Carbamazepine and topiramate modulation of transient and persistent sodium currents studied in HEK293 cells expressing the Na(v)1.3 alpha-subunit

PURPOSE: The transient and the persistent Na(+) current play a distinct role in neuronal excitability. Several antiepileptic drugs (AEDs) modulate the transient Na(+) current and block the persistent Na(+) current; both effects contribute to their antiepileptic properties. The interactions of the AEDs carbamazepine (CBZ) and topiramate (TPM) with the persistent and transient Na(+) current were investigated. METHODS: HEK293 cells stably expressing the alpha-subunit of the Na(+) channel Na(V)1.3 were used to record Na(+) currents under voltage-clamp by using the patch-clamp technique in whole-cell configuration and to investigate the effects of CBZ and TPM. RESULTS: The persistent Na(+) current was present in all cells and constituted 10.3 +/- 3.8% of the total current. CBZ partially blocked the persistent Na(+) current in a concentration-dependent manner [median effective concentration (EC(50)), 16 +/- 4 microM]. CBZ also shifted the steady-state inactivation of the transient Na(+) current to negative potentials (EC(50), 14 +/- 11 microM). TPM partially blocked the persistent Na(+) current with a much higher affinity (EC(50), 61 +/- 37 nM) than it affected the steady-state inactivation of the transient Na(+) current (EC(50), 3.2 +/- 1.8 microM). For the latter effect, TPM was at most half as effective as CBZ. CONCLUSIONS: The persistent Na(+) current flowing through the alpha-subunit of the Na(V)1.3 channel is partially blocked by CBZ at about the same therapeutic concentrations at which it modulates the transient Na(+) current, adding a distinct aspect to its anticonvulsant profile. The TPM-induced partial block of the persistent Na(+) current, already effective at low concentrations, could be the dominant action of this drug on the Na(+) current.     

K+ channel properties in cultured mouse Schwann cells: dependence on extracellular K+

In cultured Schwann cells, single-channel and whole-cell K+ currents can be activated by depolarizing the membrane to values more negative than -50 mV. In elevated extracellular K+ concentration ([K+]o), however, single-channel activity and whole-cell currents could be recorded at more negative potentials. Thus, the threshold of current activation was shifted to more negative potentials. This shift in the activation threshold was only observed with normal (50-60 mM) intracellular [K+] levels; it was not apparent when [K+]i was elevated to 145 mM. The control of [K+]o on the gating properties of K+ channels may serve to enhance the capability of the Schwann cell to take up [K+]o and thus may serve for [K+] homeostasis in the peripheral nerve.     

Changes in Na(+) channel expression and nodal persistent Na(+) currents associated with peripheral nerve regeneration in mice

Patients with peripheral neuropathy frequently suffer from positive sensory (pain and paresthesias) and motor (muscle cramping) symptoms even in the recovery phase of the disease. To investigate the pathophysiology of increased axonal excitability in peripheral nerve regeneration, we assessed the temporal and spatial expression of voltage-gated Na(+) channels as well as nodal persistent Na(+) currents in a mouse model of Wallerian degeneration. Crushed sciatic nerves of 8-week-old C57/BL6J male mice underwent complete Wallerian degeneration at 1 week. Two weeks after crush, there was a prominent increase in the number of Na(+) channel clusters per unit area, and binary or broad Na(+) channel clusters were frequently found. Excess Na(+) channel clusters were retained up to 20 weeks post-injury. Excitability testing using latent addition suggested that nodal persistent Na(+) currents markedly increased beginning at week 3, and remained through week 10. These results suggest that axonal regeneration is associated with persistently increased axonal excitability resulting from increases in the number and conductance of Na(+) channels.     

Ion channels in cultured adult human Schwann cells.

Single channel currents have been recorded from cultured adult human Schwann cells. In both cell-attached and -excised (inside-out) patches, openings from a high-conductance (360 pS) channel were observed; measurements of the zero-current potential indicated that the channel was predominantly selective for chloride. Depolarizing and hyperpolarizing voltage steps activated the anion channel, which subsequently reverted to a closed state even in the presence of the maintained step. A second channel, with a conductance near 20 pS and with a current amplitude that increased with patch hyperpolarization, passed inward K+ currents in both cell-attached and inside-out patches. The mean open times for this channel were near 20 ms at the cell resting potential and decreased with patch hyperpolarization. The presence of these anion and cation selective channels in the human Schwann cell membrane would be consistent with a role for the cells in the regulation of extracellular K+.     

Cytoskeleton mediates inhibition of the fast Na+ current in respiratory brainstem neurons during hypoxia

Whole-cell Na+ currents (INa) were recorded in inspiratory neurons in a medullary slice preparation from neonatal mouse that contains the functional respiratory network. Hypoxia and metabolic poisoning with KCN rapidly inhibited INa by reducing the number of Na+ channels available for opening during depolarization. Application of agents specific for G-proteins, protein kinase C and A, intracellular Ca2+ and pH did not prevent the hypoxic inhibition of INa. The effects of hypo-osmolarity and hypoxia were additive, whereas hyperosmolarity partially prevented a subsequent hypoxic inhibition of INa. Cytochalasin B and colchicine decreased, and taxol or phalloidin increased INa and reduced its hypoxic inhibition. We conclude that cytoskeleton rearrangements during hypoxia are responsible for suppression of a fast INa in brainstem respiratory neurons, which could be mediated by the uncoupling of channel inactivation gates from cytoskeletal elements.     

Ionic channel currents in cultured neurons from human cortex

Ionic channels in human cortical neurons have not been studied extensively. HCN-1 and HCN-1A cells, which recently were established as continuous cultures from human cortical tissue, have been shown by histochemical and immunochemical methods to exhibit a neuronal phenotype, but expression of functional ionic channels was not demonstrated. For the present study, HCN-1 and HCN-1A cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal calf serum, in some cases supplemented with 10 ng/ml nerve growth factor, 10 μM forskolin, and 1 mM dibutyryl cyclic adenosine monophosphate to promote differentiation. Cells or membrane patches were voltage clamped using conventional patch clamp techniques. In HCN-1A cells, we identified a tetrodotoxin-sensitive Na⁺ current, two types of Ca²⁺ channel current, including L-type current and a second type that in some respects resembled N-type current, and four types of K⁺ current, including a delayed outward rectifier that showed voltage-dependent inactivation, two types of noninactivating Ca²⁺-activated K⁺ channels with slope conductances of 146 and 23 pS (K⁺ _iK⁺ _o 145 mM/5 mM), and less frequently, a noninactivating, intermediate conductance channel that was not sensitive to internal Ca²⁺. When HCN-1A cells were examined after 3 days of exposure to differentiating agents, pronounced morphological changes were evident but no differences in ionic currents were apparent. HCN-1 cells also exhibited K⁺ and Ca²⁺ channel currents, but Na⁺ currents were not detected in these cells. Our data provide additional evidence indicating a functional neuronal phenotype for HCN-1A cells, and represent the most comprehensive survey to date of the variety of ionic channels expressed by human cortical neurons. © 1993 Wiley-Liss, Inc.     

Functional expression of constitutive nitric oxide synthases regulated by voltage-gated Na+ and Ca2+ channels in cultured human astrocytes

We report the functional characterization of constitutive nitric oxide synthase(s) (NOS) such as neuronal and endothelial NOS in cultured human astrocytes. Exposure of cultured human astrocytes to 1 microM veratridine or 50 mM KCl produced a pronounced increase in a calmodulin-dependent NOS activity estimated from cGMP formation. The functional expression of voltage-gated Na(+) channel, which is estimated by the response to veratridine, appeared to be earlier (at second day in culture) than that of voltage-gated Ca(2+) channels, which are estimated by the response to the KCl stimulation (at fourth day in culture). The KCl-evoked NO synthesis was totally reversed by L-type Ca(2+) channel blockers such as nifedipine and verapamil, but not by omega-conotoxin GVIA, an N-type Ca(2+) channel blocker, or omega-agatoxin IVA, a P/Q-type Ca(2+) channel blocker. In addition, verapamil abolished the KCl-induced increase in the intracellular free Ca(2+) concentration. RT-PCR analysis revealed that mRNA for neuronal and endothelial NOS was expressed in human astrocytes. In addition, Western blot analysis and double labeling of NOS and glial fibrillary acidic protein (GFAP) showed that cultured human astrocytes expressed neuronal NOS and endothelial NOS as well as the alpha(1) subunit of Ca(2+) channel. These results suggest that human astrocytes express constitutive NOS that are regulated by voltage-gated L-type Ca(2+) channel as well as Na(+) channel.     

Glutamate opens Na+/K+ channels in cultured astrocytes

Glial cells from different brain regions and species are depolarized by the neurotransmitter glutamate. The depolarization or, if voltage-clamped at the resting membrane potential, the inward current induced by glutamate could be due either to activation of receptor-coupled ion channels or electrogenic uptake of the transmitter. In the present study we applied the patch-clamp technique in the whole-cell recording mode to analyze glutamate-induced currents in cultured astrocytes from rat cerebral hemispheres. At the resting membrane potential, glutamate induced an inward current ranging from 40 to 300 pA. This current decreased in size with depolarization and reversed at about 0 mV. The resulting current-to-voltage curve was linear and depended strongly on the transmembrane Na+ but not on the Ca++ or Cl- gradient. In the presence of glutamate, current noise increased at potentials positive or negative from the reversal potential indicating that ionic channels are activated by glutamate. Both kainate and quisqualate mimicked the effect of glutamate. We conclude that glutamate opens a Na+/K+ channel in cultured astrocytes because of activation of a receptor which shares many properties with the neuronal kainate/quisqualate receptor.     

Neuropathic pain is associated with increased nodal persistent Na+ currents in human diabetic neuropathy

Abstract Peripheral nerve injury alters function and expression of voltage gated Na⁺ channels on the axolemma, leading to ectopic firing and neuropathic pain/paresthesia. Hyperglycemia also affects nodal Na⁺ currents, presumably due to activation of polyol pathway and impaired Na⁺–K⁺ pump. We investigated changes in nodal Na⁺ currents in peripheral sensory axons and their relation with pain in human diabetic neuropathy. Latent addition using computerized threshold tracking was used to estimate nodal persistent Na⁺ currents in radial sensory axons of 81 diabetic patients. Of these, 36 (44%) had chronic neuropathic pain and severe paresthesia. Compared to patients without pain, those with pain had greater nodal Na⁺ currents (p = 0.001), smaller amplitudes of sensory nerve action potentials (SNAP) (p = 0.0003), and lower hemoglobin A1c levels (p = 0.006). Higher axonal Na⁺ conductance was associated with smaller SNAP amplitudes (p = 0.03) and lower hemoglobin A1c levels (p = 0.008). These results suggest that development of neuropathic pain depends on axonal hyperexcitability due to increased nodal Na⁺ currents associated with structural changes, but the currents could also be affected by the state of glycemic control. Our findings support the view that altered Na⁺ channels could be responsible for neuropathic pain/paresthesia in diabetic neuropathy.     

Single K channel currents in Schwann cells from normal and neurofibromatosis-affected damselfish

Damselfish neurofibromatosis is a naturally occurring disease of a tropical marine fish species. Affected fish exhibit peripheral nerve sheath tumors which contain morphologically abnormal Schwann cells (SC), similar to tumors encountered in the human disease neurofibromatosis type 1. Unitary A-type K channels in cell-attached membrane patches of SC were studied. Three different K channel conductances of approximately 5, 10, and 15 pS were present in both normal SC (n = 10) and tumored SC (n = 9). The variability in K channel conductance coincided with a large range of both mean open time and open probability in patches from normal and tumored SC. Channel open time histograms were fit by a single exponential. The ranges of time constants for open times irrespective of conductance were 0.26–9.3 msec in patches from normal cells and 0.60–0.73 msec in patches from tumored cells. These ranges were not significantly different. Inactivation time constants from ensemble averages of single channel currents averaged 83 ± 46 msec for normal SC and 44 ± 26 msec for tumored SC, which were not significantly different. These results suggest that A-type K currents from fish SC are composed of channels exhibiting multiple conductances and a variety of inactivation rates, which may account for the range of inactivation observed in whole cell currents but whose activity in membrane patches may not be wholly applicable to the whole cell currents. J. Neurosci. Res. 48:342–351, 1997. © 1997 Wiley-Liss, Inc.     

Galectin-3 inhibits Schwann cell proliferation in cultured sciatic nerve

The production of galectin-3, a carbohydrate-binding mammalian lectin, is upregulated in Schwann cells after peripheral nerve injury in areas where Schwann cells proliferate. Here we tested if galectin-3 affected proliferation of Schwann cells in cultured sciatic nerve segments. Galectin-3 significantly decreased the number of bromodeoxyuridine-labelled Schwann cell nuclei. Neither lactose nor a synthetic inhibitor directed against the carbohydrate-binding region abolished the effects of galectin-3. In addition, a mutant galectin-3 unable to bind endogenous carbohydrates had similar effects as normal galectin-3. We conclude that galectin-3 reduces proliferation of Schwann cells in cultured sciatic nerve segments by a mechanism which is independent of its carbohydrate-binding moiety.     

Effects of Ca antagonists and aminoglycoside antibiotics on Ca current in isolated outer hair cells of guinea pig cochlea

The effects of various Ca²⁺ antagonists and aminoglycoside antibiotics on the Ca²⁺ channel in isolated outer hair cells of the guinea pig were investigated using a whole-cell patch-clamp technique. The inhibitory action was in the order of La³⁺Cd²⁺Ni²⁺Co²⁺ for inorganic Ca²⁺ antagonists, and flunarizine = nicardipine > ω-Conotoxin > methaxyverapamil = diltiazem amiloride for orgaini ones. Aminoglycoside antibiotics also had antagonistic effects on the Ca²⁺ channel.     

Alterations of plasma membrane fatty acid composition modify the kinetics of Na current in cultured rat diencephalic neurons

Properties of inward Na⁺⁺ currents (I_Na) were examined in dissociated diencephalic neurons whose plasma membrane fatty acid composition had been altered. These neurons were grown in a defined medium supplemented with essential fatty acids (EFA) of either the w3 class (linolenic acid, 18:3w3) or the w6 class (linoleic acid, 18:2w6), which resulted in a two-fold increase in the plasma membrane phospholipid polyunsaturated fatty acid (PUFA) concentration. The properties of the inward I_Na of these neurons were compared with those of control neurons grown in the absence of any supplemented fatty acids. The I_Na of neuronssupplemented with a non-essential fatty acid (NFA) of w9 class (oleic acid, 18:1w9) was also examined. Several properties have been modified to different degrees. The ratio of the amplitudes between the fast and the slow decay components as well as the time constant of the fast decay component changed consistently and reversibly with the membrane phospholipid PUFA composition. The current-voltage relationships, channel selectivity, rates of inactivation and recovery from inactivation did not change. Other parameters, such as time-to-peak and steady-state inactivation curves, have changed in EFA- and NFA-supplemented cultures and did not reverse completely. These findings demonstrate that the kinetics of I_Na can be modified by fatty acid supplementation. These effects can be correlated, in part, with alterations in plasma membrane phospholipid fatty acid composition.     

Dependence of axon initial segment formation on Na+ channel expression

Spinal motor neurons were isolated from embryonic rats, and grown in culture. By 2 days in vitro, the axon initial segment was characterized by colocalization and clustering of Na+ channels and ankyrinG. By 5 days, NrCAM, and neurofascin could also be detected at most initial segments. We sought to determine, as one important aim, whether Na+ channels themselves played an essential role in establishing this specialized axonal region. Small hairpin RNAs (shRNAs) were used to target multiple subtypes of Na+ channels for reduced expression by RNA interference. Transfection resulted in substantial knockdown of these channels within the cell body and also as clusters at initial segments. Furthermore, Na+ currents originating at the initial segment, and recorded under patch clamp, were strongly reduced by shRNA. Control shRNA against a nonmammalian protein was without effect. Most interestingly, targeting Na+ channels also blocked clustering of ankyrinG, NrCAM, and neurofascin at the initial segment, although these proteins were seen in the soma. Thus, both Na+ channels and ankyrinG are required for formation of this essential axonal domain. Knockdown of Na+ channels was somewhat less effective when introduced after the initial segments had formed. Disruption of actin polymerization by cytochalasin D resulted in multiple initial segments, each with clusters of both Na+ channels and ankyrinG. The results indicate that initial segment formation occurs as Na+ channels are transported into the nascent axon membrane, diffuse distally, and link to the cytoskeleton by ankyrinG. Subsequently, other components are added, and stability is increased. A computational model closely reproduced the experimental results.     

Proliferation of Schwann cells in demyelinated rat sciatic nerve

Summary Experimental demyelination was induced by intraneural injection of anti-galctocerebroside serum into the sciatic nerves of rats. Schwann cells undergoing mitotic division were observed between days 3 to 9 after the injection and demyelinated segments were still associated with macrophages. Dividing Schwann cells were often present in association with both unmyelinated and myelinated fibers. Whether or not, daughter Schwann cells migrate along the same fiber towards neighboring demyelinated segments remains unclear. When Schwann cells attached to axon membranes of demyelinated segments were studied at later time points, they were present in clusters randomly at various regions of the segments. There was no proximo-distal gradient for the wave of Schwann cell proliferation. Mean Schwann cell internuclear distances were around 40–50 m at the earliest time of remyelination. Schwann cell redistribution and remyelination progressed regardless of the length of demyelinated segments.Supported by grants for the study of disorders of peripheral nerve, subacute myelo-optic neuropathy, and immunological diseases of the nervous system from the Intractable Diseases Division, Public Health Bureau, Ministry of Health and Welfare, Japan     

K+-dependence of Na+-Ca2+ exchange in type I vestibular sensory cells of guinea-pig.

The properties of the vestibular Na+-Ca2+ exchanger in mammalian type I vestibular sensory cells were studied using fura-2 fluorescence and immunocytochemical techniques. In the absence of external Na+, the activation of Na+-Ca2+ exchange in reverse mode required the presence of external K+ (K+o) and depended on K+o concentration. Alkali cations Rb+ and NH4+ but not Li+ or Cs+ substituted for K+o to activate the exchange. For pressure applications of 10 mm K+, the contribution of voltage-sensitive calcium channels to the increase in [Ca2+]i was < 15%. The dependence of the exchange on [K+]o was also recorded when the membrane potential was clamped using carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and monensin ionophores. In these conditions, where there was no intracellular Na+, the increase in [Ca2+]i was completely blocked. These physiological results suggest that in reverse mode, Ca2+ entry is driven by both an outward transport of Na+ and an inward transport of K+. The dependence of the vestibular Na+-Ca2+ exchanger on K+ is more reminiscent of the properties of the retinal type Na+-Ca2+ exchanger than those of the more widely distributed cardiac type exchanger. Moreover, the immunocytochemical localization of both types of exchange proteins in the vestibular sensory epithelium confirmed the presence in the vestibular sensory cells of a Na+-Ca2+ exchanger which is recognized by an antibody raised against retinal type and not by an antibody raised against the cardiac type.     

Glial Na+‐dependent ion transporters in pathophysiological conditions

Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na⁺ signaling influences and regulates important glial activities, and plays a role in neuron‐glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na⁺ pumps and Na⁺‐dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na⁺ homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na⁺‐dependent ion transporters, including Na⁺/K⁺ ATPase, Na⁺/Ca²⁺ exchangers, Na⁺/H⁺ exchangers, Na⁺‐K⁺‐Cl^? cotransporters, and Na⁺‐ cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na⁺‐dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na⁺ dynamics in different neurological disorders. GLIA 2016;64:1677–1697