (300 μM) or levobupivacaine (100 μM) caused significantly more tonic block at -120 mV. These data demonstrate that low concentrations of the LAs exhibit inactivation-dependent block of NaV1.5, which may provide a rationale for their use to safely inhibit migration and invasion by metastatic cancer cells without cardiotoxicity.

A Chimeric NaV1.8 Channel Expression System Based on HEK293T Cell Line.

Among the nine voltage-gated sodium channel (NaV) subtypes, NaV1.8 is an attractive therapeutic target for pain. The heterologous expression of recombinant NaV1.8 currents is of particular importance for its electrophysiological and pharmacological studies. However, NaV1.8 expresses no or low-level functional currents when transiently transfected into non-neuronal cell lines. The present study aims to explore the molecular determinants limiting its functional expression and accordingly establish a functional NaV1.8 expression system. We conducted screening analysis of the NaV1.8 intracellular loops by constructing NaV chimeric channels and confirmed that the NaV1.8 C-terminus was the only limiting factor. Replacing this sequence with that of NaV1.4, NaV1.5, or NaV1.7 constructed functional channels (NaV1.8/1.4L5, NaV1.8/1.5L5, and NaV1.8/1.7L5, respectively), which expressed high-level NaV1.8-like currents in HEK293T cells. The chimeric channel NaV1.8/1.7L5 displayed much faster inactivation of its macroscopic currents than NaV1.8/1.4L5 and NaV1.8/1.5L5, and it was the most similar to wild-type NaV1.8 expressed in ND7/23 cells. Its currents were very stable during repetitive depolarizations, while its repriming kinetic was different from wild-type NaV1.8. Most importantly, NaV1.8/1.7L5 pharmacologically resembled wild-type NaV1.8 as revealed by testing their susceptibility to two NaV1.8 selective antagonists, APETx-2 and MrVIB. NaV chimeras study showed that at least the domain 2 and domain 4 of NaV1.8 were involved in binding with APETx-2. Our study provided new insights into the function of NaV1.8 intracellular loops, as well as a reliable and convenient expression system which could be useful in NaV1.8 studies.

Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels

Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes (Scn2b - Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with β1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with β1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i = 510nM), recombinant INa (K i = 112nM), and recombinant INa co-expressed with β1-subunits (K i = 92nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with β1-subunits. These results suggest that β1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with β1-subunits in a concentration-dependent manner.

Development of High throughput Electrophysiology Assays of Recombinant Nav1.9 Channels

Development of High throughput Electrophysiology Assays of Recombinant Nav1.9 Channels

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.

Oxidation differentially modulates the recombinant voltage-gated Na+ channel α-subunits Nav1.7 and Nav1.8

Voltage-gated Na+ channels regulate neuronal excitability by generating the upstroke of action potentials. The α-subunits Nav1.7 and Nav1.8 are required for normal function of sensory neurons and thus for peripheral pain processing, but also for an increased excitability leading to an increased pain sensitivity under several conditions associated with oxidative stress. While little is known about the direct effects of oxidants on Nav1.7 and Nav1.8, a recent study on mouse dorsal root ganglion neurons suggested that oxidant-induced alterations of nociceptor excitability are primarily driven by Nav1.8. Here we performed whole-cell patch clamp recordings to explore how oxidation modulates functional properties of recombinant Nav1.7 and Nav1.8 channels. The strong oxidant chloramine-T (ChT) at 100 and 500µM induced a shift of the voltage-dependency of activation towards more hyperpolarized potentials. While fast inactivation was stabilized by 100µM ChT, it was partially removed by 500µM ChT on both α-subunits (Nav1.7<Nav1.8) and enabled them to produce large non-inactivating persistent currents as well as prominent ramp currents. Slow inactivation of both peak and persistent currents for both Nav1.7 and Nav1.8 were stabilized by ChT. Our data demonstrate that oxidation promotes gating of Nav1.7 and Nav1.8 by reducing the threshold for activation and by abrogating fast inactivation. The resulting persistent currents are regulated by slow inactivation and appear to be more prominent for Nav1.8 as compared to Nav1.7.

Navβ4 regulates fast resurgent sodium currents and excitability in sensory neurons.

BackgroundIncreased electrical activity in peripheral sensory neurons including dorsal root ganglia (DRG) and trigeminal ganglia neurons is an important mechanism underlying pain. Voltage gated sodium channels (VGSC) contribute to the excitability of sensory neurons and are essential for the upstroke of action potentials. A unique type of VGSC current, resurgent current (INaR), generates an inward current at repolarizing voltages through an alternate mechanism of inactivation referred to as open-channel block. INaRs are proposed to enable high frequency firing and increased INaRs in sensory neurons are associated with pain pathologies. While Nav1.6 has been identified as the main carrier of fast INaR, our understanding of the mechanisms that contribute to INaR generation is limited. Specifically, the open-channel blocker in sensory neurons has not been identified. Previous studies suggest Navβ4 subunit mediates INaR in central nervous system neurons. The goal of this study was to determine whether Navβ4 regulates INaR in DRG sensory neurons.ResultsOur immunocytochemistry studies show that Navβ4 expression is highly correlated with Nav1.6 expression predominantly in medium-large diameter rat DRG neurons. Navβ4 knockdown decreased endogenous fast INaR in medium-large diameter neurons as measured with whole-cell voltage clamp. Using a reduced expression system in DRG neurons, we isolated recombinant human Nav1.6 sodium currents in rat DRG neurons and found that overexpression of Navβ4 enhanced Nav1.6 INaR generation. By contrast neither overexpression of Navβ2 nor overexpression of a Navβ4-mutant, predicted to be an inactive form of Navβ4, enhanced Nav1.6 INaR generation. DRG neurons transfected with wild-type Navβ4 exhibited increased excitability with increases in both spontaneous activity and evoked activity. Thus, Navβ4 overexpression enhanced INaR and excitability, whereas knockdown or expression of mutant Navβ4 decreased INaR generation.ConclusionINaRs are associated with inherited and acquired pain disorders. However, our ability to selectively target and study this current has been hindered due to limited understanding of how it is generated in sensory neurons. This study identified Navβ4 as an important regulator of INaR and excitability in sensory neurons. As such, Navβ4 is a potential target for the manipulation of pain sensations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12990-015-0063-9) contains supplementary material, which is available to authorized users.

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.

Potent inhibition by ropivacaine of metastatic colon cancer SW620 cell invasion and NaV1.5 channel function

Metastatic breast and colon cancer cells express neonatal and adult splice variants of NaV1.5 voltage-activated Na(+) channels (VASCs). Block of VASCs inhibits cell invasion. Local anaesthetics used during surgical tumour excision inhibit VASC activity on nociceptive neurones providing regional anaesthesia. Inhibition of VASCs on circulating metastatic cancer cells may also be beneficial during the perioperative period. However, ropivacaine, frequently used to provide analgesia during tumour resection, has not been tested on colon cancer cell VASC function or invasion. We used reverse transcription-polymerase chain reaction and sequencing to identify NaV1.5 variants in the SW620 metastatic colon cancer cell line. Recombinant adult and neonatal NaV1.5 variants were expressed in human embryonic kidney cells. Voltage-clamp recordings and invasion assays were used to examine the effects of ropivacaine on recombinant NaV1.5 channels and the metastatic potential of SW620 cells, respectively. SW620 cells expressed adult and neonatal NaV1.5 variants, which had similar steady-state inactivation profiles, but distinctive activation curves with the neonatal variant having a V1/2 of activation 7.8 mV more depolarized than the adult variant. Ropivacaine caused a concentration-dependent block of both NaV1.5 variants, with IC50 values of 2.5 and 3.9 µM, respectively. However, the reduction in available steady-state current was selective for neonatal NaV1.5 channels. Ropivacaine inhibited SW620 invasion, with a potency similar to that of inhibition of NaV1.5 channels (3.8 µM). Ropivacaine is a potent inhibitor of both NaV1.5 channel activity and metastatic colon cancer cell invasion, which may be beneficial during surgical colon cancer excision.

Voltage-gated sodium (NaV) channel blockade by plant cannabinoids does not confer anticonvulsant effects per se

Cannabidiol (CBD) is a non-psychoactive, well-tolerated, anticonvulsant plant cannabinoid, although its mechanism(s) of seizure suppression remains unknown. Here, we investigate the effect of CBD and the structurally similar cannabinoid, cannabigerol (CBG), on voltage-gated Na+ (NaV) channels, a common anti-epileptic drug target. CBG's anticonvulsant potential was also assessed in vivo. CBD effects on NaV channels were investigated using patch-clamp recordings from rat CA1 hippocampal neurons in brain slices, human SH-SY5Y (neuroblastoma) cells and mouse cortical neurons in culture. CBG effects were also assessed in SH-SY5Y cells and mouse cortical neurons. CBD and CBG effects on veratridine-stimulated human recombinant NaV1.1, 1.2 or 1.5 channels were assessed using a membrane potential-sensitive fluorescent dye high-throughput assay. The effect of CBG on pentyleneterazole-induced (PTZ) seizures was assessed in rat. CBD (10μM) blocked NaV currents in SH-SY5Y cells, mouse cortical neurons and recombinant cell lines, and affected spike parameters in rat CA1 neurons; CBD also significantly decreased membrane resistance. CBG blocked NaV to a similar degree to CBD in both SH-SY5Y and mouse recordings, but had no effect (50–200mg/kg) on PTZ-induced seizures in rat. CBD and CBG are NaV channel blockers at micromolar concentrations in human and murine neurons and recombinant cells. In contrast to previous reports investigating CBD, CBG had no effect upon PTZ-induced seizures in rat, indicating that NaV blockade per se does not correlate with anticonvulsant effects.

In Vivo and Ex Vivo Inhibition of Spinal Nerve Ligation-Induced Ectopic Activity by Sodium Channel Blockers Correlate to In Vitro Inhibition of NaV1.7 and Clinical Efficacy: A Pharmacokinetic-Pharmacodynamic Translational Approach

In vivo and ex vivo inhibition of ectopic activity of clinically used and newly developed sodium channel (NaV) blockers were quantified in the rat spinal nerve ligation (SNL) model using a pharmacokinetic-pharmacodynamic (PKPD) approach and correlated to in vitro NaV1.7 channel inhibition and clinical effective concentrations. In vivo, drug exposure and inhibition of ectopic activity were assessed in anaesthetized SNL rats at two dose levels. Ex vivo, compounds were applied at increasing concentrations to dorsal root ganglias isolated from SNL rats. The inhibitory potency (IC 50 ) was estimated using PKPD analysis. In vitro IC 50 was estimated using an electrophysiology-based assay using recombinant rat and human NaV1.7 expressing HEK293 cells. In vivo and ex vivo inhibition of ectopic activity correlated well with the in vitro inhibition on the rat NaV1.7 channel. The estimated IC 50s for inhibition of ectopic activity in the SNL model occurred at similar unbound concentrations as clinical effective concentrations in humans. Inhibition of ectopic activity in the SNL model could be useful in predicting clinical effective concentrations for novel sodium channel blockers. In addition, in vitro potency could be used for screening, characterization and selection of compounds, thereby reducing the need for in vivo testing.

Left-Shifted Nav Channels in Injured Bilayer: Primary Targets for Neuroprotective Nav Antagonists?

Mechanical, ischemic, and inflammatory injuries to voltage-gated sodium channel (Nav)-rich membranes of axon initial segments and nodes of Ranvier render Nav channels dangerously leaky. By what means? The behavior of recombinant Nav1.6 (Wang et al., 2009) leads us to postulate that, in neuropathologic conditions, structural degradation of axolemmal bilayer fosters chronically left-shifted Nav channel operation, resulting in ENa rundown. This “sick excitable cell Nav-leak” would encompass left-shifted fast- and slow-mode based persistent INa (i.e., Iwindow and slow-inactivating INa). Bilayer-damage-induced electrophysiological dysfunctions of native-Nav channels, and effects on inhibitors on those channels, should, we suggest, be studied in myelinated axons, exploiting INa(V,t) hysteresis data from sawtooth ramp clamp. We hypothesize that (like dihydropyridines for Ca channels), protective lipophilic Nav antagonists would partition more avidly into disorderly bilayers than into the well-packed bilayers characteristic of undamaged, healthy plasma membrane. Whereas inhibitors using aqueous routes would access all Navs equally, differential partitioning into “sick bilayer” would co-localize lipophilic antagonists with “sick-Nav channels,” allowing for more specific targeting of impaired cells. Molecular fine-tuning of Nav antagonists to favor more avid partitioning into damaged than into intact bilayers could reduce side effects. In potentially salvageable neurons of traumatic and/or ischemic penumbras, in inflammatory neuropathies, in muscular dystrophy, in myocytes of cardiac infarct borders, Nav-leak driven excitotoxicity overwhelms cellular repair mechanisms. Precision-tuning of a lipophilic Nav antagonist for greatest efficacy in mildly damaged membranes could render it suitable for the prolonged continuous administration needed to allow for the remodeling of the excitable membranes, and thus functional recovery.

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.

Trauma-Induced Nav Leak and Dysexcitability in Axonal Membranes: Simulating the Consequences of Mechanically-Induced Left-Shift of Transient Nav1.6 Current

Several experimental findings motivate our simulations: 1) immediately after mechanical trauma, CNS nodes of Ranvier exhibit axolemmal blebs; 2) upon mechanically-induced bleb formation, the operation of recombinant Nav1.6 channels (= node of Ranvier Nav isoform) becomes irreversibly shifted towards hyperpolarized potentials; 3) mechanically-traumatized axons (and Nav1.6-expressing HEK cells) exhibit TTX-sensitive Na+ leak; 4) mechanical trauma to axons causes diverse excitability problems. It has been assumed (without direct evidence) that trauma increases persistent Nav current but we wondered if various trauma-induced pathological states (e.g. hyperexcitability and spontaneous discharge as seen in neuropathic pain, conduction block as seen in a knock-out punch, excitotoxic rise in axoplasmic Ca++ as seen in diffuse axonal injury) could be attributed to left-shifted operation of transient Nav current. We therefore modeled excitability, ion homeostasis and action potential propagation in myelinated axons (one or several nodes of Ranvier) with Nav channels of varying fractions of the axolemma left-shifted to various extents. Left-shift of activation and availability of Nav channels shifts window current (i.e. steady-state current through transient channels) leftward. Even if persistent current left-shifted as well, window current, we found, would be the critical source of Nav leak and could trigger degenerative excitotoxic cascades. Further, we show that, depending on the extent of left-shift and the fraction of affected axolemma, displaced window current predicts conduction block as well as assorted forms of hyperexcitability (including excitatory subthreshold oscillations, spontaneous ectopic excitation, reduced threshold for stimulated firing). In voltage clamp (as opposed to action potential) simulations, we demonstrate how various protocols could be used to test whether left-shifted Nav current in a traumatized membrane patch was an all-or-none or graded process.

A-887826 is a structurally novel, potent and voltage-dependent Nav1.8 sodium channel blocker that attenuates neuropathic tactile allodynia in rats

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Na(v)1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Na(v)1.8 channel blockers. A-887826 exemplifies this series and potently (IC(50)=11nM) blocked recombinant human Na(v)1.8 channels. A-887826 was approximately 3 fold less potent to block Na(v)1.2, approximately 10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na(+)) currents and was >30 fold less potent to block Na(V)1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na(+)) currents (IC(50)=8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund's adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by approximately 4mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Na(v)1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Na(v)1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Na(v)1.8 sodium channels in pathological pain states.

Traumatic Brain Injury and Axonal Sodium Loading: Modeling the Impact of Left-Shifted Nav Channel Operation at Blebbed Nodes of Ranvier

Traumatic brain injury like stretch immediately (<2 min) and irreversibly causes a TTX-sensitive axonal [Ca2+] increase that, in situ, underlies an untreatable pathology, diffuse axonal injury. Nav1.6-expressing mammalian cells, we showed, immediately (<2 min) exhibit TTX-sensitive Na+-leak following traumatic stretch (Wang et al 2009 Am J Physiol 297: in press). In situ, even mild axonal stretch injury can trigger adverse positive feedback so that leaks progress irreversibly to lethality. Though clinical trials are underway using Nav channel blockers that might reduce the severity of this outcome, molecular understanding of Nav channel damage has been lacking. Recently, however, we showed that activation and steady-state inactivation of recombinant Nav1.6 channels both irreversibly left-shift (up to −20 mV) in traumatized membrane (Wang et al 2009) as if their voltage sensors are responding to the increased bilayer disorder of traumatized (blebbed) membrane. In axonal membrane traumatized to various extents, this should smear out the window current range leftward between the normal range toward the resting potential range, effectively degrading a well-confined window conductance into a TTX-sensitive “Na+-leak”.To assess whether minor membrane trauma could lead to Na+ (and hence Ca2+) loading of axons, we model partially left-shifted Nav operation in a free-running human node of Ranvier. Included are Kv and Nav conductances (linear and electrodiffusion driving forces, respectively) and a Na/K pump, with [ion]s (and associated ENa and EK) in realistic-sized intra- and extracellular compartments changing due to net ion fluxes. Left-shifting a fraction of the Navs immediately triggers a damped action potential burst then a voltage plateau dominated by window current. If resting conductances are large enough, pumping restores the system for several minutes, then more bursting starts and more ENa rundown occurs.

Cold‐induced disruption of Na+ channel slow inactivation underlies paralysis in highly thermosensitive paramyotonia

The Q270K mutation of the skeletal muscle Na(+) channel alpha subunit (Nav1.4) causes atypical paramyotonia with a striking sensitivity to cold. Attacks of paralysis and a drop in the compound muscle action potential (CMAP) are exclusively observed at cold. To understand the pathogenic process, we studied the consequences of this mutation on channel gating at different temperatures. WT or Q270K recombinant Nav1.4 channels fused at their C-terminal end to the enhanced green fluorescent protein (EGFP) were expressed in HEK-293 cells. Whole-cell Na(+) currents were recorded using the patch clamp technique to examine channel gating at 30 degrees C and after cooling the bathing solution to 20 degrees C. Mutant channel fast inactivation was impaired at both temperatures. Cooling slowed the kinetics and enhanced steady-state fast inactivation of both mutant and WT channels. Mutant channel slow inactivation was fairly comparable to that of the WT at 30 degrees C, but became clearly abnormal at 20 degrees C. Cooling enhanced slow inactivation in the WT by shifting the voltage dependence toward hyperpolarization, but induced the opposite effect in the mutant. Destabilization of mutant channel slow inactivation in combination with defective fast inactivation is expected to increase the susceptibility to prolonged membrane depolarization, and can ultimately lead to membrane inexcitability and paralysis at cold. Thus, abnormal temperature sensitivity of slow inactivation can be a determinant pathogenic factor, and should therefore be more widely considered in thermosensitive Na(+) channelopathies.