Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs) this AP backpropagation is supported by dendritic voltage-gated Na+ channels (Nav), whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we reveal the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability.
Voltage-gated sodium (Nav) channels are responsible for initiation and propagation of action potential in the neurons. To explore the mechanisms for chronic heart failure (CHF)-induced baroreflex dysfunction, we measured the expression and current density of Nav channel subunits (Nav1.7, Nav1.8, and Nav1.9) in the aortic baroreceptor neurons and investigated the role of Nav channels on aortic baroreceptor neuron excitability and baroreflex sensitivity in sham and CHF rats. CHF was induced by left coronary artery ligation. The development of CHF (6–8 weeks after the coronary ligation) was confirmed by hemodynamic and morphological characteristics. Immunofluorescent data indicated that Nav1.7 was expressed in A-type (myelinated) and C-type (unmyelinated) nodose neurons but Nav1.8 and Nav1.9 were expressed only in C-type nodose neurons. Real-time RT-PCR and western blot data showed that CHF reduced mRNA and protein expression levels of Nav channels in nodose neurons. In addition, using the whole cell patch-clamp technique, we found that Nav current density and cell excitability of the aortic baroreceptor neurons were lower in CHF rats than that in sham rats. Aortic baroreflex sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. Furthermore, Nav channel activator (rATX II, 100 nM) significantly enhanced Nav current density and cell excitability of aortic baroreceptor neurons and improved aortic baroreflex sensitivity in CHF rats. These results suggest that reduced expression and activation of the Nav channels is involved in the attenuation of baroreceptor neuron excitability, which subsequently contributes to the impairment of baroreflex in CHF state.
Aortic baroreceptor neuron; Baroreflex; Heart failure; Sodium channel
Voltage-gated ion channels are diverse and fundamental determinants of neuronal intrinsic excitability. Voltage-gated K+ (Kv) and Na+ (Nav) channels play complex yet fundamentally important roles in determining intrinsic excitability. The Kv and Nav channels located at the axon initial segment (AIS) play a unique and especially important role in generating neuronal output in the form of anterograde axonal and backpropagating action potentials, Aberrant intrinsic excitability in individual neurons within networks contributes to synchronous neuronal activity leading to seizures. Mutations in ion channel genes gives rise to a variety of seizure-related “Channelopathies”, and many of the ion channel subunits associated with epilepsy mutations are localized at the AIS, making this a hotspot for epileptogenesis. Here we review the cellular mechanisms that underlie the trafficking of Kv and Nav channels found at the AIS, and how Kv and Nav channel mutations associated with epilepsy can alter these processes.
Potassium; channel-Sodium; channel- Neuron-Subcellular; localization-Seizures
Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that undergo depolarization (DP) block upon moderate stimulation. Understanding DP block is important because it has been correlated with the clinical efficacy of chronic antipsychotic drug treatment. Here we describe how voltage-gated sodium (NaV) channels regulate DP block and pacemaker activity in DA neurons of the substantia nigra using rat brain slices. The distribution, density and gating of NaV currents were manipulated by blocking native channels with tetrodotoxin and by creating virtual channels and anti-channels with dynamic clamp. Although action potentials initiate in the axon initial segment (AIS) and NaV channels are distributed in multiple dendrites, selective reduction of NaV channel activity in the soma was sufficient to decrease pacemaker frequency and increase susceptibility to DP block. Conversely, increasing somatic NaV current density raised pacemaker frequency and lowered susceptibility to DP block. Finally, when NaV currents were restricted to the soma, pacemaker activity occurred at abnormally high rates due to excessive local subthreshold NaV current. Together with computational simulations, these data show that both the slow pacemaker rate and the sensitivity to DP block that characterizes DA neurons result from the low density of somatic NaV channels. More generally, we conclude that the somatodendritic distribution of NaV channels is a major determinant of repetitive spiking frequency.
The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, βIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and βIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. βIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.
βIV spectrin; sodium channel Nav1.6; neurofascin; NrCAM; axon hillock
Intrinsic excitability is a key feature dictating neuronal response to synaptic input. Here we investigate the recent observation that dentate granule neurons exhibit a more depolarized voltage threshold for action potential initiation than CA3 pyramidal neurons. We find no evidence that tonic GABA currents, leak or voltage-gated potassium conductances, or the expression of sodium channel isoform differences can explain this depolarized threshold. Axonal initial segment voltage-gated sodium channels, which are dominated by the NaV1.6 isoform in both cell types, distribute more proximally and exhibit lower overall density in granule neurons than in CA3 neurons. To test possible contributions of sodium channel distributions to voltage threshold and to test whether morphological differences participate, we performed simulations of dentate granule neurons and of CA3 pyramidal neurons. These simulations revealed that cell morphology and sodium channel distribution combine to yield the characteristic granule neuron action potential upswing and voltage threshold. Proximal axon sodium channel distribution strongly contributes to the higher voltage threshold of dentate granule neurons for two reasons. First, action potential initiation closer to the somatodendritic current sink causes the threshold of the initiating axon compartment to rise. Second, the proximity of the action potential initiation site to the recording site causes somatic recordings to more faithfully reflect the depolarized threshold of the axon than in cells like CA3 neurons, with distally initiating action potentials. Our results suggest that the proximal location of axon sodium channel in dentate granule neurons contributes to the intrinsic excitability differences between DG and CA3 neurons and may participate in the low-pass filtering function of dentate granule neurons.
seizures; hippocampus; excitability; potassium channel; principal cell
The origin of the action potential in the cochlea has been a long-standing puzzle. Since voltage-dependent Na+ (Nav) channels are essential for action potential generation, we investigated the detailed distribution of Nav1.6 and Nav1.2 in the cochlear ganglion, cochlear nerve, and organ of Corti, including the Type I and Type II ganglion cells. In most Type I ganglion cells, Nav1.6 was present at the first nodes flanking the myelinated bipolar cell body and at subsequent nodes of Ranvier. In the other ganglion cells, including Type II, Nav1.6 clustered in the initial segments of both of the axons that flank the unmyelinated bipolar ganglion cell bodies. In the organ of Corti, Nav1.6 was localized in the short segments of the afferent axons and their sensory endings beneath each inner hair cell. Surprisingly, the outer spiral fibers and their sensory endings were well labeled beneath the outer hair cells over their entire trajectory. In contrast, Nav1.2 in the organ of Corti was localized to the unmyelinated efferent axons and their endings on the inner and outer hair cells. We present a computational model illustrating the potential role of the Nav channel distribution described here. In the deaf mutant quivering mouse, the localization of Nav1.6 was disrupted in the sensory epithelium and ganglion. Taken together, these results suggest that distinct Nav channels generate and regenerate action potentials at multiple sites along the cochlear ganglion cells and nerve fibers, including the afferent endings, ganglionic initial segments, and nodes of Ranvier.
Axon initial segment; Nav1.6; Nav1.2; Spiral ganglion; Cochlear nucleus; Hair cells; Quivering mutation; Computational model
The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?
We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr+ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS.
α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.
Voltage-gated sodium channels are membrane proteins that initiate action potentials in neurons following membrane depolarization. Members of this family show differential distribution at the subcellular level. The mechanisms underlying the targeting of these isoforms are not understood. However, their specificity is important because the isoforms can change the excitability of the membrane due to differences in their electrophysiological properties. In this study, chimeras generated between Nav1.2 and Nav1.6 were used to test channel domains for sequence that would allow Nav1.2 to localize to unmyelinated axons when Nav1.6 could not. We show that the N-terminal 202 amino acids of the Nav1.2 channel can mediate membrane domain-specific sorting in polarized epithelial cells and are necessary but not sufficient for localizing the isoform to the axons of cultured neurons. The domain-sorting signal is in the region between amino acids 110-202 of the Nav1.2 channel. The C-terminal 451 amino acids of Nav1.2 likely contain determinants that interact with neuron-specific factors to direct Nav1.2 to the axon.
Sodium channels; localization; neuron
▶ The β3 subunit masks the ER retention signal of NaV1.8 and release the channel from the ER. ▶ p11 directly binds to NaV1.8 and help its translocation to the plasma membrane. ▶ PDZD2 is responsible for the functional expression of NaV1.8 on the plasma membrane. ▶ Contactin KO mice exhibit a reduction of NaV1.8 along unmyelinated axons in the sciatic nerve. ▶ PKA activation increases the NaV1.8 density on the membrane through direct phosphorylation.
The α-subunit of tetrodotoxin-resistant voltage-gated sodium channel NaV1.8 is selectively expressed in sensory neurons. It has been reported that NaV1.8 is involved in the transmission of nociceptive information from sensory neurons to the central nervous system in nociceptive  and neuropathic  pain conditions. Thus NaV1.8 has been a promising target to treat chronic pain. Here we discuss the recent advances in the study of trafficking mechanism of NaV1.8. These pieces of information are particularly important as such trafficking machinery could be new targets for painkillers.
Sodium Channel; Sensory Neuron; Pain; Trafficking
Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V½ of steady-state activation and inactivation and increased Nav1.7-mediated INa density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.
voltage-gated sodium channels (Navs); Navs β-subunits; glycosylation; biophysical properties; trafficking
Neurons are highly polarized cells with functionally distinct axonal and somatodendritic compartments. Voltage-gated sodium channels Nav1.2 and Nav1.6 are highly enriched at axon initial segments (AIS) and nodes of Ranvier, where they are necessary for generation and propagation of action potentials. Previous studies using reporter proteins in unmyelinated cultured neurons suggest that an ankyrinG-binding motif within intracellular loop 2 (L2) of sodium channels is sufficient for targeting these channels to the AIS, but mechanisms of channel targeting to nodes remain poorly understood. Using a CD4-Nav1.2/L2 reporter protein in rat dorsal root ganglion neuron-Schwann cell myelinating co-cultures, we show that the ankyrinG-binding motif is sufficient for protein targeting to nodes of Ranvier. However, reporter proteins cannot capture the complexity of full-length channels. To determine how native, full-length sodium channels are clustered in axons, and to show the feasibility of studying these channels in vivo, we constructed fluorescently-tagged and functional mouse Nav1.6 channels for in vivo analysis using in utero brain electroporation. We show here that wild-type tagged-Nav1.6 channels are efficiently clustered at nodes and AIS in vivo. Furthermore, we show that mutation of a single invariant glutamic acid residue (E1100) within the ankyrinG-binding motif blocked Nav1.6 targeting in neurons both in vitro and in vivo. Additionally, we show that caseine kinase phosphorylation sites within this motif, while not essential for targeting, can modulate clustering at the AIS. Thus, the ankyrinG- binding motif is both necessary and sufficient for the clustering of sodium channels at nodes of Ranvier and the AIS.
Ion Channel; Axon Initial Segment; Nodes of Ranvier; cytoskeleton; in utero electroporation
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.
traumatic brain injury; spinal; riluzole; ranolazine; simulation; modeling
In axon-bearing neurons, action potentials conventionally initiate at the axon initial segment (AIS) and are important for neuron excitability and cell-to-cell communication. However in axonless neurons, spike origin has remained unclear. Here we report in the axonless spiking AII amacrine cell of the mouse retina a dendritic process sharing organizational and functional similarities with the AIS. This process was revealed through viral-mediated expression of channelrhodopsin-2-GFP (ChR2-GFP) with the AIS-targeting motif of sodium channels (NavII-III). The AII-processes showed clustering of voltage-gated Na+ channel 1.1 (Nav1.1) as well as AIS markers ankyrin-G and neurofascin. Furthermore, NavII-III targeting disrupted Nav1.1 clustering in the AII-process which drastically decreased Na+ current and abolished the ability of the AII amacrine cell to generate spiking. Our findings indicate that despite lacking an axon, spiking in the axonless neuron can originate at a specialized AIS-like process.
The voltage-gated sodium channel (Nav1) plays an important role in initiating and propagating action potentials in neuronal cells. We and others have recently found that the Alzheimer’s disease-related secretases BACE1 and presenilin (PS)/γ-secretase regulate Nav1 function by cleaving auxiliary subunits of the channel complex. We have also shown that elevated BACE1 activity significantly decreases sodium current densities in neuroblastoma cells and acutely dissociated adult hippocampal neurons. For detailed molecular studies of sodium channel regulation, biochemical methods are now complementing classical electrophysiology. To understand how BACE1 regulates sodium current densities in our studies, we setup conditions to analyze surface levels of the pore-forming Nav1 α-subunits. By using a cell surface biotinylation protocol, we found that elevated BACE1 activity significantly decreases surface Nav1 α-subunit levels in both neuroblastoma cells and acutely prepared hippocampal slices. This finding would explain the decreased sodium currents shown by standard electrophysiological methods. The biochemical methods used in our studies would be applicable to analyses of surface expression levels of other ion channels as well as Nav1 in cells and adult hippocampal neurons.
Voltage-gated sodium channel; BACE1; Presenilin; γ-Secretase; Cell surface biotinylation; Neuroblastoma; Hippocampal neurons; Adult hippocampal slices
The voltage-gated sodium-channel type IX α subunit, known as Nav1.7 and encoded by the gene SCN9A, is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Nav1.7 dysfunction in three different human pain disorders. Gain-of-function missense mutations in Nav1.7 have been shown to cause primary erythermalgia and paroxysmal extreme pain disorder, while nonsense mutations in Nav1.7 result in loss of Nav1.7 function and a condition known as channelopathy-associated insensitivity to pain, a rare disorder in which affected individuals are unable to feel physical pain. This review highlights these recent developments and discusses the critical role of Nav1.7 in pain sensation in humans.
Voltage-gated sodium channels (VGSCs) are essential for the generation and propagation of action potentials in electrically excitable cells. Dominant mutations in SCN1A, which encodes the Nav1.1 VGSC α-subunit, underlie several forms of epilepsy, including Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Electrophysiological analyses of DS and GEFS+ mouse models have led to the hypothesis that SCN1A mutations reduce the excitability of inhibitory cortical and hippocampal interneurons. To more directly examine the relative contribution of inhibitory interneurons and excitatory pyramidal cells to SCN1A-derived epilepsy, we first compared the expression of Nav1.1 in inhibitory parvalbumin (PV) interneurons and excitatory neurons from P22 mice using fluorescent immunohistochemistry. In the hippocampus and neocortex, 69% of Nav1.1 immunoreactive neurons were also positive for PV. In contrast, 13% and 5% of Nav1.1 positive cells in the hippocampus and neocortex, respectively, were found to co-localize with excitatory cells identified by CaMK2α immunoreactivity. Next, we reduced the expression of Scn1a in either a subset of interneurons (mainly PV interneurons) or excitatory cells by crossing mice heterozygous for a floxed Scn1a allele to either the Ppp1r2-Cre or EMX1-Cre transgenic lines, respectively. The inactivation of one Scn1a allele in interneurons of the neocortex and hippocampus was sufficient to reduce thresholds to flurothyl- and hyperthermia-induced seizures, whereas thresholds were unaltered following inactivation in excitatory cells. Reduced interneuron Scn1a expression also resulted in the generation of spontaneous seizures. These findings provide direct evidence for an important role of PV interneurons in the pathogenesis of Scn1a-derived epilepsies.
Epilepsy; SCN1A; ion channels; interneurons; pyramidal neurons
Dravet syndrome is a severe epileptic encephalopathy mainly caused by heterozygous mutations in the SCN1A gene encoding a voltage-gated sodium channel Nav1.1. We previously reported dense localization of Nav1.1 in parvalbumin (PV)-positive inhibitory interneurons in mice and abnormal firing of those neurons in Nav1.1-deficient mice. In the present study, we investigated the physiologic consequence of selective Nav1.1 deletion in mouse global inhibitory neurons, forebrain excitatory neurons or PV cells, using vesicular GABA transporter (VGAT)-Cre, empty spiracles homolog 1 (Emx1)-Cre or PV-Cre recombinase drivers. We show that selective Nav1.1 deletion using VGAT-Cre causes epileptic seizures and premature death that are unexpectedly more severe than those observed in constitutive Nav1.1-deficient mice. Nav1.1 deletion using Emx1-Cre does not cause any noticeable abnormalities in mice; however, the severe lethality observed with VGAT-Cre-driven Nav1.1 deletion is rescued by additional Nav1.1 deletion using Emx1-Cre. In addition to predominant expression in PV interneurons, we detected Nav1.1 in subpopulations of excitatory neurons, including entorhino-hippocampal projection neurons, a subpopulation of neocortical layer V excitatory neurons, and thalamo-cortical projection neurons. We further show that even minimal selective Nav1.1 deletion, using PV-Cre, is sufficient to cause spontaneous epileptic seizures and ataxia in mice. Overall, our results indicate that functional impairment of PV inhibitory neurons with Nav1.1 haploinsufficiency contributes to the epileptic pathology of Dravet syndrome, and show for the first time that Nav1.1 haploinsufficiency in excitatory neurons has an ameliorating effect on the pathology.
Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage-gated sodium or Nav channels underlie action potential initiation and propagation, dendritic excitability, and many other aspects of neuronal excitability. Various protein kinases have been suggested to phosphorylate the primary α subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav α subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in meditating aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav α subunits affinity-purified from rat brain with two distinct monoclonal antibodies to identify 15 phosphorylation sites on Nav1.2, 12 of which have not been previously reported. We also found 3 novel phosphorylation sites on Nav1.1. In general, commonly used phosphorylation site prediction algorithms did not accurately predict these novel in vivo phosphorylation sites. Our results demonstrate that specific Nav α subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nav channel activity in mammalian brain. Identification of phosphorylation sites using monoclonal antibody-based immunopurification and mass spectrometry is an effective approach to define the phosphorylation status of Nav channels and important membrane proteins in mammalian brain.
voltage-gated sodium channels; brain; phosphorylation; tandem mass spectrometry; immunopurification; monoclonal antibody; nanoflow liquid chromatography
Voltage-gated Nav channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Nav1.5 is the predominant Nav channel, and Nav1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Nav1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Nav channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Nav1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Nav1.5 expression, abnormal Nav1.5 membrane targeting, and reduced Na+ channel current density. We define the structural requirements on ankyrin-G for Nav1.5 interactions and demonstrate that loss of Nav1.5 targeting is caused by the loss of direct Nav1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Nav channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Nav channel organization in multiple excitable tissues.
Voltage-gated sodium channels are essential for the initiation and propagation of action potentials in excitable cells and are known as a target of local anesthetics. In addition, inhibition of sodium channels by volatile anesthetics has been proposed as a mechanism of general anesthesia. The n-alcohols produce anesthesia, and their potency increases with carbon number until a “cut-off” is reached. In this study, we examined effects of a range of n-alcohols on Nav1.2 subunits to determine the alcohol cut-off for this channel. We also studied the effect of a short-chain alcohol (ethanol) and a long-chain alcohol (octanol) on Nav1.2, Nav1.4, Nav1.6, and Nav1.8 subunits, and we investigated the effects of alcohol on channel kinetics. Ethanol and octanol inhibited sodium currents of all subunits, and the inhibition of the Nav1.2 channel by n-alcohols indicated a cut-off at nonanol. Ethanol and octanol produced open-channel block, which was more pronounced for Nav1.8 than for the other sodium channels. Inhibition of Nav1.2 was due to decreased activation and increased inactivation. These results suggest that sodium channels may have a hydrophobic binding site for n-alcohols and demonstrate the differences in the kinetic mechanisms of inhibition for n-alcohols and inhaled anesthetics.
The sodium channel Nav1.6, encoded by the gene SCN8A, is one of the major voltage-gated channels in human brain. The sequences of sodium channels have been highly conserved during evolution, and minor changes in biophysical properties can have a major impact in vivo. Insight into the role of Nav1.6 has come from analysis of spontaneous and induced mutations of mouse Scn8a during the past 18 years. Only within the past year has the role of SCN8A in human disease become apparent from whole exome and genome sequences of patients with sporadic disease. Unique features of Nav1.6 include its contribution to persistent current, resurgent current, repetitive neuronal firing, and subcellular localization at the axon initial segment (AIS) and nodes of Ranvier. Loss of Nav1.6 activity results in reduced neuronal excitability, while gain-of-function mutations can increase neuronal excitability. Mouse Scn8a (med) mutants exhibit movement disorders including ataxia, tremor and dystonia. Thus far, more than ten human de novo mutations have been identified in patients with two types of disorders, epileptic encephalopathy and intellectual disability. We review these human mutations as well as the unique features of Nav1.6 that contribute to its role in determining neuronal excitability in vivo. A supplemental figure illustrating the positions of amino acid residues within the four domains and 24 transmembrane segments of Nav1.6 is provided to facilitate the location of novel mutations within the channel protein.
voltage-gated sodium channels; epilepsy; intellectual disability; SCN8A; Nav1.6; neurogenetics; genetics; exomes
Nav1.5 or SCN5A is a member of the voltage-dependent family of sodium channels. The distribution of Nav1.5 protein was investigated in the mouse brain using immunohistochemistry. Immunostaining with a Nav1.5-specific antibodyrevealed that Nav1.5 protein was localizedin certain distinctregions of brain including the cerebral cortex, thalamus, hypothalamus, basalganglia, cerebellum and brain stem. Notably, we found that Nav1.5 protein co-localized with neurofilaments and clustered at a high density in the neuronal processes, mainly axons.These results suggest that Nav1.5 protein may play a role in the physiology of the central nervous system (generation and propagation of electrical signals by axons).
Axon; Brain; Cardiac sodium channel; Nav1.5/SCN5A; Neuronal process; Seizure; Sudden death
Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation.
Voltage-gated sodium (Nav) channels are required for impulse conductance in excitable tissues. Navs have been linked to human cancers, including prostate. The expression and distribution of Nav isoforms (Nav1.1-Nav1.9) in human prostate cancer are not well established. Here, we evaluated the expression of these isoforms and investigated the expression of Nav1.8 in human prostate cancer tissues. Nav1.8 was highly expressed in all examined cells. Expression of Nav1.1, Nav1.2, and Nav1.9 were high in DU-145, PC-3 and PC-3M cells compared to LNCaP (hormone-dependent), C4-2, C4-2B, and CWR22Rv-1 cells. Nav1.5 and Nav1.6 were expressed in all cells examined. Nav1.7 expression was absent in PC-3M and CWR22Rv-1, but expressed in the other cells examined. Immunohistochemistry revealed intensive Nav1.8 staining correlated with more advanced pathologic stage of disease. Increased intensity of nuclear Nav1.8 correlated with increased Gleason grade. Our results revealed that Nav1.8 is universally expressed in human prostate cancer cells. Nav1.8 expression statistically correlated with pathologic stage (P=0.04) and Gleason score (P=0.01) of human prostate tissue specimens. The aberrant nuclear localization of Nav1.8 with advanced prostate cancer tissues warrant further investigation into use of Nav1.8 as a potential biomarker to differentiate between early and advanced disease.
Voltage-gated sodium channel; Prostate cancer; Prostate biomarker; Gleason score