Prostaglandin E2 Promotes Nav1.8 Trafficking via Its Intracellular RRR Motif Through the Protein Kinase A Pathway

Voltage‐gated sodium channels (Na_v) are essential for the initiation and propagation of action potentials in neurons. Na_v1.8 activity is regulated by prostaglandin E₂ (PGE₂). There is, however, no direct evidence showing the regulated trafficking of Na_v1.8, and the molecular and cellular mechanism of PGE₂‐induced sodium channel trafficking is not clear. Here, we report that PGE₂ regulates the trafficking of Na_v1.8 through the protein kinase A (PKA) signaling pathway, and an RRR motif in the first intracellular loop of Na_v1.8 mediates this effect. In rat dorsal root ganglion (DRG) neurons, prolonged PGE₂ treatment enhanced Na_v1.8 currents by increasing the channel density on the cell surface. Activation of PKA by forskolin had the same effect on DRG neurons and human embryonic kidney 293T cells expressing Na_v1.8. Inhibition of PKA completely blocked the PGE₂‐promoted effect on Na_v1.8. Mutation of five PKA phosphorylation sites or the RRR motif in the first intracellular loop of Na_v1.8 abolished the PKA‐promoted Na_v1.8 surface expression. Furthermore, a membrane‐tethered peptide containing the intracellular RRR motif disrupted the PGE₂‐induced promotion of the Na_v1.8 current in DRG neurons. Our data indicate that PGE₂ promotes the surface expression of Na_v1.8 via an intracellular RRR motif, and provide a novel mechanism for functional modulation of Na_v1.8 by hyperalgesic agents.     

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.     

Melatonin protects rat cerebellar granule cells against electromagnetic field‐induced increases in Na+ currents through intracellular Ca2+ release

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low‐frequency electromagnetic field (ELF‐EMF)‐induced Na_v activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF‐EMF for 60 min. significantly increased the Na_v current (I_Na) densities by 62.5%. MT (5 μM) inhibited the ELF‐EMF‐induced I_Na increase. This inhibitory effect of MT is mimicked by an MT₂ receptor agonist and was eliminated by an MT₂ receptor antagonist. The Na_v channel steady‐state activation curve was significantly shifted towards hyperpolarization by ELF‐EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF‐EMF. ELF‐EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK‐7 did not reduce the ELF‐EMF‐induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF‐EMF‐induced Na_v activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca²⁺ level, but it significantly elevated the intracellular Ca²⁺ level evoked by the high K⁺ stimulation in cerebellar GC that were either exposed or not exposed to ELF‐EMF. In the presence of ruthenium red, a ryanodine‐sensitive receptor blocker, the MT‐induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal I_Na that result from ELF‐EMF exposure through Ca²⁺ influx‐induced Ca²⁺ release.     

Two recombinant α-like scorpion toxins from Mesobuthus eupeus with differential affinity toward insect and mammalian Na channels

α-Scorpion toxins are modulators of voltage-gated Na⁺ channels (Na_vs), which bind to the receptor site 3 to inhibit the fast inactivation of the channels. MeuNaTxα-12 and MeuNaTxα-13 are two new α-scorpion toxin-like peptides identified by cDNA cloning from the scorpion Mesobuthus eupeus with unknown functions. Here, we report their recombinant production, oxidative refolding, structural and functional features. By in vitro renaturation from bacterial inclusion bodies and further purification through reverse phase high-performance liquid chromatography, we obtained high purity recombinant products with a native-like conformation identified by circular dichroism analysis. Two-electrode voltage clamp recordings on five cloned mammalian Na_v subtypes (rNa_v1.1, rNa_v1.2, rNa_v1.4, rNa_v1.5, and mNa_v1.6) and the insect counterpart DmNa_v1, all expressed in Xenopus laevis oocytes, showed that these two peptides inhibited rapid inactivation of the sensitive Na⁺ channels with significant preference for DmNa_v1. The half maximal effective concentrations (EC₅₀) of MeuNaTxα-12 and MeuNaTxα-13 for this channel are 19.95 ± 2.99 nM and 65.50 ± 7.28 nM, respectively, showing 45 and 38 folds higher affinities than for rNa_v1.1, the most sensitive mammalian channel among the five isoforms. Our functional data confirms that these two peptides belong to the α-like scorpion toxin group. A combined analysis of the site 3 sequences and the pharmacological data illuminates the importance of the loop L_D4:S5–S6 of the channel in interacting with the toxins whereas affinity variations between MeuNaTxα-12 and MeuNaTxα-13 highlight a key functional role of a cationic side chain at position 28 of MeuNaTxα-12. Successful expression together with structural and functional characterization of these two new α-like scorpion toxins lays basis for further studies of their structure–function relationship.     

Glutamate‐induced changes in the pattern of hippocampal dendrite outgrowth: A role for calcium‐dependent pathways and the microtubule cytoskeleton

Glutamate regulation of a variety of aspects of dendrite development may be involved in neuronal plasticity and neuropathology. In this study, we examine the calcium‐dependent pathways and alterations in the microtubule (MT) cytoskeleton that may mediate glutamate‐induced changes in the pattern of dendrite outgrowth. We used Fura‐2 AM and inhibitors of the calcium‐dependent proteins, calmodulin and calpain, to identify the role of specific calcium‐dependent pathways in glutamate‐regulated dendrite outgrowth. Additionally, we used a quantitative fluorescence technique to correlate changes in MT levels with glutamate‐induced changes in dendrite outgrowth. We show that the intracellular calcium concentration ([Ca²⁺ ]_i) changes in a biphasic manner over a 12‐h period in the presence of glutamate. A transient increase in [Ca²⁺ ]_i over the first hour of glutamate exposure correlated with a calmodulin‐associated increase in the rate of dendrite outgrowth, whereas a sustained increase in [Ca²⁺ ]_i was correlated with calpain‐associated dendrite retraction. Quantitative fluorescence measurements showed no net change in the level of MTs during calmodulin‐associated increases in dendrite outgrowth, but showed a significant decline in the level of MTs during calpain‐associated dendrite retraction. These findings provide insights into the intracellular mechanisms involved in activity‐dependent regulation of dendrite morphology during development and after pathology. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 159–172, 2000     

Modular Organization of α-Toxins from Scorpion Venom Mirrors Domain Structure of Their Targets,Sodium Channels

To gain success in the evolutionary “arms race,” venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Na_vs) and thereby modify the excitability of muscle and nerve cells. Although more than 100 α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Na_vs is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that despite the small size and relatively rigid structure, these toxins possess modular organization from structural, functional, and evolutionary perspectives. The more conserved and rigid “core module” is supplemented with the “specificity module” (SM) that is comparatively flexible and variable and determines the taxon (mammal versus insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of the extracellular loops of Na_vs suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Na_vs. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Na_vs.     

Arthropod toxins inhibiting Ca2+ and Na+ channels prevent AC‐1001 H3 peptide‐induced apoptosis

Peptide toxins of arthropods are one of the potential sources of bioactive substances. Toxins are able to bind to calcium channels and block them. Ca²⁺ ions play an important role in many cell processes, in particular, in apoptosis. In this work, we study the effect of some arthropod toxins on intracellular processes associated with the induction of apoptosis. Synthetic analogs of U₅‐scytotoxin‐Sth1a, ω‐hexatoxin‐Hv1a, ω‐theraphotoxin‐Hhn2a, and μ‐agatoxin‐Aa1a toxins—inhibitors of calcium L, P, and Q channels and sodium channels were used in the study. Apoptosis was induced by AC‐1001 H3 peptide. We study the effect of toxins on the level of apoptosis, ROS, mitochondrial potential, GSH, and ATP in CHO‐K1 cells. We show that all the tested toxins are able to dose dependently block the induction of apoptosis triggered by AC‐1001 H3 and reduce the level of natural apoptosis in CHO‐K1 cells. Cell incubation with apoptosis inducer AC‐1001 H3 in the presence and absence of toxins causes an increase in the intracellular concentrations of ROS, ATP, and mitochondrial potential and decreases the GSH concentration. The present study reveals the antiapoptotic effect of a number of arthropod peptide toxins. The toxins studied can represent a novel approach used in the treatment of pathologies associated with the activation of apoptotic mechanisms.     

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.     

Transmembrane Segments Prevent Surface Expression of Sodium Channel Nav1.8 and Promote Calnexin-dependent Channel Degradation

The voltage-gated sodium channel (Na_v) 1.8 contributes substantially to the rising phase of action potential in small dorsal root ganglion neurons. Na_v1.8 is majorly localized intracellularly and its expression on the plasma membrane is regulated by exit from the endoplasmic reticulum (ER). Previous work has identified an ER-retention/retrieval motif in the first intracellular loop of Na_v1.8, which prevents its surface expression. Here we report that the transmembrane segments of Na_v1.8 also cause this channel retained in the ER. Using transferrin receptor and CD8α as model molecules, immunocytochemistry showed that the first, second, and third transmembrane segments in each domain of Na_v1.8 reduced their surface expression. Alanine-scanning analysis revealed acidic amino acids as critical factors in the odd transmembrane segments. Furthermore, co-immunoprecipitation experiments showed that calnexin interacted with acidic amino acid-containing sequences through its transmembrane segment. Overexpression of calnexin resulted in increased degradation of those proteins through the ER-associated degradation pathway, whereas down-regulation of calnexin reversed the phenotype. Thus our results reveal a critical role and mechanism of transmembrane segments in surface expression and degradation of Na_v1.8.     

The discovery of benzenesulfonamide-based potent and selective inhibitors of voltage-gated sodium channel Nav1.7

The voltage gated sodium channel Na_v1.7 represents an interesting target for the treatment of pain. Human genetic studies have identified the crucial role of Na_v1.7 in pain signaling. Herein, we report the design and synthesis of a novel series of benzenesulfonamide-based Na_v1.7 inhibitors. Structural–activity relationship (SAR) studies were undertaken towards improving Na_v1.7 activity and minimizing CYP inhibition. These efforts resulted in the identification of compound 12k, a highly potent Na_v1.7 inhibitor with a thousand-fold selectivity over Na_v1.5 and negligible CYP inhibition.     

Phosphorylation of Cav1.2 on S1928 uncouples the L‐type Ca2+ channel from the β2 adrenergic receptor

Agonist‐triggered downregulation of β‐adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of β₂AR signaling highly specific for Ca_v1.2. We find that β₂‐AR binding to Ca_v1.2 residues 1923–1942 is required for β‐adrenergic regulation of Ca_v1.2. Despite the prominence of PKA‐mediated phosphorylation of Ca_v1.2 S1928 within the newly identified β₂AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the β₂AR from Ca_v1.2 upon β‐adrenergic stimulation rendering Ca_v1.2 refractory for several minutes from further β‐adrenergic stimulation. This effect is lost in S1928A knock‐in mice. Although AMPARs are clustered at postsynaptic sites like Ca_v1.2, β₂AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the β₂AR from Ca_v1.2 is a uniquely specific desensitization mechanism of Ca_v1.2 regulation by highly localized β₂AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT‐LTP) depends on Ca_v1.2 and its regulation by channel‐associated β₂AR.     

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.     

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.

     

The Sodium Channel β3-Subunit Induces Multiphasic Gating in NaV1.3 and Affects Fast Inactivation via Distinct Intracellular Regions

Electrical excitability in neurons depends on the activity of membrane-bound voltage gated sodium channels (Na_v) that are assembled from an ion conducting α-subunit and often auxiliary β-subunits. The α-subunit isoform Na_v1.3 occurs in peripheral neurons together with the Na_v β3-subunit, both of which are coordinately up-regulated in rat dorsal root ganglion neurons after nerve injury. Here we examine the effect of the β3-subunit on the gating behavior of Na_v1.3 using whole cell patch clamp electrophysiology in HEK-293 cells. We show that β3 depolarizes the voltage sensitivity of Na_v1.3 activation and inactivation and induces biphasic components of the inactivation curve. We detect both a fast and a novel slower component of inactivation, and we show that the β3-subunit increases the fraction of channels inactivating by the slower component. Using CD and NMR spectroscopy, we report the first structural analysis of the intracellular domain of any Na_v β-subunit. We infer the presence of a region within the β3-subunit intracellular domain that has a propensity to form a short amphipathic α-helix followed by a structurally disordered sequence, and we demonstrate a role for both of these regions in the selective stabilization of fast inactivation. The complex gating behavior induced by β3 may contribute to the known hyperexcitability of peripheral neurons under those physiological conditions where expression of β3 and Na_v1.3 are both enhanced.     

Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis,Axonal Conduction and Presynaptic Release

Human genetic studies show that the voltage gated sodium channel 1.7 (Na_v1.7) is a key molecular determinant of pain sensation. However, defining the Na_v1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Na_v1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Na_v1.7’s role in nociceptor physiology. We report that Na_v1.7 is the predominant functional TTX-sensitive Na_v in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Na_v1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Na_v1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.     

Effects of paraquat-induced oxidative stress on the neuronal plasma membrane Ca-ATPase

Oxidative stress leads to the disruption of calcium homeostasis in brain neurons; however, the direct effects of oxidants on proteins that regulate intracellular calcium ([Ca²⁺]_i) are not known. The calmodulin (CaM)-stimulated plasma membrane Ca²⁺-ATPase (PMCA) plays a critical role in regulating [Ca²⁺]_i. Our previous in vitro studies showed that PMCA present in brain synaptic membranes is readily inactivated by a variety of reactive oxygen species (ROS). The present studies were conducted to determine the vulnerability of PMCA to ROS generated in neurons as would probably occur in vivo. Primary cortical neurons were exposed to paraquat (PQ), a redox cycling agent that generates intracellular ROS. Low concentrations of PQ (5–10 μM) increased PMCA basal activity by two-fold but abolished its sensitivity to CaM. Higher concentrations (25–100 μM) inhibited both components of PMCA activity. Immunoblots showed the formation of high-molecular-weight PMCA aggregates. Additionally, PMCA showed evidence of proteolytic degradation. PMCA proteolysis was prevented by a calpain inhibitor, suggesting a role for calpain. Our findings suggest that PMCA is a sensitive target of oxidative stress in primary neurons. Inactivation of this Ca²⁺ transporter under prolonged oxidative stress could alter neuronal Ca²⁺ signaling.     

Effects of calpain inhibitor on the apoptosis of hepatic stellate cells induced by calcium ionophore A23187

We previously showed that changes in calcium concentrations were related to cell apoptosis in vitro. The endoplasmic reticulum (ER) is the main component of calcium storage and signal transduction, and disrupting the balance of intracellular Ca²⁺ can cause endoplasmic reticulum stress (ERS). In this process, the ER releases stored Ca ²⁺ into the cytoplasm and activates calpain-2. To further investigate the effect of calpain in hepatic stellate cells (HSCs), in the current study, we examine the effect of N-acetyl-leu-leu-norleucinal (ALLN) on apoptosis resulting from calcium ionophore A23187–induced ERS. Our findings indicate that calpain inhibition reduces calcium ionophore A23187–induced apoptosis of HSCs and decreases the expression of ER stress proteins that may be related to the calpain/caspase signaling pathway.