Loss of β1 accessory Na+ channel subunits causes failure of carbamazepine,but not of lacosamide,in blocking high‐frequency firing via differential effects on persistent Na+ currents

Purpose: In chronic epilepsy, a substantial proportion of up to 30% of patients remain refractory to antiepileptic drugs (AEDs). An understanding of the mechanisms of pharmacoresistance requires precise knowledge of how AEDs interact with their targets. Many commonly used AEDs act on the transient and/or the persistent components of the voltage‐gated Na⁺ current (I_NaT and I_NaP, respectively). Lacosamide (LCM) is a novel AED with a unique mode of action in that it selectively enhances slow inactivation of fast transient Na⁺ channels. Given that functional loss of accessory Na⁺ channel subunits is a feature of a number of neurologic disorders, including epilepsy, we examined the effects of LCM versus carbamazepine (CBZ) on the persistent Na⁺ current (I_NaP), in the presence and absence of accessory subunits within the channel complex. Methods: Using patch‐clamp recordings in intact hippocampal CA1 neurons of Scn1b null mice, I_NaP was recorded using slow voltage ramps. Application of 100 μm CBZ or 300 μm LCM reduced the maximal I_NaP conductance in both wild‐type and control mice. Key Findings: As shown previously by our group in Scn1b null mice, CBZ induced a paradoxical increase of I_NaP conductance in the subthreshold voltage range, resulting in an ineffective block of repetitive firing in Scn1b null neurons. In contrast, LCM did not exhibit such a paradoxical increase, and accordingly maintained efficacy in blocking repetitive firing in Scn1b null mice. Significance: These results suggest that the novel anticonvulsant LCM maintains activity in the presence of impaired Na⁺ channel β₁ subunit expression and thus may offer an improved efficacy profile compared with CBZ in diseases associated with an impaired expression of β sub‐units as observed in epilepsy.     

Sodium and calcium mechanisms of rhythmic bursting in excitatory neural networks of the pre‐Bötzinger complex: a computational modelling study

The neural mechanisms generating rhythmic bursting activity in the mammalian brainstem, particularly in the pre‐Bötzinger complex (pre‐BötC), which is involved in respiratory rhythm generation, and in the spinal cord (e.g. locomotor rhythmic activity) that persist after blockade of synaptic inhibition remain poorly understood. Experimental studies in rodent medullary slices containing the pre‐BötC identified two mechanisms that could potentially contribute to the generation of rhythmic bursting: one based on the persistent Na⁺ current (I_NaP), and the other involving the voltage‐gated Ca²⁺ current (I_Ca) and the Ca²⁺‐activated nonspecific cation current (I_CAN), activated by intracellular Ca²⁺ accumulated from extracellular and intracellular sources. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modelling study, we investigated Na⁺‐dependent and Ca²⁺‐dependent bursting generated in single cells and heterogeneous populations of synaptically interconnected excitatory neurons with I_NaP and I_Ca randomly distributed within populations. We analysed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca²⁺ release, and the Na⁺/K⁺ pump in rhythmic bursting generated under different conditions. We show that a heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on I_NaP and/or I_CAN, or independent of both. We demonstrate that the operating bursting mechanism may depend on neuronal excitation, synaptic interactions within the network, and the relative expression of particular ionic currents. The existence of multiple oscillatory regimes and their state dependence demonstrated in our models may explain different rhythmic activities observed in the pre‐BötC and other brainstem/spinal cord circuits under different experimental conditions.     

Propylparaben reduces the excitability of hippocampal neurons by blocking sodium channels

Propylparaben (PPB) is an antimicrobial preservative widely used in food, cosmetics, and pharmaceutics. Virtual screening methodologies predicted anticonvulsant activity of PPB that was confirmed in vivo. Thus, we explored the effects of PPB on the excitability of hippocampal neurons by using standard patch clamp techniques. Bath perfusion of PPB reduced the fast-inactivating sodium current (I_Na) amplitude, causing a hyperpolarizing shift in the inactivation curve of the I_Na, and markedly delayed the sodium channel recovery from the inactivation state. Also, PPB effectively suppressed the riluzole-sensitive, persistent sodium current (I_NaP). PPB perfusion also modified the action potential kinetics, and higher concentrations of PPB suppressed the spike activity. Nevertheless, the modulatory effects of PPB did not occur when PPB was internally applied by whole-cell dialysis. These results indicate that PPB reduces the excitability of CA1 pyramidal neurons by modulating voltage-dependent sodium channels. The mechanistic basis of this effect is a marked delay in the recovery from inactivation state of the voltage-sensitive sodium channels. Our results indicate that similar to local anesthetics and anticonvulsant drugs that act on sodium channels, PPB acts in a use-dependent manner.     

Slo2/KNa Channels in Drosophila Protect against Spontaneous and Induced Seizure-like Behavior Associated with an Increased Persistent Na+ Current

Na⁺ sensitivity is a unique feature of Na⁺-activated K⁺ (K_Na) channels, making them naturally suited to counter a sudden influx in Na⁺ ions. As such, it has long been suggested that K_Na channels may serve a protective function against excessive excitation associated with neuronal injury and disease. This hypothesis, however, has remained largely untested. Here, we examine K_Na channels encoded by the Drosophila Slo2 (dSlo2) gene in males and females. We show that dSlo2/K_Na channels are selectively expressed in cholinergic neurons in the adult brain, as well as in glutamatergic motor neurons, where dampening excitation may function to inhibit global hyperactivity and seizure-like behavior. Indeed, we show that effects of feeding Drosophila a cholinergic agonist are exacerbated by the loss of dSlo2/K_Na channels. Similar to mammalian Slo2/K_Na channels, we show that dSlo2/K_Na channels encode a TTX-sensitive K⁺ conductance, indicating that dSlo2/K_Na channels can be activated by Na⁺ carried by voltage-dependent Na⁺ channels. We then tested the role of dSlo2/K_Na channels in established genetic seizure models in which the voltage-dependent persistent Na⁺ current (I_Nap) is elevated. We show that the absence of dSlo2/K_Na channels increased susceptibility to mechanically induced seizure-like behavior. Similar results were observed in WT flies treated with veratridine, an enhancer of I_Nap. Finally, we show that loss of dSlo2/K_Na channels in both genetic and pharmacologically primed seizure models resulted in the appearance of spontaneous seizures. Together, our results support a model in which dSlo2/K_Na channels, activated by neuronal overexcitation, contribute to a protective threshold to suppress the induction of seizure-like activity.SIGNIFICANCE STATEMENT Slo2/K_Na channels are unique in that they constitute a repolarizing K⁺ pore that is activated by the depolarizing Na⁺ ion, making them naturally suited to function as a protective “brake” against overexcitation and Na⁺ overload. Here, we test this hypothesis in vivo by examining how a null mutation of the Drosophila Slo2 (dSlo2)/K_Na gene affects seizure-like behavior in genetic and pharmacological models of epilepsy. We show that indeed the loss of dSlo2/K_Na channels results in increased incidence and severity of induced seizure behavior, as well as the appearance of spontaneous seizure activity. Our results advance our understanding of neuronal excitability and protective mechanisms that preserve normal physiology and the suppression of seizure susceptibility.     

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.     

SHORT COMUNICATION Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons

We investigated the effects of oxygen (O₂)/glucose deprivation on intracellular sodium concentration ([Na⁺]_i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O₂/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na⁺]_i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (d -amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na⁺/Ca²⁺-exchanger, affected these responses to O₂/glucose. The present results demonstrate that, in cortical neurons, O₂/glucose deprivation induces an early rise in [Na⁺]_i which cannot be ascribed to the activity of voltage gated Na⁺-channels, glutamate receptors or of the Na⁺/Ca²⁺-exchanger.     

Effects of leptin on pedunculopontine nucleus (PPN) neurons

Leptin, a hormone that regulates appetite and energy expenditure, is increased in obese individuals, although these individuals often exhibit leptin resistance. Obesity is characterized by sleep/wake disturbances, such as excessive daytime sleepiness, increased REM sleep, increased nighttime arousals, and decreased percentage of total sleep time. Several studies have shown that short sleep duration is highly correlated with decreased leptin levels in both animal and human models. Arousal and rapid eye movement (REM) sleep are regulated by the cholinergic arm of the reticular activating system, the pedunculopontine nucleus (PPN). The goal of this project was to determine the role of leptin in the PPN, and thus in obesity-related sleep disorders. Whole-cell patch-clamp recordings were conducted on PPN neurons in 9- to 17-day-old rat brainstem slices. Leptin decreased action potential (AP) amplitude, AP frequency, and h-current (I _H). These findings suggest that leptin causes a blockade of Na⁺ channels. Therefore, we conducted an experiment to test the effects of leptin on Na⁺ conductance. To determine the average voltage dependence of this conductance, results from each cell were equally weighted by expressing conductance as a fraction of the maximum conductance in each cell. I _Na amplitude was decreased in a dose-dependent manner, suggesting a direct effect of leptin on these channels. The average decrease in Na⁺ conductance by leptin was ~40 %. We hypothesize that leptin normally decreases activity in the PPN by reducing I _H and I _Na currents, and that in states of leptin dysregulation (i.e., leptin resistance) this effect may be blunted, therefore causing increased arousal and REM sleep drive, and ultimately leading to sleep-related disorders.     

Different voltage-gated sodium currents are expressed by human neuroblastoma NB69 cells when cultured in defined serum-free and in astroglial-conditioned media

Voltage-gated Na⁺ currents (I_Na) were analysed with the whole-cell patch-clamp technique in human neuroblastoma NB69 cells plated in serum-free “defined” medium (DM) or in “astroglial-conditioned” medium (CM). Cells survived in both media and expressed the microtubule associated protein 1A, indicating neuron-like differentiation. Two I_Na types with different time-, voltage-dependent properties and tetrodotoxin (TTX) sensitivities were expressed in DM and CM. The I_Na in DM-plated cells was present from day 4 and its surface density increased from 11 pA/pF (days 5–7) to 68 pA/pF (days 15–30). The underlying conductance (G_Na) half-activated (V_0A) at −24 mV. I_Na inactivation was fitted by single exponentials with 7.5 ms time constant (t_h) at the −35 mV half-inactivation voltage (V_0I). I_Na was not affected by 10 nM, was reduced (65%) by 100 nM, and not completely abolished (92%) by 300 nM tetrodotoxin (TTX). The I_Na of CM-plated cells appeared at day 3–4 and its surface density increased from 14 pA/pF (days 3–6) to 28 pA/pF (days 11–14). The G_Na V_0A was −29 mV and inactivation was fitted by single exponentials with 2.6 ms t_h at the −58 mV V_0I. This I_Na was reduced (55%) by 10 nM and totally abolished by 100 nM tetrodotoxin (TTX). In conclusion, NB69 cells displayed a slow, “TTX-resistant,” or a fast, “TTX-sensitive” I_Na in DM and CM, respectively, suggesting that the CM contained diffusible trophic factors of astroglial origin that induced the expression of a different Na⁺ channel type. About half of the CM- and DM-plated cells also displayed a persistent Na⁺ current (I_NaP). © 1997 Wiley-Liss Inc.     

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta

We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na⁺]_i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na⁺]_i. In dopamine neurons voltage-clamped at a holding potential of ?60 mV elevations of [Na⁺]_i were induced by long-lasting (45–60 s) voltage jumps to more negative membrane potentials (–90 to ?120 mV) but not by corresponding voltage jumps to ?30 mV. These hyperpolarization-induced elevations of [Na⁺]_i were depressed during inhibition of I_h, a hyperpolarization-activated inward current, by Cs⁺. Hyperpolarization-induced elevations in [Na⁺]_i might occur also in other cell types which express a powerful I_h and might signal lack of postsynaptic activity.     

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.     

Effect of charybdotoxin and leiurotoxon I on potassium currents in bullfrog sympathetic ganglion and hippocampal neurons

The effects of charybdotoxin and leiurotoxin I were examined on several classes of K⁺ currents in bullfrog sympathetic ganglion and hippocampal CA1 pyramidal neurons. Highly purified preparations of charybdotoxin selectively blocked a large voltage- and Ca²⁺-dependent K⁺ current (I_c) responsible for action potential repolarization (IC₅₀ = 6 nM) while leiurotoxin I selectively blocked a small Ca²⁺-dependent K⁺ conductance (I_AHP) responsible for the slow afterhyperpolarization following an action potential (IC₅₀ = 7.5 nM) in bullfrog sympathetic ganglion neurons. Neither of the toxins had a significant effects on other K⁺ currents (M-current [I_M], A-current [I_A] and the delayed rectifier [I_KD] present in these cells. Leiurotoxin I at a concentration of 20 nM had no detectable effect on currents in hippocampal CA1 pyramidal neurons. This lack of effect on I_AHP in central neurons suggests that the channels underlying slow AHPs in those neurons are pharmacologically distinct from analogous channels in peripheral neurons.     

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.     

Two distinct types of depolarizing afterpotentials are differentially expressed in stellate and pyramidal‐like neurons of entorhinal‐cortex layer II

Two types of principal neurons, stellate cells and pyramidal‐like cells, are found in medial entorhinal‐cortex (mEC) layer II, and are believed to represent two distinct channels of information processing and transmission in the entorhinal cortex–hippocampus network. In this study, we found that depolarizing afterpotentials (DAPs) that follow single action potentials (APs) evoked from various levels of holding membrane voltage (V_h) show distinct properties in the two cells types. In both, an evident DAP followed the AP at near‐threshold V_h levels, and was accompanied by an enhancement of excitability and spike‐timing precision. This DAP was sensitive to voltage‐gated Na⁺‐channel block with TTx, but not to partial removal of extracellular Ca²⁺. Application of 5‐μM anandamide, which inhibited the resurgent and persistent Na⁺‐current components in a relatively selective way, significantly reduced the amplitude of this particular DAP while exerting poor effects on the foregoing AP. In the presence of background hyperpolarization, DAPs showed an opposite behavior in the two cell types, as in stellate cells they became even more prominent, whereas in pyramidal‐like cells their amplitude was markedly reduced. The DAP observed in stellate cells under this condition was strongly inhibited by partial extracellular‐Ca²⁺ removal, and was sensitive to the low‐voltage‐activated Ca²⁺‐channel blocker, NNC55‐0396. This Ca²⁺ dependence was not observed in the residual DAP evoked in pyramidal‐like cells from likewise negative V_h levels. These results demonstrate that two distinct mechanism of DAP generation operate in mEC layer‐II neurons, one Na⁺‐dependent and active at near‐threshold V_h levels in both stellate and‐pyramidal‐like cells, the other Ca²⁺‐dependent and only expressed by stellate cells in the presence of background membrane hyperpolarization. © 2015 Wiley Periodicals, Inc.     

Colonic inflammation up-regulates voltage-gated sodium channels in bladder sensory neurons via activation of peripheral transient potential vanilloid 1 receptors

Background Primary sensory neurons express several types of ion channels including transient receptor potential vanilloid 1 (TRPV1) and voltage‐gated Na⁺ channels. Our previous studies showed an increased excitability of bladder primary sensory and spinal neurons triggered by inflammation in the distal colon as a result of pelvic organ cross‐sensitization. The goal of this work was to determine the effects of TRPV1 receptor activation by potent agonists and/or colonic inflammation on voltage‐gated Na⁺ channels expressed in bladder sensory neurons. Methods Sprague–Dawley rats were treated with intracolonic saline (control), resiniferatoxin (RTX, 10^?7 mol L^?1), TNBS (colonic irritant) or double treatment (RTX followed by TNBS). Key Results TNBS‐induced colitis increased the amplitude of total Na⁺ current by two‐fold and of tetrodotoxin resistant (TTX‐R) Na⁺ current by 78% (P ≤ 0.05 to control) in lumbosacral bladder neurons during acute phase (3 days post‐TNBS). Instillation of RTX in the distal colon caused an enhancement in the amplitude of total Na⁺ current at ?20 mV from ?112.1 ± 18.7 pA/pF (control) to ?183.6 ± 27.8 pA/pF (3 days post‐RTX, P ≤ 0.05) without changes in TTX resistant component. The amplitude of net Na⁺ current was also increased by 119% at day 3 in the group with double treatment (RTX followed by TNBS, P ≤ 0.05 to control) which was significantly higher than in either group with a single treatment. Conclusions & Inferences These results provide evidence that colonic inflammation activates TRPV1 receptors at the peripheral sensory terminals leading to an up‐regulation of voltage gated Na⁺ channels on the cell soma of bladder sensory neurons. This mechanism may underlie the occurrence of peripheral cross‐sensitization in the pelvis and functional chronic pelvic pain.     

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K⁺ current (I_M), Ca²⁺‐gated K⁺ currents (I_Ca(K)'s) and Na⁺/K⁺‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I_M contribution to the sAHP was found in these neurons. Both I_Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α₁‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α₁‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.     

Hyperpolarization-activated ion currents in cultured rat cortical and spinal cord astrocytes

Hyperpolarization-activated currents were recorded from rat brain cortical and spinal cord astrocytes maintained in culture. Spinal cord astrocytes expressed primarily an inward rectifier potassium current characterized by time-dependent inactivation, a strong dependence on extracellular Na⁺ and insensitivity to intracellular GTP-γ-S (0.2 mM). In cortical astrocytes voltage clamp protocols aimed to elicit currents activated at, or negative to cell membrane potentials led to the development of two distinct ion currents. The most prominent current resembled the inward rectifier potassium current. This component was sensitive to blockade by extracellular cesium and was greatly reduced during recordings performed with GTP-γ-S (0.2 Mm) added to the pipette solutions. The remaining current component was similar to the endothelial I_ha current. I_ha conductance was enhanced by extracellular potassium and the current reversal potential behaved as expected for a mixed cation, Na⁺/K⁻ current, I_ha was nearly abolished after removal of extracellular Na⁺. These results are consistent with the expression of a novel mixed cation conductance in glial cells, possibly involved in extracellular potassium buffering. © 1996 Wiley-Liss, Inc.     

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.     

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.     

Modulation of the voltage-dependent sodium channel by agents affecting G-proteins: a study in Xenopus oocytes injected with brain RNA

The effects of agents known to affect G-proteins on voltage-dependent, tetrodoxin-sensitive Na⁺ channels were studied in Xenopus oocytes injected with rat brain RNA, using two-electrode voltage-clamp technique. The non-hydrolysable analogue of GTP, GTP-γ-S, Known to activate G-proteins, inhibited the Na⁺ current (I_Na). The decrease in the amplitude of I_Na was not accompanied by changes in activation or inactivation characteristics of the channel. The non-hydrolysable analogue of GDP, GDP-β-S, had no effect on I_Na. The responses to γ-aminobutyric acid and kainate in the same oocytes were also attenuated by GTP-γ-S. Pertussis toxin, which inactivates some G-proteins by catalyzing thier ADP-ribosylation, enhanced I_Na, but did not prevent the inhibition of I_Na by GTP-γ-S. We conclude that the Na⁺ channel, and possibly the GABA and kainate receptors and/or channels, are coupled to a G-protein. The activation of the G-protein modulates the channels either directly, or via activation of biochemical cascade possibly involving production of second messengers and channel phosphorylation.