Changes In Nav1 Research Articles

Proton modulation of cardiac I Na: a potential arrhythmogenic trigger.

Voltage-gated sodium (NaV) channels generate the upstroke and mediate duration of the ventricular action potential, thus they play a critical role in mediating cardiac excitability. Cardiac ischemia triggers extracellular pH to drop as low as pH 6.0, within just 10 min of its onset. Heightened proton concentrations reduce sodium conductance and alter the gating parameters of the cardiac-specific voltage-gated sodium channel, NaV1.5. Most notably, acidosis destabilizes fast inactivation, which plays a critical role in regulating action potential duration. The changes in NaV1.5 channel gating contribute to cardiac dysfunction during ischemia that can cause syncope, cardiac arrhythmia, and even sudden cardiac death. Understanding NaV channel modulation by protons is paramount to treatment and prevention of the deleterious effects of cardiac ischemia and other triggers of cardiac acidosis.

Calcium-Mediated Tailspin of Calmodulin on the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel Nav1.2

NaV1.2 is regulated by calmodulin (CaM), an essential calcium sensor with two homologous domains (N and C). CaM binds tightly to an IQ motif in the intracellular C-terminal tail of the pore-forming alpha subunit of NaV1.2 as well as to the inactivation gate. The IQ motif interacts with the “semi-open” cleft of apo CaM: I inserts into the cleft, while Q contacts the FG-turn of CaM (2KXW). To learn how CaM-IQ responds to increases in intracellular [Ca2+], we determined equilibrium constants for apo and calcium-saturated CaM binding to biosensors containing mutated IQ motif sequences sandwiched between YFP and CFP. Their quantum yields permit resolution of Kd values close to 1 nM from equilibrium titrations. Changes of NaV1.2 residues making close contacts with apo CaM were anticipated to diminish binding of both apo and calcium-saturated CaM. However, the quantitative effects differed by orders of magnitude. The affinity for calcium-saturated CaM dropped by factors of 10-100, while effects on apo CaM binding were more severe. Thus, the CaM-IQ interface differs dramatically depending on calcium-saturation of CaM. NMR studies of a complex of (Ca2+)2-CaM-C-domain bound to the IQ motif showed that calcium binding opens CaM but also causes it to pivot by 180° so that it binds to the IQ motif in the opposite direction. The I of the IQ motif contacts the “open” hydrophobic cleft of (Ca2+)2-CaM, but the Q points towards the linker between the N- and C-domains of CaM. In conjunction with 2KXW, this new structure provides the first pair of high resolution structures for apo and calcium-saturated CaM bound to a single IQ motif. Support: NIH R01 GM57001 and Carver Charitable Trust Grant 01-224.

Local Gene Expression Changes after UV-Irradiation of Human Skin

UV-irradiation is a well-known translational pain model inducing local inflammation and primary hyperalgesia. The mediators and receptor proteins specifically contributing to mechanical or heat hyperalgesia are still unclear. Therefore, we irradiated buttock skin of humans (n = 16) with 5-fold MED of UV-C and assessed the time course of hyperalgesia and axon reflex erythema. In parallel, we took skin biopsies at 3, 6 and 24 h after UVC irradiation and assessed gene expression levels (RT-PCR ) of neurotrophins (e.g. NGF, BDNF, GDNF), ion channels (e.g. NaV1.7, TRPV1), inflammatory mediators (e.g. CCL-2, CCL-3) and enzymes (e.g. PGES, COX2). Hyperalgesia to mechanical impact (12 m/s) and heat (48°C) stimuli was significant at 6 h (p<0.05 and p<0.01) and 24 h (p<0.005 and p<0.01) after irradiation. Axon reflex erythema upon mechanical and thermal stimuli was significantly increased 3 h after irradiation and particularly strong at 6 h. A significant modulation of 9 genes was found post UV-C irradiation, including NGF (3, 6, 24 h), TrkA (6, 24 h), artemin, bradykinin-1 receptor, COX-2, CCL-2 and CCL-3 (3 and 6 h each). A significant down-regulation was observed for TRPV1 and iNOS (6, 24 h). Individual one-to-one correlation analysis of hyperalgesia and gene expression revealed that changes of Nav1.7 (SCN9A) mRNA levels at 6 and 24 h correlated to the intensity of mechanical hyperalgesia recorded at 24 h post UV-irradiation (Pearson r: 0.57, p<0.04 and r: 0.82, p<0.001). Expression of COX-2 and mPGES at 6 h correlated to the intensity of heat-induced erythema 24 h post UV (r: 0.57, p<0.05 for COX-2 and r: 0.83, p<0.001 for PGES). The individual correlation analyses of functional readouts (erythema and pain response) with local expression changes provided evidence for a potential role of Nav1.7 in mechanical hyperalgesia.

Nongenomic Thyroxine Regulation of Voltage-gated Sodium Current During Embryonic Development

Thyroid hormones modulate ion flux across cellular membranes by effects on both transporters and channels. One such mechanism involves modulation of voltage-gated sodium current in spinal neurons by thyroxine. Experiments in zebrafish embryos have shown that thyroxine rapidly upregulates the current amplitude of a specific sodium channel subtype, nav1.6a. In mammals, the orthologous channel, Nav1.6, is expressed at nodes of Ranvier. In contrast to traditional thyroid hormone mechanisms that depend on nuclear receptors and regulation of gene expression, the regulation of nav1.6a channels involves a plasma membrane receptor, the integrin dimer αVβ3, indicative of a nongenomic mechanism. Although acute changes in thyroxine levels lead to rapid changes in nav1.6a function in vivo, the concentration of thyroxine in vivo is relatively stable in the absence of experimental intervention. On this basis, the endogenous concentration of thyroxine provides on-going regulation of voltage-gated sodium channel function through interaction with integrin αVβ3. This interaction is evident with blockade of the thyroxine-αVβ3 pathway, via antagonists of thyroid hormone (tetrac) or the integrin receptor (LM609 antibody). These interventions also produce significant changes in biologically relevant zebrafish behavior. In summary, thyroxine binding to integrin αVβ3 regulates voltage-gated sodium current in the embryonic nervous system and serves as an important mechanism by which thyroid hormones influence development and behavior. Keywords: Activity, embryonic, integrin, neurodevelopment, neuron, nongenomic, phosphatase, Rohon-Beard, sodium channel, thyroid, thyroxine, zebrafish

Nav1.9 Channel Contributes to Mechanical and Heat Pain Hypersensitivity Induced by Subacute and Chronic Inflammation

Inflammation is known to be responsible for the sensitization of peripheral sensory neurons, leading to spontaneous pain and invalidating pain hypersensitivity. Given its role in regulating neuronal excitability, the voltage-gated Nav1.9 channel is a potential target for the treatment of pathological pain, but its implication in inflammatory pain is yet not fully described. In the present study, we examined the role of the Nav1.9 channel in acute, subacute and chronic inflammatory pain using Nav1.9-null mice and Nav1.9 knock-down rats. In mice we found that, although the Nav1.9 channel does not contribute to basal pain thresholds, it plays an important role in heat pain hypersensitivity induced by subacute paw inflammation (intraplantar carrageenan) and chronic ankle inflammation (complete Freund's adjuvant-induced monoarthritis). We showed for the first time that Nav1.9 also contributes to mechanical hypersensitivity in both models, as assessed using von Frey and dynamic weight bearing tests. Consistently, antisense-based Nav1.9 gene silencing in rats reduced carrageenan-induced heat and mechanical pain hypersensitivity. While no changes in Nav1.9 mRNA levels were detected in dorsal root ganglia (DRGs) during subacute and chronic inflammation, a significant increase in Nav1.9 immunoreactivity was observed in ipsilateral DRGs 24 hours following carrageenan injection. This was correlated with an increase in Nav1.9 immunolabeling in nerve fibers surrounding the inflamed area. No change in Nav1.9 current density could be detected in the soma of retrolabeled DRG neurons innervating inflamed tissues, suggesting that newly produced channels may be non-functional at this level and rather contribute to the observed increase in axonal transport. Our results provide evidence that Nav1.9 plays a crucial role in the generation of heat and mechanical pain hypersensitivity, both in subacute and chronic inflammatory pain models, and bring new elements for the understanding of its regulation in those models.

The developmental changes of Na v1.1 and Na v1.2 expression in the human hippocampus and temporal lobe

Alterations of the genes encoding α1 and α2 subunits of voltage-gated sodium channels ( SCN1A, SCN2A) have been reported as causes of various types of epilepsy, most of which occur during the first year of life; as yet, however, the detailed mechanisms are unclear. We suppose that developmental changes of SCN1A and SCN2A in the human brain, which are unknown yet, may play an important role. So here, we studied the developmental changes of their corresponding proteins (Na v1.1 and Na v1.2) in the human hippocampus and temporal lobe in 28 autopsy cases, which age from 13 weeks of gestation (GW) to 63 years of age (Y). Using comparative microscopic immunohistochemical (IHC) analysis, we found that Na v1.1 and Na v1.2 immunoreactivity first appeared at 19GW, simultaneously in the hippocampus and the white matter of temporal lobe. In nearly all age groups, Na v1.1 immunoreactivity was weak and relatively homogeneous. In general, Na v1.1 immunoreactive (IR) neurons and neurites increased during the late fetal and postnatal periods, reached their peaks 7–9 months after birth (M), then decreased and remained stable at a relatively low level during childhood and adulthood. On the other hand, Na v1.2 immunoreactivity was strong and heterogeneous. In the hippocampus, Na v1.2 IR neurons increased gradually during the late fetal period, reached their peaks at 7–9M, sustained this high level during childhood, and then decreased slightly at adulthood. In the temporal lobe, Na v1.2 IR neurons reached a high level during the late fetal period, and maintained that level during subsequent developmental stages; Na v1.2 IR neurites also increased to a relatively high level during the late fetal period and continued to increase up to and during adulthood. Using double-staining IHC, we found that Na v1.1 and Na v1.2 had a relatively high colocalization rate with parvalbumin and showed distinct developmental changes. These findings extend our previous understanding of sodium channels and may help us discover the pathomechanisms of sodium channel-related age-dependent epilepsy.

Patch Trauma-Induced Kinetic Changes in Nav1.4 Channels (Oocyte Patches): Gating in Both a Nav1.4-Nav1.5 Chimera Insusceptible to β1 Modulation and a Nav1.4 Mutant Incapable of Binding G-Ankyrin is Irreversibly Modulated by Membrane Stretch

Oocytes lack endogenous Nav-modulating β subunits. Nav1.4α (unlike Nav1.5α) pore subunits expressed in oocytes exhibit anomalously slow, right-shifted voltage-dependent gating but co-expression with β1 subunits results in normal (i.e. more rapid, more left-shifted) Nav1.4 kinetics. For unknown reasons, membrane stretch due to pipette aspiration in cell-attached oocyte patches has the same effect on Nav1.4 α kinetics as co-expression of α with β1, namely, acceleration/left-shift of the activation and availability processes. This also occurs with Nav1.6 (Wang et al 2009 Am J Physiol 297:C823) where the nature of the kinetic changes in Nav1.6 suggests trauma-induced changes in bilayer mechanics as a plausible explanation. However, mechanical disruption between Navα and various protein “targets” might also be the explanation. Makita et al 1996 (J Neurosci 16:7117) generated a Nav1.4-Nav1.5 chimera in which the re-entrant S5-S6 “pore loops” of Nav1.4 domains 1 and 4 were substituted with the corresponding Nav1.5 pore loops. This rendered the chimera incapable of modulation by β1 subunits. Here we show that slow component activity in this chimera was irreversibly accelerated and left-shifted by membrane stretch. We also tested a rat Nav1.4α mutant in which the cytoplasmic loop 2 amino acids responsible for binding to G-ankyrin have been deleted. In this Nav1.4α mutant too, patch stretch irreversibly accelerated/left-shifted the anomalously slow activity of the channel. These findings rule out the possibility that the extracellular β1 interacting domains of Nav1.4α or the intracellular G-ankyrin interacting domain of Nav1.4α are required for the irreversible membrane trauma-induced acceleration/left-shift of this channel.

Nav1.7 mutations associated with paroxysmal extreme pain disorder, but not erythromelalgia, enhance Navβ4 peptide‐mediated resurgent sodium currents

Abnormal pain sensitivity associated with inherited and acquired pain disorders occurs through increased excitability of peripheral sensory neurons in part due to changes in the properties of voltage-gated sodium channels (Navs). Resurgent sodium currents (I(NaR)) are atypical currents believed to be associated with increased excitability of neurons and may have implications in pain. Mutations in Nav1.7 (peripheral Nav isoform) associated with two genetic pain disorders, inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD), enhance Nav1.7 function via distinct mechanisms. We show that changes in Nav1.7 function due to mutations associated with PEPD, but not IEM, are important in I(NaR) generation, suggesting that I(NaR) may play a role in pain associated with PEPD. This knowledge provides us with a better understanding of the mechanism of I(NaR) generation and may lead to the development of specialized treatment for pain disorders associated with I(NaR).

The Up-Regulation of Voltage-Gated Sodium Channel NaV1.6 Expression Following Fluid Percussion Traumatic Brain Injury in Rats

The influx of Na and the depolarization mediated by voltage-gated sodium channels (VGSCs) is an early event in traumatic brain injury (TBI) induced cellular abnormalities and is therefore well positioned as an upstream target for pharmacologic modulation of the pathological responses to TBI. Alteration in the expression of the VGSC alpha-subunit has occurred in a variety of neuropathological states including focal cerebral ischemia, spinal injury, and epilepsy. In this study, changes in Nav1.6 mRNA and protein expression were investigated in rat hippocampus after TBI. Forty-eight adult male Sprague Dawley rats were randomly assigned to control or TBI groups. TBI was induced with a lateral fluid percussion device. Expression of mRNA and protein for Nav1.6 in the bilateral hippocampus was examined at 2, 12, 24, and 72 hours after injury by real-time quantitative polymerase chain reaction and Western blot. Immunofluorescence was performed to localize the expression of Nav1.6 protein in the hippocampus. Expression of >Nav1.6 mRNA was significantly up-regulated in the bilateral hippocampus at 2 and 12 hours post-TBI. Significant up-regulation of Nav1.6 protein was identified in the ipsilateral hippocampus from 2 to 72 hours post-TBI and in the contralateral hippocampus from 2 to 24 hours post-TBI. Expression of Nav1.6 occurred predominantly in neurons in the hippocampus. Results of the study showed significant up-regulation of mRNA and protein for Nav1.6 in rat hippocampal neurons after TBI.

NaV1.1 channels and epilepsy

Voltage-gated sodium channels initiate action potentials in brain neurons, and sodium channel blockers are used in therapy of epilepsy. Mutations in sodium channels are responsible for genetic epilepsy syndromes with a wide range of severity, and the NaV1.1 channel encoded by the SCN1A gene is the most frequent target of mutations. Complete loss-of-function mutations in NaV1.1 cause severe myoclonic epilepsy of infancy (SMEI or Dravet's Syndrome), which includes severe, intractable epilepsy and comorbidities of ataxia and cognitive impairment. Mice with loss-of-function mutations in NaV1.1 channels have severely impaired sodium currents and action potential firing in hippocampal GABAergic inhibitory neurons without detectable effect on the excitatory pyramidal neurons, which would cause hyperexcitability and contribute to seizures in SMEI. Similarly, the sodium currents and action potential firing are also impaired in the GABAergic Purkinje neurons of the cerebellum, which is likely to contribute to ataxia. The imbalance between excitatory and inhibitory transmission in these mice can be partially corrected by compensatory loss-of-function mutations of NaV1.6 channels, and thermally induced seizures in these mice can be prevented by drug combinations that enhance GABAergic neurotransmission. Generalized epilepsy with febrile seizures plus (GEFS+) is caused by missense mutations in NaV1.1 channels, which have variable biophysical effects on sodium channels expressed in non-neuronal cells, but may primarily cause loss of function when expressed in mice. Familial febrile seizures is caused by mild loss-of-function mutations in NaV1.1 channels; mutations in these channels are implicated in febrile seizures associated with vaccination; and impaired alternative splicing of the mRNA encoding these channels may also predispose some children to febrile seizures. We propose a unified loss-of-function hypothesis for the spectrum of epilepsy syndromes caused by genetic changes in NaV1.1 channels, in which mild impairment predisposes to febrile seizures, intermediate impairment leads to GEFS+ epilepsy, and severe or complete loss of function leads to the intractable seizures and comorbidities of SMEI.

Introduction toThe Journal of Physiology's Special Issue on Neurological Channelopathies

Introduction to<i>The Journal of Physiology</i>'s Special Issue on Neurological Channelopathies

Increased peripheral nerve excitability and local NaV1.8 mRNA up-regulation in painful neuropathy.

BackgroundNeuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury.ResultsAfter unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states.ConclusionCuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.

Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis

Central nervous system plasticity is essential for normal function, but can also reinforce abnormal network behavior, leading to epilepsy and other disorders. The role of altered ion channel expression in abnormal plasticity has not been thoroughly investigated. Nav1.6 is the most abundantly expressed sodium channel in the nervous system. Because of its distribution in the cell body and axon initial segment, Nav1.6 is crucial for action potential generation. The goal of the present study was to investigate the possible role of changes in Nav1.6 expression in abnormal, activity-dependent plasticity of hippocampal circuits. We studied kindling, a form of abnormal activity-dependent facilitation. We investigated: (1) sodium channel protein expression by immunocytochemistry and sodium channel messenger RNA (mRNA) by in situ hybridization, (2) sodium current by patch clamp recordings, and (3) rate of kindling by analysis of seizure behavior. The initiation, development, and expression of kindling in wild-type mice were compared to Nav1.6 +/-med(tg) mice, which have reduced expression of Nav1.6. We found that kindling was associated with increased expression of Nav1.6 protein and mRNA, which occurred selectively in hippocampal CA3 neurons. Hippocampal CA3 neurons also showed increased persistent sodium current in kindled animals compared to sham-kindled controls. Conversely, Nav1.6 +/-med(tg) mice resisted the initiation and development of kindling. These findings suggest an important mechanism for enhanced excitability, in which Nav1.6 may participate in a self-reinforcing cycle of activity-dependent facilitation in the hippocampus. This mechanism could contribute to both normal hippocampal function and to epilepsy and other common nervous system disorders.

Axotomy does not up-regulate expression of sodium channel Na v1.8 in Purkinje cells

Aberrant expression of the sensory neuron specific (SNS) sodium channel Na v1.8 has been demonstrated in cerebellar Purkinje cells in experimental models of multiple sclerosis (MS) and in human MS. The aberrant expression of Na v1.8, which is normally present in primary sensory neurons but not in the CNS, may perturb cerebellar function, but the mechanisms that trigger it are not understood. Because axotomy can provoke changes in Na v1.8 expression in dorsal root ganglion (DRG) neurons, we tested the hypothesis that axotomy can provoke an up-regulation of Na v1.8 expression in Purkinje cells, using a surgical model that transects axons of Purkinje cells in lobules IIIb–VII in the rat. In situ hybridization and immunocytochemistry did not reveal an up-regulation of Na v1.8 mRNA or protein in axotomized Purkinje cells. Hybridization and immunostaining signals for the sodium channel Na v1.6 were clearly present, demonstrating that sodium channel transcripts and protein were present in experimental cerebella. These results demonstrate that axotomy does not trigger the expression of Na v1.8 in Purkinje cells.