Subcellular Distribution of Persistent Sodium Conductance in Cortical Pyramidal Neurons

Cortical pyramidal neurons possess a persistent Na⁺ current (I_NaP), which, in contrast to the larger transient current, does not undergo rapid inactivation. Although relatively quite small, I_NaP is active at subthreshold voltages and therefore plays an important role in neuronal input–output processing. The subcellular distribution of channels responsible for I_NaP and the mechanisms that render them persistent are not known. Using high-speed fluorescence Na⁺ imaging and whole-cell recordings in brain slices obtained from mice of either sex, we reconstructed the I_NaP elicited by slow voltage ramps in soma and processes of cortical pyramidal neurons. We found that in all neuronal compartments, the relationship between persistent Na⁺ conductance and membrane voltage has the shape of a Boltzmann function. Although the density of channels underlying I_NaP was about twofold lower in the axon initial segment (AIS) than in the soma, the axonal channels were activated by ∼10 mV less depolarization than were somatic channels. This difference in voltage dependence explains why, at functionally critical subthreshold voltages, most I_NaP originates in the AIS. Finally, we show that endogenous polyamines constrain I_NaP availability in both somatodendritic and axonal compartments of nondialyzed cortical neurons.SIGNIFICANCE STATEMENT The most salient characteristic of neuronal sodium channels is fast inactivation. However, a fraction of the sodium current does not inactivate. In cortical neurons, persistent current (I_NaP) plays a prominent role in many important functions. Its subcellular distribution and generation mechanisms are, however, elusive. Using high-speed fluorescence Na⁺ imaging and electrical recordings, we reconstructed the I_NaP in soma and processes of cortical pyramidal neurons. We found that at near-threshold voltages I_NaP originates predominately from the axon, because of the distinctive voltage dependence of the underlying channels and not because of their high density. Finally, we show that the presence of endogenous polyamines significantly constrains I_NaP availability in all compartments of nondialyzed cortical neurons.     

Cyproheptadine Regulates Pyramidal Neuron Excitability in Mouse Medial Prefrontal Cortex

Cyproheptadine (CPH), a first-generation antihistamine, enhances the delayed rectifier outward K⁺ current (I_K) in mouse cortical neurons through a sigma-1 receptor-mediated protein kinase A pathway. In this study, we aimed to determine the effects of CPH on neuronal excitability in current-clamped pyramidal neurons in mouse medial prefrontal cortex slices. CPH (10 µmol/L) significantly reduced the current density required to generate action potentials (APs) and increased the instantaneous frequency evoked by a depolarizing current. CPH also depolarized the resting membrane potential (RMP), decreased the delay time to elicit an AP, and reduced the spike threshold potential. This effect of CPH was mimicked by a sigma-1 receptor agonist and eliminated by an antagonist. Application of tetraethylammonium (TEA) to block I_K channels hyperpolarized the RMP and reduced the instantaneous frequency of APs. TEA eliminated the effects of CPH on AP frequency and delay time, but had no effect on spike threshold or RMP. The current-voltage relationship showed that CPH increased the membrane depolarization in response to positive current pulses and hyperpolarization in response to negative current pulses, suggesting that other types of membrane ion channels might also be affected by CPH. These results suggest that CPH increases the excitability of medial prefrontal cortex neurons by regulating TEA-sensitive I_K channels as well as other TEA-insensitive K⁺ channels, probably I_D and inward-rectifier Kir channels. This effect of CPH may explain its apparent clinical efficacy as an antidepressant and antipsychotic.     

Ih channels prevent overexcitability of early developmental CA1 neurons showing high input resistance in rats

Immature hippocampal neurons with high input resistances (R_in) are vulnerable to hyperexcitable or epileptogenic conditions. This phenomenon has been suggested to explain the neuroprotective roles of hyperpolarization-activated cation channels (I_h channels) to regulate membrane R_in. In the present study, we tried to electrophysiologically clarify the relationship between membrane R_in and I_h channels and determine the neuroprotective roles of these channels in development. The CA1 neurons from rats (within 3 postnatal weeks) were classified into two groups based on the onset time (shorter or longer than 20 ms) to fire the first action potential (AP) in response to a current injection (100 pA, 800 ms). Neurons with a shorter onset time (Short-OsT), exhibited higher R_in, while neurons with longer onset times (Long-OsT) revealed lower R_in. Unexpectedly, Short-OsT neurons with higher R_in exhibited larger amplitudes of I_h compared with Long-OsT neurons. Furthermore, the application of temporal depolarization stimulus (TDS, −14 mV holding for 150 s) significantly enhanced suprathreshold excitabilities of repetitive APs in Long-OsT but not Short-OsT neurons, suggesting a protective role of I_h channels under high R_in conditions. In the presence of the specific hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288, TDS also enhanced the excitability of Short-OsT neurons, suggesting that young CA1 neurons regulate I_h channel expression for neuroprotective modulation against epileptogenic conditions.     

Blockade of Na+/H+ exchanger type 3 causes intracellular acidification and hyperexcitability via inhibition of pH-sensitive K+ channels in chemosensitive respiratory neurons of the dorsal vagal nucleus in rats

Extracellular pH (pH_e) and intracellular pH (pH_i) are important factors for the excitability of chemosensitive central respiratory neurons that play an important role in respiration and obstructive sleep apnea. It has been proposed that inhibition of central Na⁺/ H⁺ exchanger 3 (NHE-3), a key pHi regulator in the brainstem, decreases the pH_i, leading to membrane depolarization for the maintenance of respiration. However, how intracellular pH affects the neuronal excitability of respiratory neurons remains largely unknown. In this study, we showed that NHE-3 mRNA is widely distributed in respiration-related neurons of the rat brainstem, including the dorsal vagal nucleus (DVN). Whole-cell patch clamp recordings from DVN neurons in brain slices revealed that the standing outward current (I _so) through pH-sensitive K⁺ channels was inhibited in the presence of the specific NHE-3 inhibitor AVE0657 that decreased the pHi. Exposure of DVN neurons to an acidified pH_e and AVE0657 (5 μmol/L) resulted in a stronger effect on firing rate and I _so than acidified pH_e alone. Taken together, our results showed that intracellular acidification by blocking NHE-3 results in inhibition of a pHsensitive K⁺ current, leading to synergistic excitation of chemosensitive DVN neurons for the regulation of respiration.     

Cannabidiol Inhibition of Murine Primary Nociceptors: Tight Binding to Slow Inactivated States of Nav1.8 Channels

The nonpsychoactive phytocannabinoid cannabidiol (CBD) has been shown to have analgesic effects in animal studies but little is known about its mechanism of action. We examined the effects of CBD on intrinsic excitability of primary pain-sensing neurons. Studying acutely dissociated capsaicin-sensitive mouse DRG neurons at 37°C, we found that CBD effectively inhibited repetitive action potential firing, from 15–20 action potentials evoked by 1 s current injections in control to 1–3 action potentials with 2 μm CBD. Reduction of repetitive firing was accompanied by a reduction of action potential height, widening of action potentials, reduction of the afterhyperpolarization, and increased propensity to enter depolarization block. Voltage-clamp experiments showed that CBD inhibited both TTX-sensitive and TTX-resistant (TTX-R) sodium currents in a use-dependent manner. CBD showed strong state-dependent inhibition of TTX-R channels, with fast binding to inactivated channels during depolarizations and slow unbinding on repolarization. CBD alteration of channel availability at various voltages suggested that CBD binds especially tightly [K_d (dissociation constant), ∼150 nm] to the slow inactivated state of TTX-R channels, which can be substantially occupied at voltages as negative as −40 mV. Remarkably, CBD was more potent in inhibiting TTX-R channels and inhibiting action potential firing than the local anesthetic bupivacaine. We conclude that CBD might produce some of its analgesic effects by direct effects on neuronal excitability, with tight binding to the slow inactivated state of Na_v1.8 channels contributing to effective inhibition of repetitive firing by modest depolarizations.SIGNIFICANCE STATEMENT Cannabidiol (CBD) has been shown to inhibit pain in various rodent models, but the mechanism of this effect is unknown. We describe the ability of CBD to inhibit repetitive action potential firing in primary nociceptive neurons from mouse dorsal root ganglia and analyze the effects on voltage-dependent sodium channels. We find that CBD interacts with TTX-resistant sodium channels in a state-dependent manner suggesting particularly tight binding to slow inactivated states of Na_v1.8 channels, which dominate the overall inactivation of Na_v1.8 channels for small maintained depolarizations from the resting potential. The results suggest that CBD can exert analgesic effects in part by directly inhibiting repetitive firing of primary nociceptors and suggest a strategy of identifying compounds that bind selectively to slow inactivated states of Na_v1.8 channels for developing effective analgesics.     

Merging Structural Motifs of Functionalized Amino Acids and α-Aminoamides Results in Novel Anticonvulsant Compounds with Significant Effects on Slow and Fast Inactivation of Voltage-Gated Sodium Channels and in the Treatment of Neuropathic Pain

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Nitric oxide as intracellular modulator: internal production of NO increases neuronal excitability via modulation of several ionic conductances

Nitric oxide (NO) has been shown to regulate neuronal excitability in the nervous system, but little is known as to whether NO, which is synthesized in certain neurons, also serves functional roles within NO‐producing neurons themselves. We investigated this possibility by using a nitric oxide synthase (NOS)‐expressing neuron, and studied the role of intrinsic NO production on neuronal firing properties in single‐cell culture. B5 neurons of the pond snail Helisoma trivolvis fire spontaneous action potentials (APs), but once the intrinsic activity of NOS was inhibited, neurons became hyperpolarized and were unable to fire evoked APs. These striking long‐term effects could be attributed to intrinsic NO acting on three types of conductances, a persistent sodium current (I_NaP), voltage‐gated Ca currents (I_Ca) and small‐conductance calcium‐activated potassium (SK) channels. We show that NOS inhibitors 7‐nitroindazole and S‐methyl‐l ‐thiocitrulline resulted in a decrease in I_NaP, and that their hyperpolarizing and inhibiting effects on spontaneous spiking were mimicked by the inhibitor of I_NaP, riluzole. Moreover, inhibition of NOS, soluble guanylate cyclase (sGC) or protein kinase G (PKG) attenuated I_Ca, and blocked spontaneous and depolarization‐induced spiking, suggesting that intrinsic NO controlled I_Ca via the sGC/PKG pathway. The SK channel inhibitor apamin partially prevented the hyperpolarization observed after inhibition of NOS, suggesting a downregulation of SK channels by intrinsic NO. Taken together, we describe a novel mechanism by which neurons utilize their self‐produced NO as an intrinsic modulator of neuronal excitability. In B5 neurons, intrinsic NO production is necessary to maintain spontaneous tonic and evoked spiking activity.     

Cocaine Sensitization Increases I h Current Channel Subunit 2 (HCN2) Protein Expression in Structures of the Mesocorticolimbic System

Alteration of the biological activity among neuronal components of the mesocorticolimbic (MCL) system has been implicated in the pathophysiology of drug abuse. Changes in the electrophysiological properties of neurons involved in the reward circuit seem to be of utmost importance in addiction. The hyperpolarization-activated cyclic nucleotide current, I _h, is a prominent mixed cation current present in neurons. The biophysical properties of the I _h and its potential modulatory role in cell excitability depend on the expression profile of the hyperpolarization-activated cyclic nucleotide gated channel (HCN) subunits. We investigated whether cocaine-induced behavioral sensitization, an animal model of drug addiction, elicits region-specific changes in the expression of the HCN₂ channel’s subunit in the MCL system. Tissue samples from the ventral tegmental area, prefrontal cortex, nucleus accumbens, and hippocampus were analyzed using Western blot. Our findings demonstrate that cocaine treatment induced a significant increase in the expression profile of the HCN₂ subunit in both its glycosylated and non-glycosylated protein isoforms in all areas tested. The increase in the glycosylated isoform was only observed in the ventral tegmental area. Together, these data suggest that the observed changes in MCL excitability during cocaine addiction might be associated with alterations in the subunit composition of their HCN channels.     

Fast Inactivation of Ca<Subscript>V</Subscript>2.2 Channels Is Prevented by the Gβ<Subscript>1</Subscript> Subunit in Rat Sympathetic Neurons

Voltage-dependent regulation of Ca_V2.2 channels by G-proteins is performed by the β (Gβ) subunit. Most studies of regulation by G-proteins have focused on channel activation; however, little is known regarding channel inactivation. This study investigated inactivation of Ca_V2.2 channels in superior cervical ganglion neurons that overexpressed Gβ subunits. Ca_V2.2 currents were recorded by whole-cell patch clamping configuration. We found that the Gβ₁ subunit reduced inactivation, while Gβ₅ subunit did not alter at all inactivation kinetics compared to control recordings. Ca_V2.2 current decay in control neurons consisted of both fast and slow inactivation; however, Gβ₁-overexpressing neurons displayed only the slow inactivation. Fast inactivation was restored by a strong depolarization of Gβ₁-overexpressing neurons, therefore, through a voltage-dependent mechanism. The Gβ₁ subunit shifted the voltage dependence of inactivation to more positive voltages and reduced the fraction of Ca_V2.2 channels resting in the inactivated state. These results support that the Gβ₁ subunit inhibits the fast inactivation of Ca_V2.2 channels in SCG neurons. They explain the long-observed sustained Ca²⁺ current under G-protein modulation.     

A sodium channel mutation linked to epilepsy increases ramp and persistent current of Nav1.3 and induces hyperexcitability in hippocampal neurons

Voltage-gated sodium channelopathies underlie many excitability disorders. Genes SCN1A, SCN2A and SCN9A, which encode pore-forming α-subunits Na_V1.1, Na_V1.2 and Na_V1.7, are clustered on human chromosome 2, and mutations in these genes have been shown to underlie epilepsy, migraine, and somatic pain disorders. SCN3A, the gene which encodes Na_V1.3, is part of this cluster, but until recently was not associated with any mutation. A charge-neutralizing mutation, K345Q, in the Na_V1.3 DI/S5-6 linker has recently been identified in a patient with cryptogenic partial epilepsy. Pathogenicity of the Na_V1.3/K354Q mutation has been inferred from the conservation of this residue in all sodium channels and its absence from control alleles, but functional analysis has been limited to the corresponding substitution in the cardiac muscle sodium channel Na_V1.5. Since identical mutations may produce different effects within different sodium channel isoforms, we assessed the K354Q mutation within its native Na_V1.3 channel and studied the effect of the mutant Na_V1.3/K354Q channels on hippocampal neuron excitability. We show here that the K354Q mutation enhances the persistent and ramp currents of Na_V1.3, reduces current threshold and produces spontaneous firing and paroxysmal depolarizing shift-like complexes in hippocampal neurons. Our data provide a pathophysiological basis for the pathogenicity of the first epilepsy-linked mutation within Na_V1.3 channels and hippocampal neurons.     

Development of resurgent and persistent sodium currents in mesencephalic trigeminal neurons

Sodium channels play multiple roles in the formation of neural membrane properties in mesencephalic trigeminal (Mes V) neurons and in other neural systems. Mes V neurons exhibit conditional robust high‐frequency spike discharges. As previously reported, resurgent and persistent sodium currents (I_NaR and I_NaP, respectively) may carry small currents at subthreshold voltages that contribute to generation of spike firing. These currents play an important role in maintaining and allowing high‐frequency spike discharge during a burst. In the present study, we investigated the developmental changes in tetrodotoxin‐sensitive I_NaR and I_NaP underlying high‐frequency spike discharges in Mes V neurons. Whole‐cell patch‐clamp recordings showed that both current densities increased one and a half times from postnatal day (P) 0–6 neurons to P7–14 neurons. Although these neurons do not exhibit subthreshold oscillations or burst discharges with high‐frequency firing, I_NaR and I_NaP do exist in Mes V neurons at P0–6. When the spike frequency at rheobase was examined in firing Mes V neurons, the developmental change in firing frequency among P7–14 neurons was significant. I_NaR and I_NaP density at ?40 mV also increased significantly among P7–14 neurons. The change to an increase in excitability in the P7–14 group could result from this quantitative change in I_NaP. In neurons older than P7 that exhibit repetitive firing, quantitative increases in I_NaR and I_NaP density may be major factors that facilitate and promote high‐frequency firing as a function of age in Mes V neurons.     

Up-regulation of A-type potassium currents protects neurons against cerebral ischemia

Excitotoxicity is the major cause of many neurologic disorders including stroke. Potassium currents modulate neuronal excitability and therefore influence the pathological process. A-type potassium current (I_A) is one of the major voltage-dependent potassium currents, yet its roles in excitotoxic cell death are not well understood. We report that, following ischemic insults, the I_A increases significantly in large aspiny (LA) neurons but not medium spiny (MS) neurons in the striatum, which correlates with the higher resistance of LA neurons to ischemia. Activation of protein kinase Cα increases I_A in LA neurons after ischemia. Cultured neurons from transgenic mice lacking both Kv1.4 and Kv4.2 subunits exhibit an increased vulnerability to ischemic insults. Increase of I_A by recombinant expression of Kv1.4 or Kv4.2 is sufficient in improving the survival of MS neurons against ischemic insults both in vitro and in vivo. These results, taken together, provide compelling evidence for a protective role of I_A against ischemia.     

Modulation of the voltage-gated sodium current in rat striatal neurons by DARPP-32, an inhibitor of protein phosphatase

DARPP-32 is a cyclic adenosine monophosphate-regulated inhibitor of protein phosphatase 1, highly enriched in striatonigral neurons. Stimulation of dopamine D₁ receptors increases phosphorylation of DARPP-32, whereas glutamate acting on N-methyl-d -aspartate receptors induces its dephosphorylation. Yet, to date, there is little direct evidence for the function of DARPP-32 in striatal neurons. Using a whole cell patch-clamp technique, we have studied the role of DARPP-32 in the regulation of voltage-gated sodium channels in rat striatal neurons maintained in primary culture. Injection of phospho-DARPP-32, but not of the unphosphorylated form, reduced the sodium current amplitude. This effect was similar to those induced by okadaic acid, with which there was no additivity and by tautomycin. Our results indicate that, in striatal neurons, sodium channels are under dynamic control by phosphorylation/dephosphorylation, and that phospho-DARPP-32 reduces sodium current by stabilizing a phosphorylated state of the channel or an associated regulatory protein. We propose that the DARPP-32-mediated modulation of sodium channels, via inhibition of phosphatase 1, contributes to the regulation of these channels by D₁ receptors and other neurotransmitters which influence the state of phosphorylation of DARPP-32.     

Functional properties and differential neuromodulation of Na(v)1.6 channels

The voltage-gated sodium channel Na_v1.6 plays unique roles in the nervous system, but its functional properties and neuromodulation are not as well established as for Na_V1.2 channels. We found no significant differences in voltage-dependent activation or fast inactivation between Na_V1.6 and Na_V1.2 channels expressed in non-excitable cells. In contrast, the voltage dependence of slow inactivation was more positive for Na_v1.6 channels, they conducted substantially larger persistent sodium currents than Na_v1.2 channels, and they were much less sensitive to inhibition by phosphorylation by cAMP-dependent protein kinase and protein kinase C. Resurgent sodium current, a hallmark of Na_v1.6 channels in neurons, was not observed for Na_V1.6 expressed alone or with the auxiliary β₄ subunit. The unique properties of Na_V1.6 channels, together with the resurgent currents that they conduct in neurons, make these channels well-suited to provide the driving force for sustained repetitive firing, a crucial property of neurons.     

Transforming growth factor-β2 selectively alters the developmental expression of the fast transient A-current in cultured rat superior cervical ganglion neurons

Cultures of neonatal rat superior cervical ganglion (SCG) were utilized to examine the ability of transforming growth factor-β2 (TGFβ2) to alter voltage-gated K⁺ channel development. Whole-cell patch clamp recordings were used to monitor changes in three separate K⁺ currents: A rapidly inactivating A-current (I_Af), a slowly inactivating A-current (I_As), and a non-inactivating current (I_K). Continuous TGFβ2 (10 ng/ml) treatment selectively altered the normal developmental decrease in I_Af expression in SCG neurons, but did not significantly change I_As or I_K expression. After 2 weeks of treatment, the mean I_Af current density in control cultures had decreased 67%, while the I_Af current density in TGFβ2 treated cultures remained near initial values (∼2.7-fold higher than control). This difference remained even after 4 weeks of exposure. TGFβ2 did not appear to change the activation kinetics or voltage-dependence of I_Af. These findings indicate that TGFβ2 may play an important role in modulating the development of neuronal excitability by regulating the expression of voltage-gated K⁺ channels. J. Neurosci. Res. 49:475–484, 1997. © 1997 Wiley-Liss, Inc.     

Reduced expression and activation of voltage‐gated sodium channels contributes to blunted baroreflex sensitivity in heart failure rats

Voltage‐gated sodium (Na_v) channels are responsible for initiation and propagation of action potential in the neurons. To explore the mechanisms of chronic heart failure (CHF)‐induced baroreflex dysfunction, we measured the expression and current density of Na_v channel subunits (Na_v1.7, Na_v1.8, and Na_v1.9) in the aortic baroreceptor neurons and investigated the role of Na_v channels in aortic baroreceptor neuron excitability and baroreflex sensitivity in sham and CHF rats. CHF was induced by left coronary artery ligation. The development of CHF (6–8 weeks after the coronary ligation) was confirmed by hemodynamic and morphological characteristics. Immunofluorescent data indicated that Na_v1.7 was expressed in A‐type (myelinated) and C‐type (unmyelinated) nodose neurons, but Na_v1.8 and Na_v1.9 were expressed only in C‐type nodose neurons. Real‐time RT‐PCR and Western blot data showed that CHF reduced mRNA and protein expression levels of Na_v channels in nodose neurons. In addition, using the whole‐cell patch‐clamp technique, we found that Na_v current density and cell excitability of the aortic baroreceptor neurons were lower in CHF rats than that in sham rats. Aortic baroreflex sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. Furthermore, Na_v channel activator (rATX II, 100 nM) significantly enhanced Na_v current density and cell excitability of aortic baroreceptor neurons and improved aortic baroreflex sensitivity in CHF rats. These results suggest that reduced expression and activation of the Na_v channels are involved in the attenuation of baroreceptor neuron excitability, which subsequently contributes to the impairment of baroreflex in CHF state. © 2010 Wiley‐Liss, Inc.     

Resveratrol Attenuates Aβ-Induced Early Hippocampal Neuron Excitability Impairment via Recovery of Function of Potassium Channels

Alzheimer’s disease (AD) is an age-related neurodegenerative disease. Amyloid-β (Aβ) is not only the morphological hallmark but also the initiator of the pathology process of AD. As a natural compound found in grapes, resveratrol shows a protective effect on the pathophysiology of AD, but the underlying mechanism is not very clear. This study was to investigate whether resveratrol could attenuate Aβ-induced early impairment in hippocampal neuron excitability and the underlying mechanism. The excitability and voltage-gated potassium currents were examined in rat hippocampal CA1 pyramidal neurons by using whole-cell patch-clamp technique. It was found that Aβ_25–35 increased the excitability of neurons. Resveratrol could reverse the Aβ_25–35-induced increase in the frequency of repetitive firing and the spike half-width of action potential (AP). Moreover, resveratrol can attenuate Aβ_25–35-induced decreases in transient potassium channel (I _A ) and delay rectifier potassium channel (I _K(DR)) of neurons. It was also found that resveratrol could decline the increase of protein kinase A (PKA) and inhibit the activation of PI3K/Akt signaling pathway induced by Aβ_25–35. The results suggest that resveratrol alleviates Aβ_25–35-induced dysfunction in hippocampal CA1 pyramidal neurons via recovery of the function of I _A and I _K(DR) by inhibiting the increase of PKA and the activation of PI3K/Akt signaling pathway.     

Growth Differentiation Factor-15 Produces Analgesia by Inhibiting Tetrodotoxin-Resistant Nav1.8 Sodium Channel Activity in Rat Primary Sensory Neurons

Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12264-021-00709-5.     

Arachidonic acid potently inhibits both postsynaptic-type Kv4.2 and presynaptic-type Kv1.4 IA potassium channels

Arachidonic acid (AA) is a free fatty acid membrane‐permeable second messenger that is liberated from cell membranes via receptor‐ and Ca²⁺‐dependent events. We have shown previously that extremely low [AA]_i (1 pm ) inhibits the postsynaptic voltage‐gated K⁺ current (I_A) in hippocampal neurons. This inhibition is blocked by some antioxidants. The somatodendritic I_A is mediated by Kv4.2 gene products, whereas presynaptic I_A is mediated by Kv1.4 channel subunits. To address the interaction of AA with these α‐subunits we studied the modulation of A‐currents in human embryonic kidney 293 cells transfected with either Kv1.4 or Kv4.2 rat cDNA, using whole‐cell voltage‐clamp recording. For both currents 1 pm [AA]_i inhibited the conductance by > 50%. In addition, AA shifted the voltage dependence of inactivation by ?9 (Kv1.4) and +6 mV (Kv4.2), respectively. Intracellular co‐application of Trolox C (10 μm ), an antioxidant vitamin E derivative, only slowed the effects of AA on amplitude. Notably, Trolox C shifted the voltage dependence of activation of Kv1.4‐mediated I_A by ?32 mV. Extracellular Trolox for > 15 min inhibited the AA effects on I_A amplitudes as well as the effect of intracellular Trolox on the voltage dependence of activation of Kv1.4‐mediated I_A. Extracellular Trolox further shifted the voltage dependence of activation for Kv4.2 by +33 mV. In conclusion, the inhibition of maximal amplitude of Kv4.2 channels by AA can explain the inhibition of somatodendritic I_A in hippocampal neurons, whereas the negative shift in the voltage dependence of inactivation apparently depends on other neuronal channel subunits. Both AA and Trolox potently modulate Kv1.4 and Kv4.2 channel α‐subunits, thereby presumably tuning presynaptic transmitter release and postsynaptic somatodendritic excitability in synaptic transmission and plasticity.     

Comparative study of lacosamide and classical sodium channel blocking antiepileptic drugs on sodium channel slow inactivation

Many antiepileptic drugs (AEDs) exert their therapeutic activity by modifying the inactivation properties of voltage‐gated sodium (Na_v) channels. Lacosamide is unique among AEDs in that it selectively enhances the slow inactivation component. Although numerous studies have investigated the effects of AEDs on Na_v channel inactivation, a direct comparison of results cannot be made because of varying experimental conditions. In this study, the effects of different AEDs on Na_v channel steady‐state slow inactivation were investigated under identical experimental conditions using whole‐cell patch‐clamp in N1E‐115 mouse neuroblastoma cells. All drugs were tested at 100 μM, and results were compared with those from time‐matched control groups. Lacosamide significantly shifted the voltage dependence of Na_v current (I_Na) slow inactivation toward more hyperpolarized potentials (by ?33 ± 7 mV), whereas the maximal fraction of slow inactivated channels and the curve slope did not differ significantly. Neither SPM6953 (lacosamide inactive enantiomer), nor carbamazepine, nor zonisamide affected the voltage dependence of I_Na slow inactivation, the maximal fraction of slow inactivated channels, or the curve slope. Phenytoin significantly increased the maximal fraction of slow inactivated channels (by 28% ± 9%) in a voltage‐independent manner but did not affect the curve slope. Lamotrigine slightly increased the fraction of inactivated currents (by 15% ± 4%) and widened the range of the slow inactivation voltage dependence. Lamotrigine and rufinamide induced weak, but significant, shifts of I_Na slow inactivation toward more depolarized potentials. The effects of lacosamide on Na_v channel slow inactivation corroborate previous observations that lacosamide has a unique mode of action among AEDs that act on Na_v channels. © 2012 Wiley Periodicals, Inc.