The effects of pumiliotoxin-B on sodium currents in guinea pig hippocampal neurons

The actions of pumiliotoxin-B, extracted from the skin of the frog Dendrobates pumilio, were examined on hippocampal slices and on acutely dissociated hippocampal neurons from the adult guinea pig. Application of 0.5-1 microM pumiliotoxin-B to hippocampal slices caused spontaneous, repetitive field discharges in the CA3 subfield. In whole-cell patch-clamp recordings of isolated CA1 and CA3 neurons, 1-2 microM pumiliotoxin-B shifted the midpoint of Na+ current activation by -11.4 +/- 1.1 mV. This shift was not dependent upon prior activation of the sodium channel. Pumiliotoxin-B did not block macroscopic Na+ inactivation but did reduce the apparent voltage-dependence of inactivation such that currents decayed faster at membrane potentials more negative than -30 mV. Single-channel recordings of sodium currents from excised membrane patches indicated that pumiliotoxin-B had little or no effect on channel closings due to entry into inactivated state(s) but did increase the rate of channel closings due to reversal of channel opening. The increase in the channel closing rate was consistent with a +8.7 mV shift in voltage sensitivity. Negative shifts in activation and positive shifts in closing rates implied a negative shift in the voltage-dependence of channel opening, suggesting that pumiliotoxin-B increases the rate of Na+ channel opening and closing in cells at rest, which could result in spontaneous activity in the neurons.     

Voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671

A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay. The rates of early activation and subsequent inactivation accelerate and approach a maximum in response to test potentials, V, of greater depolarization. The magnitude of peak sodium current increased from negligible values below V = -50 mV and reached a maximum at V = -3.6 mV +/- 2.7 mV (mean +/- S.E.M., n = 12). Sodium currents reversed at V = + 70 mV, near the predicted Nernst equilibrium potential for a Na+ selective channel. The peak sodium conductance, gpeak increased with depolarizing voltages to a maximum at V = approximately 0 mV, exhibiting half-activation voltage at V approximately equal to -36.8 mV and an e-fold change in gpeak/9.5 mV. The Hodgkin-Huxley inactivation parameter h infinity indicates that at V = -73.6 mV half of the sodium currents were inactivated. Single channel current recordings demonstrated the occurrence of discrete events: the latency for first opening was shorter as the depolarizing pulse became more positive. The single-channel current amplitude was ohmic with a slope conductance, gamma = 17.13 pS +/- 0.66 pS. Sodium channel currents were reversibly blocked by tetrodotoxin (TTX).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the involvement of more than one mRNA species in controlling the inactivation process of rat and rabbit brain Na channels expressed in Xenopus oocytes

The properties of rat and rabbit brain sodium (Na) channels expressed in Xenopus oocytes following either unfractionated or high-molecular-weight mRNA injections were compared to assess the relative contribution of different size messages to channel function. RNA was size-fractionated on a sucrose gradient and a high-molecular-weight fraction (7-10 kilobase) encoding the alpha-subunit gave rise to functional voltage-dependent Na channels in the oocyte membrane. Single-channel conductance, mean open time, and time to first opening were all similar to the values for channels following injection of unfractionated RNA. In contrast, inactivation properties were markedly different; Na currents from high-molecular-weight RNA inactivated with a several-fold smaller macroscopic inactivation rate and showed a steady-state voltage dependence that was shifted in the depolarizing direction by at least 10 mV relative to that for unfractionated RNA. Single-channel recording revealed that the kinetic difference arose from a greater probability for high-molecular-weight RNA induced channels to reopen during a depolarizing voltage step. Pooling all gradient fractions and injecting this RNA into oocytes led to the appearance of Na channels with inactivation properties indistinguishable from those following injection of unfractionated RNA. These results suggest that mRNA species not present in the high-molecular-weight fraction can influence the inactivation process of rat brain Na channels expressed in Xenopus oocytes. This mRNA may encode beta-subunits or other proteins that are involved in posttranslational processing of voltage-dependent Na channels.     

Single inward rectifier channels in horizontal cells

The ion channels responsible for inward rectification in horizontal cells were studied using the patch clamp technique applied to isolated cells from goldfish retina. Inward currents recorded from these cells were identified as due to the opening of inward rectifier channels based on their ion selectivity, channel gating behavior, and the effects of external blocking ions. The single channel conductance was 20 pS in 125 mM external K+. The null current potential shifted with changes in the K+ concentration as expected for a channel permeable to K+, and the channel appeared to have little permeability to Na+. The probability of a channel being in an open state increased as the membrane was hyperpolarized from the K+ equilibrium potential (0 to -10 mV) over potentials ranging to -80 mV, in the presence of external Na+. The closing rate was insensitive to membrane potential in the presence of external Na+. The opening rate of the channel increased as the membrane was hyperpolarized. The increase in the probability of a channel being open at negative potentials was therefore caused by the voltage sensitivity of the rate of channel opening.     

Resveratrol inhibits Na+ currents in rat dorsal root ganglion neurons

Resveratrol, a phytoalexin found in grapevines, exerts neuroprotective, cancer chemopreventive, antiinflammatory and cardioprotective activities. Studies have also shown that resveratrol exhibits analgesic effects. Cyclooxygenase inhibition and K+ channel opening have been suggested as underlying mechanisms for the resveratrol-induced analgesia. Here, we investigated the effects of resveratrol on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na+ currents in rat dorsal root ganglion (DRG) neurons. Resveratrol suppressed both Na+ currents evoked at 0 mV from -80 mV. TTX-S Na+ current (K(d), 72 microM) was more susceptible to resveratrol than TTX-R Na+ current (K(d), 211 microM). Although the activation voltage of TTX-S Na+ current was shifted in the depolarizing direction by resveratrol, that of TTX-R Na+ current was not. Resveratrol caused a hyperpolarizing shift of the steady-state inactivation voltage and slowed the recovery from inactivation of both Na+ currents. However, no frequency-dependent inhibition of resveratrol on either type of Na+ current was observed. The suppression and the unfavorable effects on the kinetics of Na+ currents in terms of the excitability of DRG neurons may make a great contribution to the analgesia by resveratrol.     

Block of two subtypes of sodium channels in cockroach neurons by indoxacarb insecticides

Indoxacarb, a novel insecticide, and its decarbomethoxyllated metabolite, DCJW, are known to block voltage-gated Na(+) channels in insects and mammals, but the mechanism of block is not yet well understood. The present study was undertaken to characterize the action of indoxacarb and DCJW on cockroach Na(+) channels. Na(+) currents were recorded using the whole-cell patch clamp technique from neurons acutely dissociated from thoracic ganglia of the American cockroach Periplaneta americana L. Two types of tetrodotoxin-sensitive Na(+) currents were observed, with different voltage dependencies of channel inactivation. Type-I Na(+) currents were inactivated at more negative potentials than type-II Na(+) currents. As a result, these two types of Na(+) channels responded to indoxacarb compounds differentially. At a holding potential of -100 mV, type-I Na(+) currents were inhibited reversibly by 1 microM indoxacarb and irreversibly by 1 microM DCJW in a voltage-dependent manner, whereas type-II Na(+) currents were not affected by either of the compound. However, type-II Na(+) currents were inhibited by indoxacarb or DCJW at more depolarizing membrane potentials, ranging from -60 to -40 mV. The slow inactivation curves of type-I and type-II Na(+) channels were significantly shifted in the hyperpolarizing direction by indoxacarb and DCJW, suggesting that these compounds have high affinities for the inactivated state of the Na(+) channels. It was concluded that the differential blocking actions of indoxacarb insecticides on type-I and type-II Na(+) currents resulted from their different voltage dependence of Na(+) channel inactivation. The irreversible nature of DCJW block may be partially responsible for its potent action in insects.     

Voltage clamp analysis of tetrodotoxin-sensitive and -insensitive sodium channels in rat muscle cells developing in vitro

Sodium currents in cultured rat muscle cells converted to myoballs by treatment with colchicine were recorded using a giga-ohm seal voltage clamp procedure in the whole cell configuration. The mean peak Na+ conductance of the myoballs was 90 pS/microns2 of surface membrane. Half-maximal activation of Na+ currents was observed for test pulses to -31 mV and half-maximal inactivation was observed for prepulses to -74 mV. Titration of the inhibition of Na+ currents by tetrodotoxin (TTX) yielded a biphasic inhibition curve consistent with the presence of two classes of Na+ channels differing in affinity for TTX. The TTX-sensitive channels carried 28% of the Na+ current and had an apparent KD for TTX of 13 nM at 20 degrees C. The TTX-insensitive Na+ channels had an apparent KD for TTX of 3.2 microns. Inhibition of TTX-insensitive Na+ channels by TTX was enhanced by repetitive stimulation of the myoballs at 2 Hz, whereas the inhibition of TTX-sensitive Na+ channels by TTX was not frequency dependent. We conclude that rat muscle cells developing in vitro synthesize physiologically functional, TTX-sensitive Na+ channels in the absence of innervation. These channels, which are characteristic of adult skeletal muscle, function in parallel with TTX-insensitive Na+ channels that are present in embryonic muscle.     

Anandamide suppression of Na+ currents in rat dorsal root ganglion neurons

Anandamide, the ethanolamide of arachidonic acid, is an endogenous cannabinoid. It is an agonist at CB1 and CB2 cannabinoid receptors as well as the vanilloid receptor, VR1. It is analgesic in inflammatory and neuropathic pain. Both central and peripheral mechanisms are considered to participate in its analgesia. Primary sensory neurons express Na+ currents that are involved in the pathogenesis of pain. We examined the effect of anandamide on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na+ currents in rat dorsal root ganglion neurons. Anandamide inhibited both Na+ currents in a concentration-dependent manner. At a membrane potential of -80 mV, the current inhibition was greater in TTX-S than TTX-R currents (K(d); 5.4 microM vs. 38.4 microM). The activation and inactivation became faster in TTX-R current but not in TTX-S current. Anandamide did not alter the activation voltage in either type of current. It, however, produced a hyperpolarizing shift of the steady-state inactivation voltage in both types of currents. The maximum availability at a large negative potential was not reduced by anandamide. Thus, anandamide seems to affect inactivated Na+ channels rather than resting channels. The inhibition of Na+ currents was not reversed by AM 251 (a CB1 antagonist), AM 630 (a CB2 antagonist) or capsazepine (a VR1 antagonist), suggestive of a direct action of anandamide on Na+ channels. The inhibition of Na+ currents in sensory neurons may contribute to the anandamide analgesia.     

Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons.

Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in acutely isolated hippocampal neurons through phosphorylation of the alpha subunit of the Na+ channel by cAMP-dependent protein kinase (PKA). Here we report that neuromodulation of Na+ currents by DA receptors via PKA is voltage-dependent in the range of -110 to -70 mV and is also sensitive to concurrent activation of protein kinase C (PKC). Depolarization enhanced the ability of D1-like DA receptors to reduce peak Na+ currents via the PKA pathway. Similar voltage-dependent modulation was observed when PKA was activated directly with the membrane-permeant PKA activator DCl-cBIMPS (cBIMPS; 20 microM), indicating that the membrane potential dependence occurs downstream of PKA. PKA activation caused only a small (-2.9 mV) shift in the voltage dependence of steady-state inactivation and had no effect on slow inactivation or on the rates of entry into the fast or slow inactivated states, suggesting that another mechanism is responsible for coupling of membrane potential changes to PKA modulation. Activation of PKC with a low concentration of the membrane-permeant diacylglycerol analog oleylacetyl glycerol also potentiated modulation by SKF 81297 or cBIMPS, and these effects were most striking at hyperpolarized membrane potentials where PKA modulation was not stimulated by membrane depolarization. Thus, activation of D1-like DA receptors causes a strong reduction in Na+ current via the PKA pathway, but it is effective primarily when it is combined with depolarization or activation of PKC. The convergence of these three distinct signaling modalities on the Na+ channel provides an intriguing mechanism for integration of information from multiple signaling pathways in the hippocampus and CNS.     

Two components of calcium channel current in embryonic chick skeletal muscle cells developing in culture

The properties of the Ca channel currents in chick skeletal muscle cells (myoballs) in culture were studied using a suction pipette technique which allows internal perfusion and voltage clamp. The Ca channel currents as carried by Ba ions were recorded, after suppression of currents through ordinary Na, K and Cl channels by absence of Na, K and Cl ions, by external TEA, by internal EGTA and by observing the Ba currents instead of the Ca currents. Two components of Ba current could be distinguished. One was present only if the myoballs were held at relatively negative holding potentials below -50 mV. This component first became detectable at clamp potentials of about -50 mV and reached a maximum between -10 and -20 mV. During long clamp steps, it became inactivated completely. The inactivation process of this component at a clamp potential of -30 mV was well fitted to a single exponential with a time constant of about -20 ms. Half-maximal steady-state inactivation was observed at -63 mV. The other component persisted even at relatively positive holding potentials above -40 mV, was observed during clamp pulses to -20 mV and above, and reached a maximum between +10 and +20 mV. This component inactivated very little; a substantial fraction of this component remained at the end of clamp pulses lasting 1 s. The inactivation process of this component at a clamp potential of -10 mV apparently followed a single exponential with a time constant of about 1 s. Half-maximal steady-state inactivation was attained at -33 mV. Both components of Ba current were blocked by Co ions, but organic Ca channel blocker D600 preferentially blocked the high-threshold, slowly inactivating component. The relationship between the current amplitude and the concentration of the external Ba ions was different between the two components. Furthermore, the two components of Ba current also differed in their developmental profile. These findings demonstrate the existence of two distinct types of Ca channels in the early stages of chick muscle cell development.     

Single K+ channel properties in cultured mouse Schwann cells: conductance and kinetics

Cultured Schwann cells are characterized by a strong outward rectification of the membrane; the threshold of the outward currents is close to the resting membrane potential of about -50 mV (Gray et al.: In Ritchie, Keynes (eds): Ion Channels in Neural Membranes. New York: Alan R. Liss, Inc., pp 145-157, 1986). These outward currents show up a heterogeneity among the cultured Schwann cells: some cells displayed inactivating, others non-inactivating outward currents (Hoppe et al.: Pflügers Arch 415:22-28, 1989). In this study we characterized the single channel currents using the patch-clamp technique in the intact patch recording configuration. The conductance of all recorded channels was 10-12 pS (5.6 mM [K+]o). These channels were K+ selective since changes in extracellular [K+] resulted in changes of the reversal potential as predicted for an exclusively K+ selective pore. The reversal potentials also predicted an intracellular [K+] of 60 mM indicating that the K+ equilibrium potential is slightly negative to the membrane potential. Analysis of the kinetic behavior of the channels resolved two different types of behaviour: 40% inactivated during a depolarizing voltage step, the others showing no sign of inactivation. The analysis of open probability and gating properties in the steady state showed up more differences between these two channel types: mean open probability peaked at about 10 mV for inactivating channels, while it continuously increased for non-inactivating channels. The inactivation time constants of averaged single channel and whole cell currents were similar and showed both a similar voltage dependency. We conclude that cultured Schwann cells express either two types of K+ channels with similar conductance or a channel which can acquire two functional states and that these channels can account for the different types of K+ currents observed in these cells.     

ATP modulates Na+ channel gating and induces a non-selective cation current in a neuronal hippocampal cell line

Extracellular ATP evoked two excitatory responses in hippocampal neuroblastoma cells (HN2). The first, an opening of a receptor-operated non-selective cation channel and the second was a leftward shift in Na+ channel activation. Both ATP (5-1000 microM) and 2',3'-(4-benzoyl)-benzoyl-ATP (Bb-ATP, 50 microM) activated a non-selective cation current reversing near 0 mV and shifted the Na+ activation and inactivation curves to the left. Based on a comparison of a series of agonists and antagonists, the inward current appeared to be partially mediated by activation of a P2X7 receptor, although hybrid channels cannot be ruled out. The shift in Na+ channel gating could be separated from the opening of the cation channel, as application of the P2Y antagonist Reactive Blue-2 and GTP shifted the Na+ current activation to the left but failed to elicit the inward cation current. Both responses to ATP and Bb-ATP were insensitive to block by the P2X antagonist suramin (300 microM) but were prevented by incubation in oxidized ATP (200 microM); a putative P2X7 receptor antagonist. Prior screening of the surface negative charge of the membrane with a high concentration of divalent cations prevented both responses. We suggest that ATP4- activates a P2X receptor and becomes trapped on a site, on or near the Na+ channel. Activation of the P2X receptor leads to the opening of a non-specific cation channel, while the binding of ATP4- leads to a modified charge sensed by the Na+ channel, similar to what occurs in the presence of charged amphiphiles as well as a number of beta-scorpion toxins.     

Lidocaine stabilizes the open state of CNS voltage-dependent sodium channels

We have previously reported that the lidocaine action is different between CNS and muscle batrachotoxin-modified Na+ channels [Salazar et al., J. Gen. Physiol. 107 (1996) 743-754; Brain Res. 699 (1995) 305-314]. In this study we examined lidocaine action on CNS Na+ currents, to investigate the mechanism of lidocaine action on this channel isoform and to compare it with that proposed for muscle Na+ currents. Na+ currents were measured with the whole cell voltage clamp configuration in stably transfected cells expressing the brain alpha-subunit (type IIA) by itself (alpha-brain) or together with the brain beta(1)-subunit (alphabeta(1)-brain), or the cardiac alpha-subunit (hH1) (alpha-cardiac). Lidocaine (100 microM) produced comparable levels of Na+ current block at positive potentials and of hyperpolarizing shift of the steady-state inactivation curve in alpha-brain and alphabeta(1)-brain Na+ currents. Lidocaine accelerated the rates of activation and inactivation, produced an hyperpolarizing shift in the steady-state activation curve and increased the current magnitude at negative potentials in alpha-brain but not in alphabeta(1)-brain Na+ currents. The lidocaine action in alphabeta(1)-brain resembled that observed in alpha-cardiac Na+ currents. The lidocaine-induced increase in current magnitude at negative potentials and the hyperpolarizing shift in the steady-state activation curve of alpha-brain, are novel effects and suggest that lidocaine treatment does not always lead to current reduction/block when it interacts with Na+ channels. The data are explained by using a modified version of the model proposed by Vedantham and Cannon [J. Gen. Physiol., 113 (1999) 7-16] in which we postulate that the difference in lidocaine action between alpha-brain and alphabeta(1)-brain Na+ currents could be explained by differences in the lidocaine action on the open channel state.     

Ionic currents of cultured olfactory receptor neurons from antennae of male Manduca sexta.

Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the ionic currents of insect olfactory receptor neurons (ORNs) in vitro. The cells were isolated from the antennae of male Manduca sexta pupae at stages 3-5 of adult development and maintained in primary cell culture. After 2-3 weeks in vitro, the presumptive ORNs had resting potentials of -62 +/- 12 mV (n = 18) and expressed at least 1 type of Na+ channel and at least 3 types of K+ channels. Na+ currents, recorded in the whole-cell mode, were reversibly blocked by 0.1 microM tetrodotoxin. The predominant type of K+ channel observed was a voltage-activated K+ channel (gamma = 30 pS) with characteristics similar to those of the delayed rectifier. The activity of the 30-pS K+ channel could be inhibited by the application of nucleotides to the cytoplasmic face of inside-out patches of membrane. The nucleotides had relative potencies as follows: ATP greater than cGMP greater than cAMP, with an inhibition constant for ATP of Ki = 0.18 mM. Raising the intracellular Ca2+ concentration from 0.1 to 5 microM induced the opening of a Ca2(+)-activated K+ channel (gamma = 66 pS at 0 mV) that had a low voltage sensitivity. A third, transient type of K+ channel (gamma = 12-18 pS) could be activated by depolarizing voltage steps from very negative resting potentials. Properties of this channel were similar to those of the "A-channel." These results support the conclusion that M. sexta ORNs differentiate in vitro and provide the basis for studying primary mechanisms of olfactory transduction.     

Voltage clamp analysis of sodium channels in normal and scorpion toxin-resistant neuroblastoma cells

Sodium currents mediated by voltage-sensitive sodium channels in normal and scorpion toxin-resistant neuroblastoma cells were measured using a giga-ohm seal recording method in the whole cell patch configuration. The voltage and time dependence of sodium currents were similar in normal and mutant cell lines. Half-maximal activation occurred for test depolarizations in the range of -7 to -11 mV. Half-maximal inactivation occurred for pre-pulses in the range of -62 to -69 mV. Scorpion toxin from Leiurus quinquestriatus (100 to 200 nM) increased the time constant for sodium channel inactivation 6- to 9-fold, increased the peak sodium current 2.0 +/- 0.5-fold, shifted the voltage dependence of sodium channel activation 7 to 11 mV to more negative potentials, and made the voltage dependence of inactivation less steep. These effects were observed for both normal and scorpion toxin-resistant neuroblastoma cells. However, the effect of Leiurus toxin on the rate of inactivation was half-maximal at 1.7 nM for the parental cell line N18, in contrast to 5.4 or 39 nM for the scorpion toxin-resistant clone LV30 and 24 or 51 nM for LV10. These results show that scorpion toxin resistance results from a specific change in channel properties that does not impair normal function but causes an increase in the apparent KD for Leiurus toxin action on sodium channels.     

Voltage-gated ionic currents in an identified modulatory cell type controlling molluscan feeding

An important modulatory cell type, found in all molluscan feeding networks, was investigated using two-electrode voltage- and current-clamp methods. In the cerebral giant cells of Lymnaea, a transient inward Na+ current was identified with activation at -58 +/- 2 mV. It was sensitive to tetrodotoxin only in high concentrations (approximately 50% block at 100 microm), a characteristic of Na+ channels in many molluscan neurons. A much smaller low-threshold persistent Na+ current (activation at < -90 mV) was also identified. Two purely voltage-sensitive outward K+ currents were also found: (i) a transient A-current type which was activated at -59 +/- 4 mV and blocked by 4-aminopyridine; (ii) a sustained tetraethylammonium-sensitive delayed rectifier current which was activated at -47 +/- 2 mV. There was also evidence that a third, Ca2+-activated, K+ channel made a contribution to the total outward current. No inwardly rectifying currents were found. Two Ca2+ currents were characterized: (i) a transient low-voltage (-65 +/- 2 mV) activated T-type current, which was blocked in NiCl2 (2 mm) and was completely inactivated at approximately -50 mV; (ii) A sustained high voltage (-40 +/- 1 mV) activated current, which was blocked in CdCl2 (100 microm) but not in omega-conotoxin GVIA (10 microm), omega-agatoxin IVA (500 nm) or nifedipine (10 microm). This current was enhanced in Ba2+ saline. Current-clamp experiments revealed how these different current types could define the membrane potential and firing properties of the cerebral giant cells, which are important in shaping the wide-acting modulatory influence of this neuron on the rest of the feeding network.     

Neurotoxins targetting receptor site 5 of voltage-dependent sodium channels increase the nodal volume of myelinated axons

The effects of a C57 type ciguatoxin (CTX-3C) and two types of brevetoxins (PbTx-1 and PbTx-3), known to bind to receptor site 5 of the neuronal voltage-dependent Na+ channel-protein, were studied on the morphology of living frog myelinated axons using confocal laser scanning microscopy. During the action of CTX-3C, PbTx-1, and PbTx-3 (10-50 nM), a marked swelling of nodes of Ranvier was observed without apparent modification of internodal parts of axons. In all cases, toxin-induced nodal swelling attained a steady-state within 75-100 min that was well maintained during an additional 90-115 min. The nodal swelling was reversed by an external hyperosmotic solution containing 100 mM D-mannitol and could be completely prevented by blocking voltage-dependent Na+ channels with 1 microM tetrodotoxin. It is suggested that CTX-3C, PbTx-1, and PbTx-3 by activating Na+ channels cause a continuous Na+ entry into axons, increasing internal Na+ concentration. Such an increase directly or indirectly disturbs the osmotic equilibrium between intra- and extra-axonal media, resulting in an influx of water, which is responsible for the long-lasting nodal swelling. Similar results were previously reported with two C60 type ciguatoxins (CTX-1B and CTX-4B). Thus, it is concluded that the four types of toxins targetting receptor site 5 of neuronal voltage-dependent Na+ channels, not only enhance nerve membrane excitability but also, on a long-term basis, cause a marked increase in the axonal volume.     

Characterization of large conductance Ca2+-activated K+ channels in cerebellar Purkinje neurons

We investigated the role of large conductance, calcium-activated potassium channels (BK channels) in regulation of the excitability of cerebellar Purkinje neurons. Block of BK channels by iberiotoxin reduced the afterhyperpolarization of spontaneous action potentials in Purkinje neurons in acutely prepared cerebellar slices. To establish the conditions required for activation of BK channels in Purkinje neurons, the dependence of BK channel open probability on calcium concentration and membrane voltage were investigated in excised patches from soma of acutely prepared Purkinje cells. Single channel currents were studied under conditions designed to select for potassium currents and in which voltage-activated currents were largely inactivated. Micromolar calcium concentrations activated channels with a mean single channel conductance of 266 pS. BK channels were activated by both calcium and membrane depolarization, and showed no sign of inactivation. At a given calcium concentration, depolarization over a 60-mV range increased the mean open probability (P(O)) from < 0.1 to > 0.8. Increasing the calcium concentration shifted the voltage required for half maximal activation to more hyperpolarized potentials. The apparent affinity of the channels for calcium increased with depolarization. At -60 mV the apparent affinity was approximately 35 micro m decreasing to approximately 3 micro M at +40 mV. These results suggest that BK channels are unlikely to be activated at resting membrane potentials and calcium concentrations. We tested the hypothesis that Purkinje cell BK channels may be activated by calcium entry during individual action potentials. Significant BK channel activation could be detected when brief action potential-like depolarizations were applied to patches under conditions in which the sole source of calcium was flux across the plasma membrane via the endogenous voltage-gated calcium channels. It is proposed that BK channels regulate the excitability of Purkinje cells by contributing to afterhyperpolarizations and perhaps by shaping individual action potentials.     

Identification and characterization of an L-type Cav1.2 channel in spiral ligament fibrocytes of gerbil inner ear

Intracellular free Ca2+ levels are critical to the activity of BK channels in inner ear type I spiral ligament fibrocytes. However, the mechanisms for regulating intracellular Ca2+ levels in these cells are currently poorly understood. Using patch-clamp technique, we have identified a voltage-dependent L-type Ca2+ channel in type I spiral ligament fibrocytes cultured from gerbil inner ear. With 10 mM Ba2+ as the conductive cation, an inwardly rectifying current was elicited with little inactivation by membrane depolarization. The voltage activation threshold and the half-maximal voltage activation were -40 and -6 mV, respectively. This inward whole-cell current reached its peak at around 10 mV of membrane potential. The amplitude of the peak current varied among cells ranging from 50 to 274 pA with an average of 132.4 +/- 76.2 pA (n = 19); 10(-6) M nifedipine significantly inhibited the inward currents by 90.3 +/- 1.2% (n = 11). RT-PCR analysis revealed that cultured type I spiral ligament fibrocytes express the alpha1C isoform of the L-type Ca2+ channels encoded by the Cav1.2 gene. The expression of this channel in gerbil inner ear was confirmed by RT-PCR analysis using freshly isolated spiral ligament tissues. The Cav1.2 channel may function in conjunction with a previously identified intracellular Ca-ATPase (SERCA) to regulate intracellular free Ca2+ levels in type I spiral ligament fibrocytes, and thus modulate BK channel activity in these cells.     

Two types of Na(+)-currents in cultured rat optic nerve astrocytes: changes with time in culture and with age of culture derivation.

Na+ channel expression was studied in cultures of rat optic nerve astrocytes using whole-cell voltage-clamp recordings. Astrocytes from postnatal day 7 rat optic nerve (RON) expressed two distinct types of Na+ currents, which had significantly different h infinity curves. Stellate, GFAP+/A2B5+ astrocytes showed currents with h infinity curve midpoints close to -65 mV, similar to Na+ currents in most neurons. In contrast, flat fibroblast-like GFAP+/A2B5- astrocytes showed Na+ currents with h infinity midpoints around -85 mV, almost 20 mV more hyperpolarized than in neurons or A2B5+ astrocytes. Interestingly, Na+ current expression was maintained in A2B5+ astrocytes but began to decrease in A2B5- astrocytes after 6 days in vitro (DIV) and fell to or below the level of detection (i.e., 1 pA/pF) at 12 DIV. Astrocytes cultured from neonatal rats (P0) are almost exclusively GFAP+/A2B5-. These cells did not display measurable Na+ currents when studied at 2 DIV; however, Na+ current was observed after 5 DIV in A2B5- astrocytes from these neonatal (P0) cultures. These findings were substantiated by immunocytochemical experiments using 7493, an antibody raised against purified rat brain Na+ channels; in P0-derived astrocyte cultures 7493 antibody staining was initially lacking (up to 3 DIV), but it was prominent in cultures after 5 DIV, suggesting that Na+ current expression in RON astrocytes occurs postnatally.