Functional up-regulation of Nav1.8 sodium channel in A? afferent fibers subjected to chronic peripheral inflammation.
ABSTRACT: BACKGROUND: Functional alterations in the properties of A? afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Na(v)1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Na(v)1.8 in controlling A?-fiber excitability following persistent inflammation. METHODS: Distribution and expression of Na(v)1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund's adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Na(v)1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Na(v)1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats. RESULTS: Our findings revealed that Na(v)1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Na(v)1.8 peak current densities are enhanced in inflamed large myelinated A?-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in A?-fiber neuron excitability by shifting the voltage-dependent activation of Na(v)1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Na(v)1.8 currents in A?-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Na(v)1.8 regulation in A?-fibers contributes to inflammatory pain. CONCLUSIONS: Collectively, these findings support a key role for Na(v)1.8 in controlling the excitability of A?-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.
Project description:Tetrodotoxin-resistant (TTX-R) sodium channels Na(V)1.8 and Na(V)1.9 in sensory neurons were known as key pain modulators. Comparing with the widely reported Na(V)1.8, roles of Na(V)1.9 on inflammatory pain are poorly studied by antisense-induced specific gene knockdown. Here, we used molecular, electrophysiological and behavioral methods to examine the effects of antisense oligodeoxynucleotide (AS ODN) targeting Na(V)1.8 and Na(V)1.9 on inflammatory pain. Following complete Freund's adjuvant (CFA) inflammation treatment, Na(V)1.8 and Na(V)1.9 in rat dorsal root ganglion (DRG) up-regulated mRNA and protein expressions and increased sodium current densities. Immunohistochemical data demonstrated that Na(V)1.8 mainly localized in medium and small-sized DRG neurons, whereas Na(V)1.9 only expressed in small-sized DRG neurons. Intrathecal (i.t.) delivery of AS ODN was used to down-regulate Na(V)1.8 or Na(V)1.9 expressions confirmed by immunohistochemistry and western blot. Unexpectedly, behavioral tests showed that only Na(V)1.8 AS ODN, but not Na(V)1.9 AS ODN could reverse CFA-induced heat and mechanical hypersensitivity. Our data indicated that TTX-R sodium channels Na(V)1.8 and Na(V)1.9 in primary sensory neurons played distinct roles in CFA-induced inflammatory pain and suggested that antisense oligodeoxynucleotide-mediated blocking of key pain modulator might point toward a potential treatment strategy against certain types of inflammatory pain.
Project description:Voltage-gated sodium channels (VGSCs) play a key role in the initiation and propagation of action potentials in neurons. Na(V)1.8 is a tetrodotoxin (TTX) resistant VGSC expressed in nociceptors, peripheral small-diameter neurons able to detect noxious stimuli. Na(V)1.8 underlies the vast majority of sodium currents during action potentials. Many studies have highlighted a key role for Na(V)1.8 in inflammatory and chronic pain models. Lipid rafts are microdomains of the plasma membrane highly enriched in cholesterol and sphingolipids. Lipid rafts tune the spatial and temporal organisation of proteins and lipids on the plasma membrane. They are thought to act as platforms on the membrane where proteins and lipids can be trafficked, compartmentalised and functionally clustered. In the present study we investigated Na(V)1.8 sub-cellular localisation and explored the idea that it is associated with lipid rafts in nociceptors. We found that Na(V)1.8 is distributed in clusters along the axons of DRG neurons in vitro and ex vivo. We also demonstrated, by biochemical and imaging studies, that Na(V)1.8 is associated with lipid rafts along the sciatic nerve ex vivo and in DRG neurons in vitro. Moreover, treatments with methyl-?-cyclodextrin (M?CD) and 7-ketocholesterol (7KC) led to the dissociation between rafts and Na(V)1.8. By calcium imaging we demonstrated that the lack of association between rafts and Na(V)1.8 correlated with impaired neuronal excitability, highlighted by a reduction in the number of neurons able to conduct mechanically- and chemically-evoked depolarisations. These findings reveal the sub-cellular localisation of Na(V)1.8 in nociceptors and highlight the importance of the association between Na(V)1.8 and lipid rafts in the control of nociceptor excitability.
Project description:The impact of persistent inflammation on voltage-activated Ca(2+) channels in cutaneous DRG neurons from adult rats was assessed with whole cell patch clamp techniques, sqRT-PCR and Western blot analysis. Inflammation was induced with a subcutaneous injection of complete Freund's adjuvant (CFA). DiI was used to identify DRG neurons innervating the site of inflammation. Three days after CFA injection, high threshold Ca(2+) current (HVA) density was significantly reduced in small and medium, but not large diameter neurons, reflecting a decrease in N-, L- and P/Q-type currents. This decrease in HVA current was associated with an increase in mRNA encoding the ?2?1-subunit complex, but no detectable change in N-type subunit (Ca(V)2.2) mRNA. An increase in both ?2?1 and Ca(V)2.2 protein was detected in the central nerves arising from L4 and L5 ganglia ipsilateral to the site of inflammation. In current clamp experiments on small and medium diameter cutaneous DRG neurons from naïve rats, blocking ?40% of HVA current with Cd(2+) (5?M), had opposite effects on subpopulations of cutaneous DRG neurons (increasing excitability and action potential duration in some and decreasing excitability in others). The alterations in the density and distribution of voltage-activated Ca(2+) channels in subpopulations of cutaneous DRG neurons that develop following CFA injection should contribute to changes in sensory transmission observed in the presence of inflammation.
Project description:Voltage-dependent sodium (Na<sub>V</sub>) 1.8 channels regulate action potential generation in nociceptive neurons, identifying them as putative analgesic targets. Here, we show that Na<sub>V</sub>1.8 channel plasma membrane localization, retention, and stability occur through a direct interaction with the postsynaptic density-95/discs large/zonula occludens-1-and WW domain-containing scaffold protein called membrane-associated guanylate kinase with inverted orientation (Magi)-1. The neurophysiological roles of Magi-1 are largely unknown, but we found that dorsal root ganglion (DRG)-specific knockdown of Magi-1 attenuated thermal nociception and acute inflammatory pain and produced deficits in Na<sub>V</sub>1.8 protein expression. A competing cell-penetrating peptide mimetic derived from the Na<sub>V</sub>1.8 WW binding motif decreased sodium currents, reduced Na<sub>V</sub>1.8 protein expression, and produced hypoexcitability. Remarkably, a phosphorylated variant of the very same peptide caused an opposing increase in Na<sub>V</sub>1.8 surface expression and repetitive firing. Likewise, <i>in vivo</i>, the peptides produced diverging effects on nocifensive behavior. Additionally, we found that Magi-1 bound to sequence like a calcium-activated potassium channel sodium-activated (Slack) potassium channels, demonstrating macrocomplexing with Na<sub>V</sub>1.8 channels. Taken together, these findings emphasize Magi-1 as an essential scaffold for ion transport in DRG neurons and a central player in pain.-Pryce, K. D., Powell, R., Agwa, D., Evely, K. M., Sheehan, G. D., Nip, A., Tomasello, D. L., Gururaj, S., Bhattacharjee, A. Magi-1 scaffolds Na<sub>V</sub>1.8 and Slack K<sub>Na</sub> channels in dorsal root ganglion neurons regulating excitability and pain.
Project description:Cardiac Nav1.5 and Kir2.1-2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems - rat ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) - demonstrated that endoplasmic reticulum (ER) trafficking-defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking-defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking-defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.
Project description:BACKGROUND AND PURPOSE:7-[2-[4-(2-Chlorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) is a xanthine-based derivative. It has soluble GC activation and K(+) -channel opening activity. Effects of this compound on ion currents in pituitary GH3 cells were investigated in this study. EXPERIMENTAL APPROACH:The aim of this study was to evaluate effects of KMUP-1 on the amplitude and gating of voltage-gated Na(+) current (INa ) in pituitary GH3 cells and in HEKT293T cells expressing SCN5A. Both the amplitude of Ca(2+) -activated K(+) current and the activity of large-conductance Ca(2+) -activated K(+) (BKCa ) channels were also studied. KEY RESULTS:KMUP-1 depressed the transient and late components of INa with different potencies. The IC50 values required for its inhibitory effect on transient and late INa were 22.5 and 1.8 ?M respectively. KMUP-1 (3 ?M) shifted the steady-state inactivation of INa to a hyperpolarized potential by -10 mV, despite inability to alter the recovery of INa from inactivation. In cell-attached configuration, KMUP-1 applied to bath increased BKCa -channel activity; however, in inside-out patches, this compound applied to the intracellular surface had no effect on it. It prolonged the latency in the generation of action currents elicited by triangular voltage ramps. Additionally, KMUP-1 decreased the peak INa with a concomitant increase of current inactivation in HEKT293T cells expressing SCN5A. CONCLUSIONS AND IMPLICATIONS:Apart from activating BKCa channels, KMUP-1 preferentially suppresses late INa . The effects of KUMP-1 on ion currents presented here constitute an underlying ionic mechanism of its actions.
Project description:Opiates are potent analgesics for moderate to severe pain. Paradoxically, patients under chronic opiates have reported hypernociception, the mechanisms of which are unknown. Using standard patch-clamp technique, we examined the excitability, biophysical properties of tetrodotoxin-resistant (TTX-R) Na(+) and transient receptor potential vanilloid 1 (TRPV1) channels of dorsal root ganglia neurons (DRG) (L(5)-S(1)) from mice pelleted with morphine (75 mg) or placebo (7 days). Hypernociception was confirmed by acetic acid-writhing test following 7-day morphine. Chronic morphine enhanced the neuronal excitability, since the rheobase for action potential (AP) firing was significantly (P < 0.01) lower (38 ± 7 vs. 100 ± 15 pA) while the number of APs at 2× rheobase was higher (4.4 ± 0.8 vs. 2 ± 0.5) than placebo (n = 13-20). The potential of half-maximum activation (V(1/2)) of TTX-R Na(+) currents was shifted to more hyperpolarized potential in the chronic morphine group (-37 ± 1 mV) vs. placebo (-28 ± 1 mV) without altering the V(1/2) of inactivation (-41 ± 1 vs. -33 ± 1 mV) (n = 8-11). Recovery rate from inactivation of TTX-R Na(+) channels or the mRNA level of any Na(+) channel subtypes did not change after chronic morphine. Also, chronic morphine significantly (P < 0.05) enhanced the magnitude of TRPV1 currents (-64 ± 11 pA/pF) vs. placebo (-18 ± 6 pA/pF). The increased excitability of sensory neurons by chronic morphine may be due to the shift in the voltage threshold of activation of TTX-R Na(+) currents. Enhanced TRPV1 currents may have a complementary effect, with TTX-R Na(+) currents on opiate-induced hyperexcitability of sensory neurons causing hypernociception. In conclusion, chronic morphine-induced hypernociception is associated with hyperexcitability and functional remodeling of TTX-R Na(+) and TRPV1 channels of sensory neurons.
Project description:High-frequency spontaneous firing in myelinated sensory neurons plays a key role in initiating pain behaviors in several different models, including the radicular pain model in which the rat lumbar dorsal root ganglia (DRG) are locally inflamed. The sodium channel isoform NaV1.6 contributes to pain behaviors and spontaneous activity in this model. Among all isoforms in adult DRG, NaV1.6 is the main carrier of tetrodotoxin-sensitive resurgent Na currents that allow high-frequency firing. Resurgent currents flow after a depolarization or action potential, as a blocking particle exits the pore. In most neurons, the regulatory ?4 subunit is potentially the endogenous blocker. We used in vivo siRNA-mediated knockdown of NaV?4 to examine its role in the DRG inflammation model. NaV?4 but not control siRNA almost completely blocked mechanical hypersensitivity induced by DRG inflammation. Microelectrode recordings in isolated whole DRG showed that NaV?4 siRNA blocked the inflammation-induced increase in spontaneous activity of A? neurons and reduced repetitive firing and other measures of excitability. NaV?4 was preferentially expressed in larger diameter cells; DRG inflammation increased its expression, and this was reversed by NaV?4 siRNA, based on immunohistochemistry and Western blotting. NaV?4 siRNA also reduced immunohistochemical NaV1.6 expression. Patch-clamp recordings of tetrodotoxin-sensitive Na currents in acutely cultured medium diameter DRG neurons showed that DRG inflammation increased transient and especially resurgent current, effects blocked by NaV?4 siRNA. NaV?4 may represent a more specific target for pain conditions that depend on myelinated neurons expressing NaV1.6.
Project description:Class 1 antiarrhythmic drugs are highly effective in restoring and maintaining sinus rhythm in atrial fibrillation patients but carry a risk of ventricular tachyarrhythmia. The antianginal agent ranolazine is a prototypic atrial-selective voltage-gated Na+ channel blocker but the mechanisms underlying its atrial-selective action remain unclear.The present study examined the mechanisms underlying the atrial-selective action of ranolazine.Whole-cell voltage-gated Na+ currents (INa) were recorded at room temperature (?22°C) from rabbit isolated left atrial and right ventricular myocytes.INa conductance density was ?1.8-fold greater in atrial than in ventricular cells. Atrial INa was activated at command potentials ?7 mV more negative and inactivated at conditioning potentials ?11 mV more negative than ventricular INa. The onset of inactivation of INa was faster in atrial cells than in ventricular myocytes. Ranolazine (30 ?M) inhibited INa in atrial and ventricular myocytes in a use-dependent manner consistent with preferential activated/inactivated state block. Ranolazine caused a significantly greater negative shift in voltage of half-maximal inactivation in atrial cells than in ventricular cells, the recovery from inactivation of INa was slowed by ranolazine to a greater extent in atrial myocytes than in ventricular cells, and ranolazine produced an instantaneous block that showed marked voltage dependence in atrial cells.Differences exist between rabbit atrial and ventricular myocytes in the biophysical properties of INa. The more negative voltage dependence of INa activation and inactivation, together with trapping of the drug in the inactivated channel, underlies an atrial-selective action of ranolazine.
Project description:The ventrolateral periaqueductal gray (vlPAG) is a key structure in the descending pain modulatory circuit. Activation of the circuit occurs via disinhibition of GABAergic inputs onto vlPAG output neurons. In these studies, we tested the hypothesis that GABAergic inhibition is increased during persistent inflammation, dampening activation of the descending circuit from the vlPAG. Our results indicate that persistent inflammation induced by Complete Freund's adjuvant (CFA) modulates GABA signaling differently in male and female rats. CFA treatment results in increased presynaptic GABA release but decreased high-affinity tonic GABAA currents in female vlPAG neurons. These effects are not observed in males. The tonic currents in the vlPAG are dependent on GABA transporter activity and are modulated by agonists that activate GABAA receptors containing the ? subunit. The GABAA ? agonist THIP (gaboxadol) induced similar amplitude currents in naive and CFA-treated rats. In addition, a positive allosteric modulator of the GABAA ? subunit, DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide), increased tonic currents. These results indicate that GABAA ? receptors remain on the cell surface but are less active in CFA-treated female rats. In vivo behavior studies showed that morphine induced greater antinociception in CFA-treated females that was reversed with microinjections of DS2 directly into the vlPAG. DS2 did not affect morphine antinociception in naive or CFA-treated male rats. Together, these data indicate that sex-specific adaptations in GABAA receptor signaling modulate opioid analgesia in persistent inflammation. Antagonists of GABAA ? receptors may be a viable strategy for reducing pain associated with persistent inflammation, particularly in females.These studies demonstrate that GABA signaling is modulated in the ventrolateral periaqueductal gray by persistent inflammation differently in female and male rats. Our results indicate that antagonists or negative allosteric modulators of GABAA ? receptors may be an effective strategy to alleviate chronic inflammatory pain and promote opioid antinociception, especially in females.