The voltage-gated sodium channel (Nav) 1.8 contributes substantially to the rising phase of action potential in small dorsal root ganglion neurons. Nav1.8 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 Nav1.8, which prevents its surface expression. Here we report that the transmembrane segments of Nav1.8 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 Nav1.8 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 Nav1.8. 相似文献
The tetrodotoxin‐resistant (TTX‐R) voltage‐gated sodium channel Nav1.8 is predominantly expressed in peripheral afferent neurons, but in case of neuronal injury an ectopic and detrimental expression of Nav1.8 occurs in neurons of the CNS. In CNS neurons, Nav1.2 and Nav1.6 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 Nav1.8 and ankG. GST pull‐down assay and surface plasmon resonance technology revealed that Nav1.8 strongly and constitutively interacts with ankG, in comparison to what observed for Nav1.2. An ion channel bearing the ankyrin‐binding motif of Nav1.8 displaced the endogenous Nav1 accumulation at the axon initial segment of hippocampal neurons. Finally, Nav1.8 and ankG co‐localized in skin nerves fibers. Altogether, these results indicate that Nav1.8 carries all the information required for its localization at ankG micro‐domains. The constitutive binding of Nav1.8 with ankG could contribute to the pathological aspects of illnesses where Nav1.8 is ectopically expressed in CNS neurons.
Aquaporin-1 (AQP1) water channels are expressed in the plasma membrane of dorsal root ganglion (DRG) neurons. We found reduced osmotic water permeability in freshly isolated DRG neurons from AQP1−/−versus AQP1+/+ mice. Behavioral studies showed greatly reduced thermal inflammatory pain perception in AQP1−/− mice evoked by bradykinin, prostaglandin E2, and capsaicin as well as reduced cold pain perception. Patch clamp of freshly isolated DRG neurons showed reduced action potential firing in response to current injections. Single action potentials after pulse current injections showed reduced maximum inward current, suggesting impaired Nav1.8 Na+ function. Whole-cell Nav1.8 Na+ currents in Nav1.8-expressing ND7-23 cells showed slowed frequency-dependent inactivation after AQP1 transfection. Immunoprecipitation studies showed AQP1- Nav1.8 Na+ interaction, which was verified in live cells by single-particle tracking of quantum dot-labeled AQP1. Our results implicate the involvement of AQP1 in DRG neurons for the perception of inflammatory thermal pain and cold pain, whose molecular basis is accounted for, in part, by reduced Nav1.8-dependent membrane Na+ current. AQP1 is, thus, a novel target for pain management. 相似文献
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 Nav1.8.
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 V1/2 values of -73 and -37 mV. These values are similar to the V1/2 values for steady-state fast inactivation of tetrodotoxin-sensitive (TTX-s) sodium channels, and the tetrodotoxin-resistant (TTX-r) Nav1.8 sodium channel, respectively. Addition of TTX eliminates the more hyperpolarized V1/2 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 Nav1.8(-/-) mice, the voltage-dependent decrease in AP amplitude is characterized by a single Boltzmann equation with a V1/2 value of -55 mV, suggesting a shift in the steady-state fast inactivation properties of TTX-s sodium channels. Transfection of Nav1.8(-/-) DRG neurons with DNA encoding Nav1.8 results in a membrane potential-dependent decrease in AP amplitude that recapitulates WT properties.
Conclusion
We conclude that the presence of Nav1.8 allows AP amplitude to be maintained in DRG neurons and their centrally projecting axons even when depolarized within the dorsal horn. 相似文献
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 Nav1.8 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 Nav1.8 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 Nav1.8 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 Nav1.8 current density by promoting the phosphorylation of Nav1.8 and its expression. 相似文献
Sodium channel Nav1.6 is essential for neuronal excitability in central and peripheral nervous systems. Loss-of-function mutations in Nav1.6 underlie motor disorders, with homozygous-null mutations causing juvenile lethality. Phosphorylation of Nav1.6 by the stress-induced p38 MAPK at a Pro-Gly-Ser553-Pro motif in its intracellular loop L1 reduces Nav1.6 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 Scn8amedtg mice that lack Nav1.6, reduces tetrodotoxin-S sodium currents, suggesting isoform-specific modulation of Nav1.6 by p38 in these neurons. Pharmacological block of endocytosis completely abolishes p38-mediated Nav1.6 current reduction, supporting our hypothesis that channel internalization underlies current reduction. We also report that the ubiquitin ligase Nedd4-2 interacts with Nav1.6 via a Pro-Ser-Tyr1945 motif in the C terminus of the channel and reduces Nav1.6 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 Nav1.6 current, whereas abrogating binding of the ubiquitin ligase Nedd4-2 to the Pro-Ser-Tyr motif results in stress-mediated increase in Nav1.6 current density. Thus, phosphorylation of the Pro-Gly-Ser-Pro motif within L1 of Nav1.6 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. 相似文献
Nav1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Nav1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Nav1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Nav1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration. 相似文献
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 Nav activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF‐EMF for 60 min. significantly increased the Nav current (INa) densities by 62.5%. MT (5 μM) inhibited the ELF‐EMF‐induced INa increase. This inhibitory effect of MT is mimicked by an MT2 receptor agonist and was eliminated by an MT2 receptor antagonist. The Nav 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 Nav 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 INa that result from ELF‐EMF exposure through Ca2+ influx‐induced Ca2+ release. 相似文献
Increased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na+ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions.
Results
Using the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,β-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,β-meATP-induced Na+ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,β-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,β-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Nav1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Nav1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude.
Conclusion
Neuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Nav1.3 subunit, and the function of the Nav1.3 channel is regulated by PKC. 相似文献
Previous data showed that prostaglandin E2 (PGE2) mediates the inhibitory effect of bradykinin (BK) on proximal tubule (PT) Na+-ATPase activity. The aim of this work was to investigate the molecular mechanisms involved in the effect of PGE2 on PT Na+-ATPase. We used isolated basolateral membrane (BLM) from pig PT, which expresses several components of different signaling pathways. The inhibitory effect of PGE2 on PT Na+-ATPase activity involves G-protein and the activation of protein kinase A (PKA) because: (1) PGE2 increased [35S]GTPγS binding; (2) GDPβS abolished the inhibitory effect of PGE2; (3) PGE2 increased PKA activity; (4) the inhibitory effect of PGE2 was abolished by PKA inhibitor peptide. We observed that the PKA-mediated inhibitory effect of PGE2 on PT Na+-ATPase activity requires previous activation of protein kinase C. In addition, we observed that PGE2 stimulates Ca2+-independent phospholipase A2 activity representing an important positive feedback to maintain the inhibition of the enzyme. These results open new perspectives to understanding the mechanism involved in the effect of PGE2 on proximal tubule sodium reabsorption. 相似文献
Prostaglandin E2 (PGE2) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na+,K+‐ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE2 decreases Na+,K+‐ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE2. Na+,K+‐ATPase activity was determined by assessing ouabain‐sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE2 (0.1–10 μM) for 30 min decreased Na+,K+‐ATPase activity in a concentration‐dependent manner. However, PGE2 did not alter Na+,K+‐ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE2 on Na+,K+‐ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic α subunit of Na+,K+‐ATPase. We found that the inhibitory effect of PGE2 (1 μM) on Na+,K+‐ATPase activity was receptor‐mediated, as incubation with selective antagonists for EP1 (SC‐19220, 10 μM), EP3 (L‐826266, 1 μM) or EP4 (L‐161982, 1 μM) receptors prevented the PGE2‐induced decrease of Na+,K+‐ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1–10 μM) increased enzyme activity per se in a concentration‐dependent manner, but did not prevent the inhibitory effect of PGE2. Incubation with a protein kinase A (PKA) inhibitor (H‐89, 1 μM) and a protein kinase C (PKC) inhibitor (GF‐109203X, 300 nM) also prevented PGE2‐induced decrease of Na+,K+‐ATPase activity. Accordingly, PGE2 increased phosphorylation of Ser943 at the α subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE2 decreases Na+,K+‐ATPase activity in vivo. The results presented here imply Na+,K+‐ATPase as a target for PGE2‐mediated signaling, which may underlie PGE2‐induced increase of brain excitability. 相似文献
The cardiac sodium channel Nav1.5 is essential for the physiological function of the heart and causes cardiac arrhythmias and sudden death when mutated. Many disease-causing mutations in Nav1.5 cause defects in protein trafficking, a cellular process critical to the targeting of Nav1.5 to cell surface. However, the molecular mechanisms underlying the trafficking of Nav1.5, in particular, the exit from the endoplasmic reticulum (ER) for cell surface trafficking, remain poorly understood.
Methods and results
Here we investigated the role of the SAR1 GTPases in trafficking of Nav1.5. Overexpression of dominant-negative mutant SAR1A (T39N or H79G) or SAR1B (T39N or H79G) significantly reduces the expression level of Nav1.5 on cell surface, and decreases the peak sodium current density (INa) in HEK/Nav1.5 cells and neonatal rat cardiomyocytes. Simultaneous knockdown of SAR1A and SAR1B expression by siRNAs significantly reduces the INa density, whereas single knockdown of either SAR1A or SAR1B has minimal effect. Computer modeling showed that the three-dimensional structure of SAR1 is similar to RAN. RAN was reported to interact with MOG1, a small protein involved in regulation of the ER exit of Nav1.5. Co-immunoprecipitation showed that SAR1A or SAR1B interacted with MOG1. Interestingly, knockdown of SAR1A and SAR1B expression abolished the MOG1-mediated increases in both cell surface trafficking of Nav1.5 and the density of INa.
Conclusions
These data suggest that SAR1A and SAR1B are the critical regulators of trafficking of Nav1.5. Moreover, SAR1A and SAR1B interact with MOG1, and are required for MOG1-mediated cell surface expression and function of Nav1.5. 相似文献
During axonal maturation, voltage-gated sodium (Nav) 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 Nav 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 Nav1.6 construct containing an extracellular biotinylation domain we demonstrate that Nav1.6 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 Nav1.6 channel lacking the ankyrin-binding motif nor a chimeric Kv2.1 channel containing the Nav ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Nav1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS. 相似文献
Phosphorylation on the activation loop of AGC kinases is typically mediated by PDK1. The precise mechanism for this in‐trans phosphorylation is unknown; however, docking of a hydrophobic (HF) motif in the C‐tail of the substrate kinase onto the N‐lobe of PDK1 is likely an essential step. Using a peptide array of PKA to identify other PDK1‐interacting sites, we discovered a second AGC‐conserved motif in the C‐tail that interacts with PDK1. Since this motif [FD(X)1‐2Y/F] lies in the active site tether region and in PKA contributes to ATP binding, we call it the Adenosine binding (Ade) motif. The Ade motif is conserved as a PDK1‐interacting site in Akt and PRK2, and we predict it will be a PDK1‐interacting site for most AGC kinases. In PKA, the HF motif is only recognized when the turn motif Ser338 is phosphorylated, possibly serving as a phosphorylation “switch” that regulates how the Ade and HF motifs interact with PDK1. These results demonstrate that the extended AGC C‐tail serves as a polyvalent element that trans‐regulates PDK1 for catalysis. Modeling of the PKA C‐tail onto PDK1 structure creates two chimeric sites; the ATP binding pocket, which is completed by the Ade motif, and the C‐helix, which is positioned by the HF motif. Together, they demonstrate substrate‐assisted catalysis involving two kinases that have co‐evolved as symbiotic partners. The highly regulated turn motifs are the most variable part of the AGC C‐tail. Elucidating the highly regulated cis and trans functions of the AGC tail is a significant future challenge. 相似文献
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. 相似文献
We examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE2 from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE2 to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freund''s adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE2 to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE2-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE2-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE2-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE2-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE2-induced hypersensitivity during inflammation. 相似文献
The treatment of neuropathic pain remains a major challenge to pain clinicians. Certain nociceptive and non-nociceptive dorsal root ganglion (DRG) neurons may develop abnormal spontaneous activities following peripheral nerve injury, which is believed to be a major contributor to chronic pain. Subthreshold membrane potential oscillation (SMPO) observed in injured DRG neurons was reported to be involved in the generation of abnormal spontaneous activity. Tetrodotoxin-sensitive sodium (Na+) channels were testified to be involved in the generation of SMPO, but their specific subunits have not been clarified. We hypothesize that the subunits of voltage-gated sodium channel, Nav1.3 and Nav1.6, are involved in the generation of SMPO. An attempt to test this hypothesis may lead to a new therapeutic strategy for neuropathic pain. 相似文献