Differential regulation of Na(v)beta subunits during myogenesis

Voltage-gated sodium channels (Na_v) consist of a pore-forming α subunit (Na_vα) associated with β regulatory subunits (Na_vβ). Adult skeletal myocytes primarily express Na_v1.4 channels. We found, however, using neonatal L6E9 myocytes, that myofibers acquire a Na_v1.5-cardiac-like phenotype efficiently. Differentiated myotubes elicited faster Na_v1.5 currents than those recorded from myoblasts. Unlike myoblasts, I_Na recorded in myotubes exhibited an accumulation of inactivation after the application of trains of pulses, due to a slower recovery from inactivation. Since Na_vβ subunits modulate channel gating and pharmacology, the goal of the present work was to study Na_vβ subunits during myogenesis. All four Na_vβ (Na_vβ1-4) isoforms were present in L6E9 myocytes. While Na_vβ1-3 subunits were up-regulated by myogenesis, Na_vβ4 subunits were not. These results show that Na_vβ genes are strongly regulated during muscle differentiation and further support a physiological role for voltage-gated Na⁺ channels during development and myotube formation.     

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.     

Sialic acids attached to N- and O-glycans within the Nav1.4 D1S5–S6 linker contribute to channel gating

Background

Voltage-gated Na⁺ channels (Na_v) are responsible for the initiation and conduction of neuronal and muscle action potentials. Na_v gating can be altered by sialic acids attached to channel N-glycans, typically through isoform-specific electrostatic mechanisms.

Methods

Using two sets of Chinese Hamster Ovary cell lines with varying abilities to glycosylate glycoproteins, we show for the first time that sialic acids attached to O-glycans and N-glycans within the Na_v1.4 D1S5–S6 linker modulate Na_v gating.

Results

All measured steady-state and kinetic parameters were shifted to more depolarized potentials under conditions of essentially no sialylation. When sialylation of only N-glycans or of only O-glycans was prevented, the observed voltage-dependent parameter values were intermediate between those observed under full versus no sialylation. Immunoblot gel shift analyses support the biophysical data.

Conclusions

The data indicate that sialic acids attached to both N- and O-glycans residing within the Na_v1.4 D1S5-S6 linker modulate channel gating through electrostatic mechanisms, with the relative contribution of sialic acids attached to N- versus O-glycans on channel gating being similar.

General significance

Protein N- and O-glycosylation can modulate ion channel gating simultaneously. These data also suggest that environmental, metabolic, and/or congenital changes in glycosylation that impact sugar substrate levels, could lead, potentially, to changes in Na_v sialylation and gating that would modulate AP waveforms and conduction.     

Modulation of human Nav1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogs

Nine different voltage-gated sodium channel isoforms are responsible for inducing and propagating action potentials in the mammalian nervous system. The Na_v1.7 channel isoform plays an important role in conducting nociceptive signals. Specific mutations of this isoform may impair gating behavior of the channel resulting in several pain syndromes. In addition to channel mutations, similar or opposite changes in gating may be produced by spider and scorpion toxins binding to different parts of the voltage-gated sodium channel. In the present study, we analyzed the effects of the α-scorpion toxin OD1 and 2 synthetic toxin analogs on the gating properties of the Na_v1.7 sodium channel. All toxins potently inhibited channel inactivation, however, both toxin analogs showed substantially increased potency by more than one order of magnitude when compared with that of wild-type OD1. The decay phase of the whole-cell Na⁺ current was substantially slower in the presence of toxins than in their absence. Single-channel recordings in the presence of the toxins revealed that Na⁺ current inactivation slowed due to prolonged flickering of the channel between open and closed states. Our findings support the voltage-sensor trapping model of α-scorpion toxin action, in which the toxin prevents a conformational change in the domain IV voltage sensor that normally leads to fast channel inactivation.     

Structural modeling of human cardiac sodium channel pore domain

The pore domain of human voltage-dependent cardiac sodium channel Na_v1.5 (hNa_v1.5) is the crucial binding targets for anti-arrhythmics drugs and some local anesthetic drugs but its three-dimensional structure is still lacking. This has affected the detailed studies of the binding features and mechanism of these drugs. In this paper, we present a structural model for open-state pore domain of hNa_v1.5 built using single template ROSETTA-membrane homology modeling with the crystal structure of Na_vMs. The assembled structural models are evaluated by rosettaMP energy and locations of binding sites. The modeled structures of the pore domain of hNa_v1.5 in open state will be helpful to explore molecular mechanism of a state-dependent drug binding and help designing new drugs.     

Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na+ channels

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na⁺ channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na⁺ current (I_Na). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in I_Na. Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4 min), there is little effect on I_Na. However, with longer exposures (4–8 min), there is a large decrease in I_Na, but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) — MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na⁺ channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.     

Selectivity signatures of three isoforms of recombinant T-type Ca channels

Voltage-gated Ca²⁺ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca²⁺ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca²⁺ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca²⁺-channel α1 subunits, Ca_v3.1, Ca_v3.2 and Ca_v3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca_v3.1, Ca_v3.2, and Ca_v3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca_v3 channels select Ca²⁺ over Na⁺ by affinity. Ca_v3.1 and Ca_v3.2 discriminate Ca²⁺, Sr²⁺ and Ba²⁺ based on the ion's effects on the open channel probability, whilst Ca_v3.3 discriminates based on the ion's intrapore binding affinity. All Ca_v3s were characterized by much smaller difference in the K_D values for Na⁺ current blockade by Ca²⁺ (K_D1 ∼ 6 μM) and for Ca²⁺ current saturation (K_D2 ∼ 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na⁺/Ca²⁺ current at close to physiological Ca²⁺ concentrations, which was the strongest for Ca_v3.3, smaller for Ca_v3.2 and the smallest for Ca_v3.1. In addition to intrapore Ca²⁺ binding site(s) Ca_v3.2, but not Ca_v3.1 and Ca_v3.3, is likely to possess an extracellular Ca²⁺ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca²⁺ current in native cells.     

Molecular dynamics simulations on the interaction of the transmembrane NavAb channel with cholesterol and lipids in the membrane

Increased cholesterol levels are associated with multiple pathological conditions. In this work, molecular dynamics simulations were applied to observe the influence of membrane cholesterol levels on a voltage-gated sodium channel. Different lipid compositions are modeled around the channel to obtain information about the possible effects by which cholesterol influences Na_vAb channels. Cholesterol was normally not directly interacting with either the closed or inactivated conformation. Cholesterol increased lipid packing implying that it plays a crucial role in restricting lipid movement in the region around 1 nm of the channel in a 1-palmitoyl-2-oeleoyl phosphatidylcholine matrix. Our results provide the first computational indication of an indirect modulation of Na_vAb channels by membrane cholesterol.     

Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Na_v1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Na_v1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Na_v1.4. Here, we determine the NMR structure of a selectively ¹³C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By ¹³C NMR, we obtain NMR structural constraints as ¹³C chemical shifts and the ¹H-²H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the ¹H-²H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel Na_vRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Na_v1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.     

Block of Tetrodotoxin-sensitive,Nav1.7, and Tetrodotoxin-resistant,Nav1.8, Na+ channels by Ranolazine

Evidence supports a role for the tetrodotoxin-sensitive Nav1.7 and the tetrodotoxin-resistant Nav1.8 in the pathogenesis of pain. Ranolazine, an anti-ischemic drug, has been shown to block cardiac (Nav1.5) late sodium current (I_Na). In this study, whole-cell patch-clamp techniques were used to determine the effects of ranolazine on human Nav1.7 (hNav1.7+β1 subunits) and rat Nav1.8 (rNav1.8) channels expressed in HEK293 and ND7-23 cells, respectively. Ranolazine reduced hNav1.7 and rNav1.8 I_Na with IC₅₀ values of 10.3 and 21.5 μM (holding potential=-120 or -100 mV, respectively). The potency of I_Na block by ranolazine increased to 3.2 and 4.3 μM when 5-sec depolarizing prepulses to -70 (hNav1.7) and -40 (rNav1.8) mV were applied. Ranolazine caused a preferential hyperpolarizing shift of the steady-state fast, intermediate and slow inactivation of hNav1.7 and and intermediate and slow inactivation of rNav1.8, suggesting preferential interaction of the drug with the inactivated states of both channels. Ranolazine (30 μM) caused a use-dependent block (10-msec pulses at 1, 2 and 5 Hz) of hNav1.7 and rNav1.8 I_Na and significantly accelerated the onset of, and slowed the recovery from inactivation, of both channels. An increase of depolarizing pulse duration from 3 to 200 msec did not affect the use-dependent block of I_Na by 100 μM ranolazine. Taken together, the data suggest that ranolazine blocks the open state and may interact with the inactivated states of Nav1.7 and Nav1.8 channels. The state-and use-dependent modulation of hNav1.7 and rNav1.8 Na⁺ channels by ranolazine could lead to an increased effect of the drug at high firing frequencies, as in injured neurons.     

Block of Persistent Late Na+ Currents by Antidepressant Sertraline and Paroxetine

Antidepressants, such as traditional tricyclic antidepressants (TCAs), are the first-line treatment for various pain syndromes. Available evidence indicates that TCAs may target Na⁺ channels for their analgesic action. In this report, we examined the effects of contemporary antidepressants sertraline and paroxetine on (1) neuronal Na⁺ channels expressed in GH₃ cells and (2) muscle rNav1.4 Na⁺ channels heterologously expressed in Hek293t cells. Our results showed that both antidepressants blocked Na⁺ channels in a highly state-dependent manner. The 50% inhibitory concentrations (IC₅₀) for sertraline and paroxetine ranged ∼18–28 μm for resting block and ∼2–8 μm for inactivated block of neuronal and rNav1.4 Na⁺ channels. Surprisingly, the IC₅₀ values for both drugs were about 0.6–0.7 μm for the open channel block of persistent late Na⁺ currents generated through inactivation-deficient rNav1.4 mutant Na⁺ channels. For comparison, the open channel block in neuronal hNav1.7 counterparts yielded IC₅₀ values around 0.3–0.4 μm for both drugs. Receptor mapping using fast inactivation-deficient rNav1.4-F1579A/K mutants with reduced affinities toward local anesthetics (LAs) and TCAs indicated that the F1579 residue is not involved in the binding of sertraline and paroxetine. Thus, sertraline and paroxetine are potent open channel blockers that target persistent late Na⁺ currents preferentially, but their block is not mediated via the phenylalanine residue at the known LA/TCA receptor site.     

Heterologous expression and purification of neurotoxic Hainantoxin-III in E. coli

In the present study, we used Escherichia coli to produce recombinant Hainantoxin-III (rHNTX-III), a 33-amino acid peptic toxin from the tarantula spider Haplopelma hainanum. The toxin has three pairs of disulfide bonds. A pET-HS-HNTX-III vector was constructed and transformed into the E. coli strain SHuffle^TM. rHNTX-III was expressed using auto-induction medium. After using a Ni–NTA column, the expressed fusion protein was digested using SUMO protease (ULP1) to remove the HIS–SUMO tag, and then RP–HPLC and ultrafiltration were used for further purification. Then the rHNTX-III was identified by MALDI–TOF/TOF mass spectrometry. The purified rHNTX-III was further analyzed using a whole-cell patch-clamp assay. It was shown that the rHNTX-III was able to block currents generated by human Na_v1.7 (hNa_v1.7) at an IC₅₀ of 225?nM and also have high selectivity for different voltage-gated sodium channels. Therefore, it has very similar activity to the natural one.     

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

Ca_v3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca_v3.2 and modulation by redox agents. His¹⁹¹ is found on the extracellular face of the Ca_v3.2 channel on the IS3-S4 linker and is not conserved in other Ca_v3 channels. Mutation of the corresponding residue in Ca_v3.1 to histidine, Gln¹⁷², significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca_v3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3–S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.     

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms

Voltage-gated sodium channels, Na_Vs, are responsible for the rapid rise of action potentials in excitable tissues. Na_V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-Na_V nanobodies (Nbs), Nb17 and Nb82, that recognize the Na_V1.4 (skeletal muscle) and Na_V1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na_V1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na_V isoform has been challenging because they usually show undesired crossreactivity for different Na_V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa_V1.4 or CTNa_V1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa_V1.4 and CTNa_V1.5 with high affinity (K_D ∼ 40–60 nM). In addition, as proof of concept, we show that Nb82 could detect Na_V1.4 and Na_V1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na_V1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na_V reagents to capture Na_Vs from cell lysates and as molecular visualization agents for Na_Vs.     

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.     

Potency optimization of Huwentoxin-IV on hNav1.7: A neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider Selenocosmia huwena

The spider venom peptide Huwentoxin-IV (HwTx-IV) 1 is a potent antagonist of hNa_v1.7 (IC₅₀ determined herein as 17 ± 2 nM). Na_v1.7 is a voltage-gated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. We prepared a number of HwTx-IV analogs as part of a structure–function study into Na_v1.7 antagonism. The inhibitory potency of these analogs was determined by automated electrophysiology and is reported herein. In particular, the native residues Glu¹, Glu⁴, Phe⁶ and Tyr³³ were revealed as important activity modulators and several peptides bearing mutations in these positions showed significantly increased potency on hNa_v1.7 while maintaining the original selectivity profile of the wild-type peptide 1 on hNa_v1.5. Peptide 47 (Gly¹, Gly⁴, Trp³³-HwTx) demonstrated the largest potency increase on hNa_v1.7 (IC₅₀ 0.4 ± 0.1 nM).     

Effects of Calmodulin and Ca<Superscript>2+</Superscript> Channel Blockers on ω-conotoxin GVI A Binding to Crude Membranes from α<Subscript>1B</Subscript> Subunit (Ca<Subscript>v</Subscript>2.2) Expressed BHK Cells and Mice Brain Lacking the α<Subscript>1B</Subscript> Subunits

Characteristics for the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to crude membranes from BHKN101 cells expressing the α_1B subunits of Ca_v2.2 channels and from mice brain lacking the α_1B subunits of Ca_v2.2 channels, particularly, the effects of CaM and various Ca²⁺ channel blockers on these specific bindings were investigated. Specific binding of ¹²⁵I-ω-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. ω-CTX GVIA, ω-CTX MVIIC and ω-CTX SVIB inhibited the specific binding of ¹²⁵I-ω-CTX GVIA to crude membranes from BHKN101 cells, and the IC₅₀ values for ω-CTXGVIA, ω-CTX MVIIC and ω-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, ω-agatoxin IVA and calciseptine at concentrations of 10⁻⁹–10⁻⁶ M did not inhibit specific binding. Specific binding was also about 80% inhibited by 20 μg protein/ml CaM. The amount of ¹²⁵I-ω-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking α_1B subunits of Ca_v2.2 channels was about 30% of that to the crude membranes from brain of wild-type. On the other hand, specific binding of ¹²⁵I-ω-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of ¹²⁵I-ω-CTX MVIIC (200 pM) was not inhibited by ω-CTX GVIA and ω-CTX SVIB, and also ω-Aga IVA and calciseptine at concentrations of 10⁻⁹–10⁻⁷ M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled ω-CTX MVIIC (IC₅₀ value was about 0.1 nM). 20 μg protein/ml CaM did not inhibit specific binding. Therefore, these results suggest that BHKN101 cells have a typical Ca_v2.2 channels which are also inhibited by CaM and have not specific binding sites for ω-CTX MVIIC, although ω-CTX MVIIC is a blocker for both Ca_v2.1 (α_1A; P/Q-type) and Ca_v2.2 channels.     

PKC–NF-κB are involved in CCL2-induced Nav1.8 expression and channel function in dorsal root ganglion neurons

CCL2 [chemokine (C–C motif) ligand 2] contributes to the inflammation-induced neuropathic pain through activating VGSC (voltage-gated sodium channel)-mediated nerve impulse conduction, but the underlying mechanism is currently unknown. Our study aimed to investigate whether PKC (protein kinase C)–NF-κB (nuclear factor κB) is involved in CCL2-induced regulation of voltage-gated sodium Nav1.8 currents and expression. DRG (dorsal root ganglion) neurons were prepared from adult male Sprague–Dawley rats and incubated with various concentration of CCL2 for 24 h. Whole-cell patch-clamps were performed to record the Nav1.8 currents in response to the induction by CCL2. After being pretreated with 5 and10 nM CCL2 for 16 h, CCR2 [chemokine (C–C motif) receptor 2] and Nav1.8 expression significantly increased and the peak currents of Na_v1.8 elevated from the baseline 46.53±4.53 pA/pF to 64.28±3.12 pA/pF following 10 nM CCL2 (P<0.05). Compared with the control, significant change in Na_v1.8 current density was observed when the CCR2 inhibitor INCB3344 (10 nM) was applied. Furthermore, inhibition of PKC by AEB071 significantly eliminated CCL2-induced elevated Nav1.8 currents. In vitro PKC kinase assays and autoradiograms suggested that Nav1.8 within DRG neurons was a substrate of PKC and direct phosphorylation of the Na_v1.8 channel by PKC regulates its function in these neurons. Moreover, p65 expression was significantly higher in CCL2-induced neurons (P<0.05), and was reversed by treatment with INCB3344 and AEB071. PKC–NF-κB are involved in CCL2-induced elevation of Na_v1.8 current density by promoting the phosphorylation of Na_v1.8 and its expression.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.