Sodium Current Amplitude Research Articles

Activation of protein kinase C and inositol 1,4,5-triphosphate receptors antagonistically modulate voltage-gated sodium channels in striatal neurons

Regulation of voltage-gated sodium channels is crucial to firing patterns that constitute the output of medium spiny neurons (MSN), projecting neurons of the striatum. This modulation is thus critical for the final integration of information processed within the striatum. It has been shown that the adenylate cyclase pathway reduces sodium currents in MSN through channel phosphorylation by cAMP-dependent protein kinase. However, it is unknown whether a phospholipase C (PLC)-mediated signaling cascade could also modulate voltage-gated sodium channels within MSN. Using the whole-cell patch clamp technique, we investigated the effects of activation of two key components in PLC-mediated signaling cascades: protein kinase C (PKC) and inositol-1,4,5-triphosphate (IP 3) receptors on voltage-dependent sodium current. Cellular dialysis with phorbol 12-myristate 13-acetate, an activator of PKC, significantly reduced peak sodium current amplitude, while adenophostin A, an activator of IP 3 receptors, significantly increased peak sodium current amplitude. This effect of adenophostin was abolished by calcium chelation or by FK506, an inhibitor of calcineurin. These results suggest an antagonistic role of PKC and IP 3 in the modulation of striatal voltage-gated sodium channels, peak current amplitude being decreased through phosphorylation by PKC and increased through dephosphorylation by calcineurin.

Effect of clonidine on potential-dependent sodium currents in sensory neurons

In experiments on rat pups we studied the effect of clonidine on potential-dependent Na+ currents in dorsal root ganglia by the voltage clamp method. Clonidine decreased the amplitude of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents. The range of acting concentrations and the absence of modulatory effect of norepinephrine on the efficiency of clonidine-induced blockade of sodium currents suggest that this blockade results from a direct interaction of clonidine with sodium channels.

Functional characterization of the pentapeptide QYNAD on rNav1.2 channels and its NMR structure.

The endogenous pentapeptide QYNAD (Gln-Tyr-Asn-Ala-Asp) is present in human cerebrospinal fluid (CSF), and its concentration is increased in demyelinating diseases. QYNAD was synthesized and its action on the rNav1.2 voltage-gated sodium channel alpha-subunit was studied using whole-cell recordings in a heterologous expression system. The effects were seen only upon equilibration of the peptide in the external bath solution for at least 10 min before the commencement of whole-cell experiments. The steady-state activation curve showed a rightward shift of 10 mV, while the steady-state inactivation curve showed a leftward shift of 5 mV. Frequency-dependent inhibition of the sodium current amplitude was observed at 2-10 Hz, in the presence of external QYNAD, but was not seen when applied internally. Fits of the whole-cell sodium current traces by Hodgkin-Huxley equations revealed subtle changes in the voltage-dependent rate constants governing the transition of the activation and the inactivation gates. Two dimensional NMR spectroscopy revealed the absence of medium and long-range Nuclear Overhauser effects (NOEs), which indicates that the peptide does not adopt any canonical secondary structure in solution. In summary, our studies show that although the pentapeptide QYNAD does not have a defined structure in solution, it has defined actions on the rNav1.2 voltage-gated sodium channel isoform.

Interleukin-1β protects neurons via the interleukin-1 (IL-1) receptor-mediated Akt pathway and by IL-1 receptor-independent decrease of transmembrane currents in vivo

Recently, we have demonstrated that tumor necrosis factor-α (TNF-α) rescues retinal ganglion cells (RGCs) from retrograde cell death in vivo after axotomy of the optic nerve. The mechanism of RGC rescue was dependent on TNF-receptor I-mediated potassium current reduction and consecutive activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Here, we present evidence that interleukin-1β (IL-1β) also promotes RGC survival, but shows distinct differences with respect to its neuroprotective mechanisms. Using whole-cell and outside-out patch-clamp techniques, we observed that IL-1β decreased both inward sodium current amplitudes and outward potassium current amplitudes. Counteracting these effects by sodium or potassium channel opening inhibited the survival-promoting effects of this cytokine. IL-1β-induced current reduction could not be abolished by the interleukin-1 receptor antagonist, indicating that the electrophysiological effects of IL-1β are independent of interleukin-1 receptor I (IL-1RI) activation. Western blot analysis revealed an IL-1β-induced IL-1RI-dependent upregulation of phospho-Akt. Antagonism of the survival-promoting effects of IL-1β by PI3-K inhibition revealed the functional relevance of the PI3-K/Akt pathway in IL-1β-induced signal transduction in vivo.

Ceramide, a putative second messenger for nerve growth factor, modulates the TTX-resistant Na(+) current and delayed rectifier K(+) current in rat sensory neurons.

Because nerve growth factor (NGF) is elevated during inflammation and is known to activate the sphingomyelin signalling pathway, we examined whether NGF and its putative second messenger, ceramide, could modulate the excitability of capsaicin-sensitive adult and embryonic sensory neurons. Using the whole-cell patch-clamp recording technique, exposure of isolated sensory neurons to either 100 ng ml(-1) NGF or 1 microM N-acetyl sphingosine (C2-ceramide) produced a 3- to 4-fold increase in the number of action potentials (APs) evoked by a ramp of depolarizing current in a time-dependent manner. Intracellular perfusion with bacterial sphingomyelinase (SMase) also increased the number of APs suggesting that the release of native ceramide enhanced neuronal excitability. Glutathione, an inhibitor of neutral SMase, completely blocked the NGF-induced augmentation of AP firing, whereas dithiothreitol, an inhibitor of acidic SMase, was without effect. In the presence of glutathione and NGF, exogenous ceramide still enhanced the number of evoked APs, indicating that the sensitizing action of ceramide was downstream of NGF. To investigate the mechanisms of action for NGF and ceramide, isolated membrane currents were examined. Both NGF and ceramide facilitated the peak amplitude of the TTX-resistant sodium current (TTX-R I(Na)) by approximately 1.5-fold and shifted the activation to more hyperpolarized voltages. In addition, NGF and ceramide suppressed an outward potassium current (I(K)) by approximately 35 %. Ceramide reduced I(K) in a concentration-dependent manner. Isolation of the NGF- and ceramide-sensitive currents indicates that they were delayed rectifier types of I(K). The inflammatory prostaglandin, PGE(2), produced an additional suppression of I(K) after exposure to ceramide (approximately 35 %), suggesting that these agents might act on different targets. Thus, our findings indicate that the pro-inflammatory agent, NGF, can rapidly enhance the excitability of sensory neurons. This NGF-induced sensitization is probably mediated by activation of the sphingomyelin signalling pathway to liberate ceramide(s), wherein ceramide appears to be the second messenger involved in modulating neuronal excitability.

Hypoxia and persistent sodium current.

During prolonged depolarization of excitable cells, some voltage-activated, tetrodotoxin-sensitive sodium channels are resistant to inactivation and can continue to open for long periods of time, generating a "persistent" sodium current ( I(NaP)). The amplitude of I(NaP) is small [generally less than 1% of the peak amplitude of the transient sodium current ( I(NaT))], activates at potentials close to the resting membrane potential, and is more sensitive to Na channel blocking drugs than I(NaT). It is thought that persistent Na channels are generated by a change in gating of transient Na channels, possibly because of a change in phosphorylation or protein structure, e.g. loss of the inactivation gate. Drugs that block Na channels can prevent the increase in [Ca(2+)](i) in cardiac cells during hypoxia. Hypoxia increases the amplitude of I(NaP). Paradoxically, NO causes a similar increase in I(NaP) and the effects of both can be inhibited by reducing agents such as dithiothreitol and reduced glutathione. It is proposed that an increased inflow of Na(+) during hypoxia increases [Na(+)](i), which in turn reverses the Na/Ca exchanger so that [Ca(2+)](i) rises. An increase in I(NaP) and [Ca(2+)](i) could cause arrhythmias and irreversible cell damage.

Localization of cardiac sodium channels in caveolin-rich membrane domains: regulation of sodium current amplitude.

This study demonstrates that caveolae, omega-shaped membrane invaginations, are involved in cardiac sodium channel regulation by a mechanism involving the alpha subunit of the stimulatory heterotrimeric G-protein, Galpha(s), via stimulation of the cell surface beta-adrenergic receptor. Stimulation of beta-adrenergic receptors with 10 micromol/L isoproterenol in the presence of a protein kinase A inhibitor increased the whole-cell sodium current by a "direct" cAMP-independent G-protein mechanism. The addition of antibodies against caveolin-3 to the cell's cytoplasm via the pipette solution abrogated this direct G protein-induced increase in sodium current, whereas antibodies to caveolin-1 or caveolin-2 did not. Voltage-gated sodium channel proteins were found to associate with caveolin-rich membranes obtained by detergent-free buoyant density separation. The purity of the caveolar membrane fraction was verified by Western blot analyses, which indicated that endoplasmic/sarcoplasmic reticulum, endosomal compartments, Golgi apparatus, clathrin-coated vesicles, and sarcolemmal membranes were excluded from the caveolin-rich membrane fraction. Additionally, the sodium channel was found to colocalize with caveolar membranes by immunoprecipitation, indirect immunofluorescence, and immunogold transmission electron microscopy. These results suggest that stimulation of beta-adrenergic receptors, and thereby Galpha(s), promotes the presentation of cardiac sodium channels associated with caveolar membranes to the sarcolemma.

Novel sodium channel gene mutations in Blattella germanica reduce the sensitivity of expressed channels to deltamethrin

Pyrethroid insecticides alter the normal gating of voltage-gated sodium channels in the nervous system. Three sodium channel mutations (E434K, C764R, L993F) were recently identified in pyrethroid resistant German cockroach populations. In this report, we show that the L993F mutation decreased sodium channel sensitivity to the pyrethroid, deltamethrin, by five-fold in Xenopus oocytes. In contrast, neither E434K nor C764R alone decreased channel sensitivity to deltamethrin. However, E434K or C764R combined with L993F reduced deltamethrin sensitivity by 100-fold. Furthermore, concomitant presence of all three mutations (KRF) reduced channel sensitivity to deltamethrin by 500-fold. None of the mutations significantly affected channel gating. However, sodium current amplitudes from the mutant sodium channel carrying either E434K or C764R alone were much reduced compared to those of the wild-type channel or the channel carrying the double or triple mutations (KF, RF and KRF). These results indicated that evolution of sodium channel insensitivity in the German cockroach is achieved by sequential selection of a primary mutation L993F and two secondary mutations E434K and C764R, and concomitant presence of all three mutations dramatically reduced sodium channel sensitivity to deltamethrin.

A possible explanation for a neurotoxic effect of the anticancer agent oxaliplatin on neuronal voltage-gated sodium channels.

Oxaliplatin, a new widely used anticancer drug, displays frequent, sometimes severe, acute sensory neurotoxicity accompanied by neuromuscular signs that look like the symptoms observed in tetany and myotonia. The whole cell patch-clamp technique was employed to investigate the oxaliplatin effects on the electrophysiological properties of short-term cultured dorsal unpaired median (DUM) neurons isolated from the CNS of the cockroach Periplaneta americana. Within the clinical concentration range, oxaliplatin (40-500 microM), applied intracellularly, decreased the amplitude of the voltage-gated sodium current resulting in a reduction of half the amplitude of the action potential. For comparison, two other platinum derivatives, cisplatin and carboplatin, were found to be ineffective at reducing the sodium current amplitude. In addition, we compared the oxaliplatin action to those of its metabolites dichloro-diaminocyclohexane platinum (dach-Cl(2)-platin) and oxalate. Oxalate (500 microM) was found to be effective, like oxaliplatin, at reducing the inward sodium current amplitude, whereas dach-Cl(2)-platin (500 microM) failed to change the current amplitude. Interestingly, the effect of oxalate or oxaliplatin could be mimicked by using intracellularly applied 10 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), known as chelator of calcium ions. We concluded that oxaliplatin was capable of altering the voltage-gated sodium channels through a pathway involving calcium ions probably immobilized by its metabolite oxalate. The medical interest of preventing acute neurotoxic side effects of oxaliplatin by infusing Ca(2+) and Mg(2+) is discussed.

Potent Actions of the Pyrethroid Insecticides Cismethrin and Cypermethrin on Rat Tetrodotoxin-Resistant Peripheral Nerve (SNS/PN3) Sodium Channels Expressed in Xenopus Oocytes

Abstract The rat peripheral nerve tetrodotoxin (TTX)-resistant voltage-sensitive sodium channel (designated SNS/PN3) was expressed in Xenopus laevis oocytes, and the responses of the expressed channels to two pyrethroid insecticides, cismethrin and cypermethrin, were assessed by recording macroscopic sodium currents under voltage clamp conditions. Each pyrethroid produced two distinct modifications of sodium currents carried by SNS/PN3 channels: a sustained, slowly inactivating current evident during a depolarizing pulse and a prominent tail current following repolarization. In addition, high concentrations of cismethrin produced a reduction of the amplitude of the peak transient sodium current. Cismethrin-induced tail currents decayed with a first-order decay constant (τ) of 14.7 ms, whereas cypermethrin-induced tail currents were much more persistent (τ = 558 ms). Modified currents were obtained in the presence of nanomolar concentrations of each compound, and the amplitude of the modified currents exhibited a strong concentration dependence. High concentrations of cismethrin and cypermethrin shifted the voltage dependence of activation of SNS/PN3 sodium channels by ∼5 mV in the direction of hyperpolarization and the voltage dependence of steady-state inactivation by ∼8–10 mV in the direction of depolarization. The high sensitivity of the SNS/PN3 sodium channel to pyrethroids is consistent with the identification of this channel isoform as the principal carrier of the TTX-resistant, pyrethroid-sensitive sodium current of dorsal root ganglion neurons.

Protein kinase C-dependent modulation of Na+ currents increases the excitability of rat neocortical pyramidal neurones.

The effect of the protein kinase C (PKC) activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) on TTX-sensitive Na+ currents in neocortical pyramidal neurones was evaluated using voltage-clamp and intracellular current-clamp recordings. In pyramid-shaped dissociated neurones, the addition of OAG to the superfusing medium consistently led to a 30% reduction in the maximal peak amplitude of the transient sodium current (I(Na,T)) evoked from a holding potential of -70 mV. We attributed this inhibitory effect to a significant negative shift of the voltage dependence of steady-state channel inactivation (of approximately 14 mV). The inhibitory effect was completely prevented by hyperpolarising prepulses to potentials that were more negative than -80 mV. A small but significant leftward shift of INa,T activation was also observed, resulting in a slight increase of the currents evoked by test pulses at potentials more negative then -35 mV. In the presence of OAG, the activation of the persistent fraction of the Na+ current (INa,P) evoked by means of slow ramp depolarisations was consistently shifted in the negative direction by 3.9+/-0.5 mV, while the peak amplitude of the current was unaffected. In slice experiments, the OAG perfusion enhanced a subthreshold depolarising rectification affecting the membrane response to the injection of positive current pulses, and thus led the neurones to fire in response to significantly lower depolarising stimuli than those needed under control conditions. This effect was attributed to an OAG-induced enhancement of INa,P, since it was observed in the same range of potentials over which I(Na,P) activates and was completely abolished by TTX. The qualitative firing characteristics of both the intrinsically bursting and regular spiking neurones were unaffected when OAG was added to the physiological perfusing medium, but their firing frequency increased in response to slight suprathreshold depolarisations. The obtained results suggest that physiopathological events working through PKC activation can increase neuronal excitability by directly amplifying the I(Na,P)-dependent subthreshold depolarisation, and that this facilitating effect may override the expected reduction in neuronal excitability deriving from OAG-induced inhibition of the maximal INa, T peak amplitude.

Regulation of sodium currents through oxidation and reduction of thiol residues

Changes in redox state are involved in several physiological and pathophysiological processes. Previous experiments have demonstrated that nitric oxide can function as a reactive oxygen species, inhibiting neuronal sodium currents by nitrosylation of thiol residues. We hypothesized that nitric oxide and thiol oxidizers similarly modulate voltage-dependent sodium currents. Voltage-dependent sodium currents were studied with the whole-cell patch-clamp technique in NB41A3 neuroblastoma cells. The nitric oxide donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine did not affect sodium currents. In contrast, the thiol oxidizers thimerosal and 4,4′-dithiopyridine significantly inhibited sodium currents. The effect of thimerosal persisted after washout, but could be fully reversed by the reducing agent dithiothreitol. Reduced glutathione did not restore the sodium current amplitude when given extracellularly, while intracellular glutathione prevented the inhibitory effect of thimerosal. Pretreatment with the alkylating agent N-ethylmaleimide blocked the inhibitory action of thimerosal. Thiol oxidation caused a shift in the voltage dependence of fast and slow inactivation to more hyperpolarized potentials without concomitant effects on the voltage dependence of activation. Mercaptoethanol and reduced glutathione enhanced sodium currents by shifting the voltage dependence of inactivation to depolarized potentials. These results demonstrate that the oxidation and reduction of thiol residues alters the properties of voltage-sensitive sodium channels and may play an important role in the regulation of membrane excitability.

Potentiation of rat brain sodium channel currents by PKA in Xenopus oocytes involves the I-II linker.

Functional modulation of voltage-gated sodium channels affects the electrical excitability of neurons. Protein kinase A (PKA) can decrease sodium currents by phosphorylation at consensus sites in the cytoplasmic I-II linker. Once the sites are phosphorylated, however, additional PKA activity can increase sodium currents by an unknown mechanism. When the PKA sites were eliminated by substitutions of alanine for serine, peak sodium current amplitudes were increased by 20-80% when PKA was activated in Xenopus oocytes either by stimulation of a coexpressed beta(2)-adrenergic receptor or by perfusion with reagents that increase cAMP. Potentiation required the I-II linker of the brain channel, in that a chimeric channel in which the brain linker was replaced with the comparable linker from the skeletal muscle channel did not demonstrate potentiation. Using a series of chimeric and deleted channels, we demonstrate that potentiation is not dependent on any single region of the linker and that the extent of potentiation varies depending on the total length and the residues throughout the linker. These data are consistent with the hypothesis that potentiation by PKA is an indirect process involving phosphorylation of an accessory protein that interacts with the I-II linker of the sodium channel.

Isolation and pharmacological characterisation of δ-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin

δ-Atracotoxins (δ-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion α-toxins. We have isolated and determined the amino acid sequence of a novel δ-ACTX, designated δ-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with δ-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other δ-ACTXs. However, δ-ACTX-Hv1b is approximately 15–30-fold less potent than other δ-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between δ-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.

The Anticonvulsant SGB‐017 (ADCI) Blocks Voltage‐Gated Sodium Channels in Rat and Human Neurons: Comparison with Carbamazepine

SGB-017 (ADCI) is a novel anticonvulsant that blocks both voltage-activated sodium channels and N-methyl-D-aspartate (NMDA)-receptor-gated channels. Results by Rogawski et al. suggested that SGB-017 produces its anticonvulsant action primarily by inhibition of NMDA-receptor channels. However, SGB-017 is effective in several animal models of epilepsy that are unresponsive to NMDA antagonists. These results indicate that block of NMDA-receptor channels is not the only mechanism contributing to its anticonvulsant activity. Thus the effects of SGB-017 on neuronal sodium channels were investigated. Whole cell voltage-clamp techniques were used to record sodium currents in freshly dissociated rat superior cervical ganglion (SCG) and hippocampal neurons and cultured human NT2 neurons. The effects of SGB-017 on the amplitude of sodium currents, elicited by a depolarizing pulse to 0 mV from different holding potentials, were measured and compared with those of carbamazepine (CBZ). SGB-017 inhibited sodium currents in rat SCG and hippocampal neurons with a similar potency to CBZ. Like CBZ, the inhibition of sodium channels by SGB-017 was voltage dependent. Its median inhibitory concentration (IC50) for inhibition of sodium channels at depolarized holding potentials is similar to that for its inhibition of NMDA receptor channels. In human hNT2 neurons, SGB-017 was more potent than CBZ at inhibiting sodium currents. SGB-017 produces its anticonvulsant activity by blocking both sodium- and NMDA-receptor channels in a voltage- and use-dependent manner. The combination of these two mechanisms of action makes SGB-017 an effective AED in several different animal models of epilepsy.

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of −80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below −10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance–voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.

Cellular physiology of the neonatal rat cerebral cortex: intrinsic membrane properties, sodium and calcium currents.

The cellular physiology of the primary somatosensory cortex was studied in postnatal day (P) 0 to P5 rats using whole-cell patch-clamp recordings. Visually identified Cajal-Retzius, subplate, bifurcated pyramidal, and immature, putatively migrating neurons showed resting membrane potentials between -44 and -50 mV and TTX-sensitive action potentials. Immature pyramidal neurons with the smallest surface area ( approximately 1,600 microm(2)) revealed the largest input resistance ( approximately 1.8 GOmega), and subplate cells with the largest surface area ( approximately 6,200 microm(2)) showed an input resistance of approximately 1 GOmega. Ontogenetically older Cajal-Retzius and subplate cells revealed shorter and larger action potentials compared to bifurcated and immature pyramidal neurons. Whereas Cajal-Retzius and subplate cells responded to injection of depolarizing current pulses with a repetitive nonadapting and fast spiking firing pattern, immature pyramidal neurons showed strong adaptation. Subplate cells revealed the fastest action potentials, largest sodium current amplitude (-714 pA), and highest sodium current density (-38 microA/cm(2)), enabling these cells to transmit afferent activity faithfully to postsynaptic neurons. Whereas all cell types expressed a high-voltage-activated (HVA) calcium current, none of them showed a significant low-voltage-activated calcium current. The largest peak (-25.5 microA/cm(2)) and steady-state (-7.6 microA/cm(2)) HVA calcium current density could be observed in immature presumed migrating neurons. In contrast, Cajal-Retzius and subplate neurons showed a significantly lower peak (-4.9 microA/cm(2)) and steady-state (<-3.3 microA/cm(2)) HVA calcium current density. Whereas a large HVA calcium current may promote neuronal migration of immature neurons, low intracellular calcium levels may provoke apoptosis in Cajal-Retzius and subplate cells.

Differential role of GDNF and NGF in the maintenance of two TTX-resistant sodium channels in adult DRG neurons

Following sciatic nerve transection, the electrophysiological properties of small dorsal root ganglion (DRG) neurons are markedly altered, with attenuation of TTX-R sodium currents and the appearance of rapidly repriming TTX-S currents. The reduction in TTX-R currents has been attributed to a down-regulation of sodium channels SNS/PN3 and NaN. While infusion of exogenous NGF to the transected nerve restores SNS/PN3 transcripts to near-normal levels in small DRG neurons, TTX-R sodium currents are only partially rescued. Binding of the isolectin IB4 distinguishes two subpopulations of small DRG neurons: IB4 + neurons, which express receptors for the GDNF family of neurotrophins, and IB4 − neurons that predominantly express TrkA. We show here that SNS/PN3 is expressed in approximately one-half of both IB4 + and IB4 − DRG neurons, while NaN is preferentially expressed in IB4 + neurons. Whole-cell patch-clamp studies demonstrate that TTX-R sodium currents in IB4 + neurons have a more hyperpolarized voltage-dependence of activation and inactivation than do IB4 − neurons, suggesting different electrophysiological properties for SNS/PN3 and NaN. We confirm that NGF restores SNS/PN3 mRNA levels in DRG neurons in vitro and demonstrate that the trk antagonist K252a blocks this rescue. The down-regulation of NaN mRNA is, nevertheless, not rescued by NGF-treatment in either IB4 + or IB4 − neurons and NGF-treatment in vitro does not significantly increase the peak amplitude of the TTX-R current in small DRG neurons. In contrast, GDNF-treatment causes a twofold increase in the peak amplitude of TTX-R sodium currents and restores both SNS/PN3 and NaN mRNA to near-normal levels in IB4 + neurons. These observations provide a mechanism for the partial restoration of TTX-R sodium currents by NGF in axotomized DRG neurons, and demonstrate that the neurotrophins NGF and GDNF differentially regulate sodium channels SNS/PN3 and NaN.

Aluminum enhances the voltage activated sodium currents in the neurons of the pond snail Lymnaea stagnalis L.

1. The effects of aluminum on voltage activated sodium currents (VASCs) were investigated by using the conventional two-electrode voltage clamp technique in Lymnaea stagnalis L. neurons. The peak amplitude, kinetics, and voltage-dependence of activation and inactivation of the sodium currents were studied in the presence of 5-500 microM AlCl3, at pH = 7.7. 2. There was a significant concentration-dependent increase in the peak amplitude of sodium currents after Al treatment, ED50 = 67 microM. The threshold concentration of the enhancement was 50 microM. The maximal peak increase of 143% was caused by a 500 microM aluminum. The action of aluminum on VASCs developed slowly, and it is not recovered by washing within 20 min. 3. There was little alteration of the voltage-dependence of the current. It was not a significant effect on the activation- and inactivation time constants of INa, but the steady-state inactivation curve shifted to negative direction on the voltage axis in the presence of Al. 4. The leak currents were not influenced by aluminum up to the highest concentration applied.

Zebrafish Touch-Insensitive Mutants Reveal an Essential Role for the Developmental Regulation of Sodium Current

Developmental changes in neuronal connectivity and membrane properties underlie the stage-specific appearance of embryonic behaviors. The behavioral response of embryonic zebrafish to tactile stimulation first appears at 27 hr postfertilization. Because the touch response requires the activation of mechanosensory Rohon-Beard neurons, we have used whole-cell recordings in semi-intact preparations to characterize Rohon-Beard cell electrical membrane properties in several touch-insensitive mutants and then to correlate the development of excitability in these cells with changes in wild-type behavior. Electrophysiological analysis of mechanosensory neurons of touch-insensitive zebrafish mutants indicates that in three mutant lines that have been examined the sodium current amplitudes are reduced, and action potentials either have diminished overshoots or are not generated. In macho mutants the action potential never overshoots, and the sodium current remains small; alligator and steifftier show similar but weaker effects. The effects are specific to sodium channel function; resting membrane potentials are unaffected, and outward currents of normal amplitude are present. Developmental analysis of sodium current expression in mechanosensory neurons of wild-type embryos indicates that, during the transition from a touch-insensitive to a touch-sensitive embryo, action potentials acquire larger overshoots and briefer durations as both sodium and potassium currents increase in amplitude. However, in macho touch-insensitive mutants, developmental changes in action potential overshoot and sodium current are absent despite the normal regulation of action potential duration and potassium current. Thus, the maturation of a voltage-dependent sodium current promotes a behavioral response to touch. A study of these mutants will allow insight into the genes controlling the maturation of the affected sodium current.