Functional Expression of Rat Nav1.6 Voltage-Gated Sodium Channels in HEK293 Cells: Modulation by the Auxiliary β1 Subunit

The Na_v1.6 voltage-gated sodium channel α subunit isoform is abundantly expressed in the adult rat brain. To assess the functional modulation of Na_v1.6 channels by the auxiliary β1 subunit we expressed the rat Na_v1.6 sodium channel α subunit by stable transformation in HEK293 cells either alone or in combination with the rat β1 subunit and assessed the properties of the reconstituted channels by recording sodium currents using the whole-cell patch clamp technique. Coexpression with the β1 subunit accelerated the inactivation of sodium currents and shifted the voltage dependence of channel activation and steady-state fast inactivation by approximately 5–7 mV in the direction of depolarization. By contrast the β1 subunit had no effect on the stability of sodium currents following repeated depolarizations at high frequencies. Our results define modulatory effects of the β1 subunit on the properties of rat Na_v1.6-mediated sodium currents reconstituted in HEK293 cells that differ from effects measured previously in the Xenopus oocyte expression system. We also identify differences in the kinetic and gating properties of the rat Na_v1.6 channel expressed in the absence of the β1 subunit compared to the properties of the orthologous mouse and human channels expressed in this system.     

Dexamethasone effects on Na<Subscript>v</Subscript>1.6 in tooth pulp,dental nerves,and alveolar osteoclasts of adult rats

Dexamethasone causes extensive physiologic reactions including the reduction of inflammation and pain. Here, we asked whether it also affected dental or periodontal cells or dental innervation by altering voltage-gated sodium channel Na_v1.6 immunoreactivity (IR) or neural synaptophysin. Daily dexamethasone (0.2 mg/kg) given for 1 week to rats caused 12-fold increased intensity of Na_v1.6-IR in dendritic pulpal cells of normal molars and incisors compared with vehicle treatment. These cells also co-localized monocyte (ED-1) or dendritic cell (CD11b/Ox42) markers, and their location in molars expanded during dexamethasone treatment to include deeper pulp. Furthermore, dexamethasone caused a 10-fold decrease in the number of Na_v1.6-immunoreactive multinucleate osteoclasts along the alveolar bone of molar root sockets. No changes occurred for neural Na_v1.6 at axonal nodes of Ranvier, even though IR for calcitonin gene-related peptide was greatly decreased, as expected, and neural synaptophysin-IR was decreased 59% by dexamethasone. At 4 days after tooth injury, pulpal vasodilation and increased Na_v1.6-immunoreactive pulp cells were similar for all groups. Thus, dexamethasone changes dental pulp cell and alveolar osteoclast Na_v1.6-IR in normal teeth, but different mechanisms occur after tooth injury when tissue reactions were similar for dexamethasone- and vehicle-treated rats. Steroid-induced alterations of dental pain and inflammation coincide with altered exocytic capability in dental nerve fibers as shown by synaptophysin-IR and with altered pulp cell Na_v1.6-IR and osteoclast number, but not with any changes in Na_v1.6-IR for nodes of Ranvier in myelinated dental axons.     

Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary β1 and β2 subunits

The Na_v1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Na_v1.6 exist predominantly as Na_v1.6 + β1 + β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 subunits on the gating and kinetic properties of rat Na_v1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The β1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The β2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The β1 and β2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Na_v1.6 sodium channels. These results identify unique effects of the β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.     

Localization of receptor site on insect sodium channel for depressant β-toxin BmK IT2

Background

BmK IT2 is regarded as a receptor site-4 modulator of sodium channels with depressant insect toxicity. It also displays anti-nociceptive and anti-convulsant activities in rat models. In this study, the potency and efficacy of BmK IT2 were for the first time assessed and compared among four sodium channel isoforms expressed in Xenopus oocytes. Combined with molecular approach, the receptor site of BmK IT2 was further localized.

Principal Findings

2 µM BmK IT2 strongly shifted the activation of DmNa_v1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNa_v1.2, rNa_v1.3 and mNa_v1.6, three mammalian central neuronal sodium channel subtypes. (1) Mutations of Glu₈₉₆, Leu₈₉₉, Gly₉₀₄ in extracellular loop Domain II S3–S4 of DmNa_v1 abolished the functional action of BmK IT2. (2) BmK IT2-preference for DmNa_v1 could be conferred by Domain III. Analysis of subsequent DmNa_v1 mutants highlighted the residues in Domain III pore loop, esp. Ile₁₅₂₉ was critical for recognition and binding of BmK IT2.

Conclusions/Significance

In this study, BmK IT2 displayed total insect-selectivity. Two binding regions, comprising domains II and III of DmNa_v1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Na_v1.2, Na_v1.3 and Na_v1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.     

Preferential Targeting of Nav1.6 Voltage-Gated Na+ Channels to the Axon Initial Segment during Development

During axonal maturation, voltage-gated sodium (Na_v) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Na_v reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Na_v1.6 construct containing an extracellular biotinylation domain we demonstrate that Na_v1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Na_v1.6 channel lacking the ankyrin-binding motif nor a chimeric K_v2.1 channel containing the Na_v ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Na_v1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS.     

Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation

Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3′-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.     

The Mechanism of Na+/K+ Selectivity in Mammalian Voltage-Gated Sodium Channels Based on Molecular Dynamics Simulation

Voltage-gated sodium (Na_v) channels and their Na⁺/K⁺ selectivity are of great importance in the mammalian neuronal signaling. According to mutational analysis, the Na⁺/K⁺ selectivity in mammalian Na_v channels is mainly determined by the Lys and Asp/Glu residues located at the constriction site within the selectivity filter. Despite successful molecular dynamics simulations conducted on the prokaryotic Na_v channels, the lack of Lys at the constriction site of prokaryotic Na_v channels limits how much can be learned about the Na⁺/K⁺ selectivity in mammalian Na_v channels. In this work, we modeled the mammalian Na_v channel by mutating the key residues at the constriction site in a prokaryotic Na_v channel (Na_vRh) to its mammalian counterpart. By simulating the mutant structure, we found that the Na⁺ preference in mammalian Na_v channels is collaboratively achieved by the deselection from Lys and the selection from Asp/Glu within the constriction site.     

Two Nedd4-binding Motifs Underlie Modulation of Sodium Channel Nav1.6 by p38 MAPK

Sodium channel Na_v1.6 is essential for neuronal excitability in central and peripheral nervous systems. Loss-of-function mutations in Na_v1.6 underlie motor disorders, with homozygous-null mutations causing juvenile lethality. Phosphorylation of Na_v1.6 by the stress-induced p38 MAPK at a Pro-Gly-Ser⁵⁵³-Pro motif in its intracellular loop L1 reduces Na_v1.6 current density in a dorsal root ganglion-derived cell line, without changing its gating properties. Phosphorylated Pro-Gly-Ser⁵⁵³-Pro motif is a putative binding site to Nedd4 ubiquitin ligases, and we hypothesized that Nedd4-like ubiquitin ligases may contribute to channel ubiquitination and internalization. We report here that p38 activation in hippocampal neurons from wild-type mice, but not from Scn8a^medtg mice that lack Na_v1.6, reduces tetrodotoxin-S sodium currents, suggesting isoform-specific modulation of Na_v1.6 by p38 in these neurons. Pharmacological block of endocytosis completely abolishes p38-mediated Na_v1.6 current reduction, supporting our hypothesis that channel internalization underlies current reduction. We also report that the ubiquitin ligase Nedd4-2 interacts with Na_v1.6 via a Pro-Ser-Tyr¹⁹⁴⁵ motif in the C terminus of the channel and reduces Na_v1.6 current density, and we show that this regulation requires both the Pro-Gly-Ser-Pro motif in L1 and the Pro-Ser-Tyr motif in the C terminus. Similarly, both motifs are necessary for p38-mediated reduction of Na_v1.6 current, whereas abrogating binding of the ubiquitin ligase Nedd4-2 to the Pro-Ser-Tyr motif results in stress-mediated increase in Na_v1.6 current density. Thus, phosphorylation of the Pro-Gly-Ser-Pro motif within L1 of Na_v1.6 is necessary for stress-induced current modulation, with positive or negative regulation depending upon the availability of the C-terminal Pro-Ser-Tyr motif to bind Nedd4-2.     

Tetrodotoxin‐resistant voltage‐gated sodium channel Nav1.8 constitutively interacts with ankyrin G

The tetrodotoxin‐resistant (TTX‐R) voltage‐gated sodium channel Na_v1.8 is predominantly expressed in peripheral afferent neurons, but in case of neuronal injury an ectopic and detrimental expression of Na_v1.8 occurs in neurons of the CNS. In CNS neurons, Na_v1.2 and Na_v1.6 channels accumulate at the axon initial segment, the site of the generation of the action potential, through a direct interaction with the scaffolding protein ankyrin G (ankG). This interaction is regulated by protein kinase CK2 phosphorylation. In this study, we quantitatively analyzed the interaction between Na_v1.8 and ankG. GST pull‐down assay and surface plasmon resonance technology revealed that Na_v1.8 strongly and constitutively interacts with ankG, in comparison to what observed for Na_v1.2. An ion channel bearing the ankyrin‐binding motif of Na_v1.8 displaced the endogenous Na_v1 accumulation at the axon initial segment of hippocampal neurons. Finally, Na_v1.8 and ankG co‐localized in skin nerves fibers. Altogether, these results indicate that Na_v1.8 carries all the information required for its localization at ankG micro‐domains. The constitutive binding of Na_v1.8 with ankG could contribute to the pathological aspects of illnesses where Na_v1.8 is ectopically expressed in CNS neurons.

     

Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

NaChBac is a bacterial voltage-gated sodium (Na_v) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Na_v channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of Na_vRh, a marine bacterial NaChBac ortholog. Two Na⁺ binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na⁺ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca²⁺ ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na⁺ and Ca²⁺ to the vestibule of the SF. In addition, Na⁺ permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Na_v orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na⁺ over Ca²⁺ in mammalian Na_v channels.     

Role of Calcium and Calpain in the Downregulation of Voltage‐Gated Sodium Channel Expression by the Pyrethroid Pesticide Deltamethrin

Voltage‐gated sodium channels (Na_v) are essential for initiation and propagation of action potentials. Previous in vitro studies reported that exposure to the Na_v toxins veratridine and α scorpion toxin cause persistent downregulation of Na_v mRNA in vitro. However the mechanism of this downregulation is not well characterized. Here, we report that the type‐II pyrethroid deltamethrin, which has a similar mechanism as these toxins, elicited an approximate 25% reduction in Na_v 1.2 and Na_v 1.3 mRNA in SK‐N‐AS cells. Deltamethrin‐induced decreases of Na_v mRNA were blocked with the Na_v antagonist tetrodotoxin, demonstrating a primary role for interaction with Na_v. Pre–treatment with the intracellular calcium chelator BAPTA‐AM and the calpain inhibitor PD‐150606 also prevented these decreases, identifying a role for intracellular calcium and calpain activation. Because alterations in Na_v expression and function can result in neurotoxicity, additional studies are warranted to determine whether or not such effects occur in vivo.     

Being there: cellular targeting of voltage-gated sodium channels in the heart

Voltage-gated sodium (Na_v) channels in cardiomyocytes are localized in specialized membrane domains that optimize their functions in propagating action potentials across cell junctions and in stimulating voltage-gated calcium channels located in T tubules. Mutation of the ankyrin-binding site of Na_v1.5, the principal Na_v channel in the heart, was previously known to cause cardiac arrhythmia and the retention of Na_v1.5 in an intracellular compartment in cardiomyocytes. Conclusive evidence is now provided that direct interaction between Na_v1.5 and ankyrin-G is necessary for the expression of Na_v1.5 at the cardiomyocyte cell surface.     

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels Na<Subscript>V</Subscript>1.1, Na<Subscript>V</Subscript>1.4 and Na<Subscript>V</Subscript>1.7

Human voltage-gated sodium (Na_V) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane β-subunits. Na_V pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all Na_V isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of Na_V1.2, Na_V1.5, and Na_V1.6 showed that CaM binding was mediated by the C-domain of CaM (CaM_C), while the N-domain (CaM_N) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other Na_V isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of Na_V1.1, Na_V1.4, and Na_V1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaM_C; resonances of CaM_N were identical to free CaM. Thus, CaM_C residues contribute to the interface with the IQ motif, while CaM_N is available to interact elsewhere on the channel.     

Mutant SOD1 protein increases Na<Subscript>v</Subscript>1.3 channel excitability

Amyotrophic lateral sclerosis (ALS) is a lethal paralytic disease caused by the degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) are present in ~20% of familial ALS and ~2% of all ALS cases. The most common SOD1 gene mutation in North America is a missense mutation substituting valine for alanine (A4V). In this study, we analyze sodium channel currents in oocytes expressing either wild-type or mutant (A4V) SOD1 protein. We demonstrate that the A4V mutation confers a propensity to hyperexcitability on a voltage-dependent sodium channel (Na_v1.3) mediated by heightened total Na⁺ conductance and a hyperpolarizing shift in the voltage dependence of Na_v1.3 activation. To estimate the impact of these channel effects on excitability in an intact neuron, we simulated these changes in the program NEURON; this shows that the changes induced by mutant SOD1 increase the spontaneous firing frequency of the simulated neuron. These findings are consistent with the view that excessive excitability of neurons is one component in the pathogenesis of this disease.     

Re‐engineering the μ‐conotoxin SIIIA scaffold

Voltage‐gated sodium (Na_v) channels are responsible for generation and propagation of action potentials throughout the nervous system. Their malfunction causes several disorders and chronic conditions including neuropathic pain. Potent subtype specific ligands are essential for deciphering the molecular mechanisms of Na_v channel function and development of effective therapeutics. µ‐Conotoxin SIIIA is a potent mammalian Na_v1.2 channel blocker that exhibits analgesic activity in rodents. We undertook to reengineer loop 1 through a strategy involving charge alterations and truncations which led to the development of µ‐SIIIA mimetics with novel selectivity profiles. A novel [N5K/D15A]SIIIA(3–20) mutant with enhanced net positive charge showed a dramatic increase in its Na_v1.2 potency (IC₅₀ of 0.5 nM vs. 9.6 nM for native SIIIA) though further truncations led to loss of potency. Unexpectedly, it appears that SIIIA loop 1 significantly influences its Na_v channel interactions despite loop 2 and 3 residues constituting the pharmacophore. This minimal functional conotoxin scaffold may allow further development of selective Na_V blockers. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 347–354, 2014.     

Understanding LTP in pain pathways

Background

Small neurons of the dorsal root ganglion (DRG) express five of the nine known voltage-gated sodium channels. Each channel has unique biophysical characteristics which determine how it contributes to the generation of action potentials (AP). To better understand how AP amplitude is maintained in nociceptive DRG neurons and their centrally projecting axons, which are subjected to depolarization within the dorsal horn, we investigated the dependence of AP amplitude on membrane potential, and how that dependence is altered by the presence or absence of sodium channel Na_v1.8.

Results

In small neurons cultured from wild type (WT) adult mouse DRG, AP amplitude decreases as the membrane potential is depolarized from -90 mV to -30 mV. The decrease in amplitude is best fit by two Boltzmann equations, having V_1/2 values of -73 and -37 mV. These values are similar to the V_1/2 values for steady-state fast inactivation of tetrodotoxin-sensitive (TTX-s) sodium channels, and the tetrodotoxin-resistant (TTX-r) Na_v1.8 sodium channel, respectively. Addition of TTX eliminates the more hyperpolarized V_1/2 component and leads to increasing AP amplitude for holding potentials of -90 to -60 mV. This increase is substantially reduced by the addition of potassium channel blockers. In neurons from Na_v1.8(-/-) mice, the voltage-dependent decrease in AP amplitude is characterized by a single Boltzmann equation with a V_1/2 value of -55 mV, suggesting a shift in the steady-state fast inactivation properties of TTX-s sodium channels. Transfection of Na_v1.8(-/-) DRG neurons with DNA encoding Na_v1.8 results in a membrane potential-dependent decrease in AP amplitude that recapitulates WT properties.

Conclusion

We conclude that the presence of Na_v1.8 allows AP amplitude to be maintained in DRG neurons and their centrally projecting axons even when depolarized within the dorsal horn.     

Sodium Channel Carboxyl-terminal Residue Regulates Fast Inactivation

The Na_v1.2 and Na_v1.3 voltage-gated sodium channel isoforms demonstrate distinct differences in their kinetics and voltage dependence of fast inactivation when expressed in Xenopus oocytes. Co-expression of the auxiliary β1 subunit accelerated inactivation of both the Na_v1.2 and Na_v1.3 isoforms, but it did not eliminate the differences, demonstrating that this property is inherent in the α subunit. By constructing chimeric channels between Na_v1.2 and Na_v1.3, we demonstrate that the carboxyl terminus is responsible for the differences. The Na_v1.2 carboxyl terminus caused faster inactivation in the Na_v1.3 backbone, and the Na_v1.3 carboxyl terminus caused slower inactivation in the Na_v1.2 channel. Through analysis of truncated channels, we identified a homologous 60-amino acid region within the carboxyl terminus of the Na_v1.2 and the Na_v1.3 channels that is responsible for this modulation of fast inactivation. Site-directed replacement of Na_v1.3 lysine 1826 in this region to its Na_v1.2 analogue glutamic acid 1880 (K1826E) shifted the voltage dependence of inactivation toward that of Na_v1.2. The K1826E mutation also accelerated the inactivation kinetics to a level comparable with that of Na_v1.2. The reverse Na_v1.2 E1880K mutation exhibited much slower inactivation kinetics and depolarized inactivation voltage dependence. A complementary mutation located within the inactivation linker of Na_v1.3 (K1453E) caused inactivation changes mirroring those caused by the K1826E mutation in Na_v1.3. Therefore, we have identified a homologous carboxyl-terminal residue that regulates the kinetics and voltage dependence of fast inactivation in sodium channels, possibly via a charge-dependent interaction with the inactivation linker.     

Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly

The vertebrate sodium (Na_v) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Na_v channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Na_v 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Na_v channel cross-linking and provide new structural insights into some pathological Na_v channel mutations.