The action of pyrethroids on sodium channels in myelinated nerve fibres and spinal ganglion cells of the frog

The interaction of pyrethroids with the voltage-dependent sodium channel was studied in voltage-clamped nodes of Ranvier and isolated spinal ganglion neurons of the clawed frog, Xenopus laevis. In the node, pyrethroids prolonged the sodium tail current associated with a step repolarization of the membrane. It was found that the amplitude of the slow, pyrethroid-induced, sodium tail current (PIT) first increased and then decreased as a function of the duration of membrane depolarization (to -5 mV). This decrease of the PIT amplitude was absent when depolarizations to the sodium equilibrium potential (+40 mV) were used. Measurements of changes in sodium reversal potential indicated that sodium ion depletion in the perinodal space is largely responsible for the inactivation of the pyrethroid-modified sodium current. Inactivation is not completely abolished by pyrethroid treatment since the probability of channel opening, measured in membrane patches excised from spinal ganglion cells, decreased slowly during prolonged depolarization. Analysis of unitary currents indicated that both activation and inactivation are retarded by pyrethroids. The arrival of sodium channels in the pyrethroid-modified open state followed a time course that was slower than both activation and inactivation of unmodified sodium channels. Our findings indicate that sodium channels are modified when in the closed resting state and that both opening and closing kinetics are delayed by pyrethroids.     

Sodium depletion in the periaxonal space of the squid axon treated with pyrethroids

Pyrethriods are known to increase the steady-state sodium current during a step depolarization and to increase and prolong the tail sodium current associated with a step repolarization of the membrane. The pyrethroid-induced tail sodium current of squid axons developed as a function of the duration of the conditioning depolarizing pulse. However, with further lengthening the conditioning pulse duration, it decreased, further increased, or remained constant depending on the direction of sodium current during the depolarization, irrespective of the membrane potential per se. The depletion or accumulation of sodium in the periaxonal space during a 200-ms conditioning depolarizing pulse in the axon internally treated with pronase, pyrethroids, or both, was demonstrated by measurements of the changes in sodium reversal potential. Thus the observed changes in tail current amplitude as a function of the conditioning pulse duration are explicable in terms of changes in sodium concentration in the periaxonal space without assuming inactivation of the pyrethroid-modified channel.     

Increase of sodium current after pyrethroid insecticides in mouse neuroblastoma cells

The effects of 4 different pyrethroid insecticides on sodium channel gating in internally perfused, cultured mouse neuroblastoma cells (N1E-115) were studied using the suction pipette, voltage clamp technique. Pyrethroids increased the amplitude of the sodium current, sometimes by more than 200%. Activation of the sodium current occurred at more hyperpolarized potentials than under control conditions. The declining phase of the sodium current during depolarization was markedly slowed down and after repolarization of the membrane a large, slowly decaying sodium tail current developed. Pyrethroids did not affect the sodium current reversal potential, steady-state sodium inactivation or recovery from sodium channel inactivation. The amplitude of the pyrethroid-induced slow tail current was always proportional to the sodium current at the end of the preceding depolarizing pulse. The rate of decay of the slow tail current strongly depended on pyrethroid structure and increased in the order deltamethrin, cyphenothrin, fenfluthrin and phenothrin. The rate of decay further depended on membrane potential and temperature. Below -85 m V the instantaneous current-voltage relationship of the slow tail current showed a negative slope conductance. The tail current decayed more slowly at low temperatures. Arrhenius plots indicated that the relaxation of open sodium channels to a closed state involved a higher energy barrier for pyrethroid-affected than for normal channels. The energy barrier was higher after deltamethrin than after the non-cyano pyrethroid fenfluthrin. It is concluded that in mammalian neuronal membrane pyrethroids selectively reduce the rate of closing of sodium channels both during depolarization and after repolarization of the nerve membrane.     

Differential state-dependent modification of inactivation-deficient Nav1.6 sodium channels by the pyrethroid insecticides S-bioallethrin, tefluthrin and deltamethrin

Pyrethroid insecticides disrupt nerve function by modifying the gating kinetics of transitions between the conducting and nonconducting states of voltage-gated sodium channels. Pyrethroids modify rat Na(v)1.6+β1+β2 channels expressed in Xenopus oocytes in both the resting state and in one or more states that require channel activation by repeated depolarization. The state dependence of modification depends on the pyrethroid examined: deltamethrin modification requires repeated channel activation, tefluthrin modification is significantly enhanced by repeated channel activation, and S-bioallethrin modification is unaffected by repeated activation. Use-dependent modification by deltamethrin and tefluthrin implies that these compounds bind preferentially to open channels. We constructed the rat Na(v)1.6Q3 cDNA, which contained the IFM/QQQ mutation in the inactivation gate domain that prevents fast inactivation and results in a persistently open channel. We expressed Na(v)1.6Q3+β1+β2 sodium channels in Xenopus oocytes and assessed the modification of open channels by pyrethroids by determining the effect of depolarizing pulse length on the normalized conductance of the pyrethroid-induced sodium tail current. Deltamethrin caused little modification of Na(v)1.6Q3 following short (10ms) depolarizations, but prolonged depolarizations (up to 150ms) caused a progressive increase in channel modification measured as an increase in the conductance of the pyrethroid-induced sodium tail current. Modification by tefluthrin was clearly detectable following short depolarizations and was increased by long depolarizations. By contrast modification by S-bioallethrin following short depolarizations was not altered by prolonged depolarization. These studies provide direct evidence for the preferential binding of deltamethrin and tefluthrin (but not S-bioallethrin) to Na(v)1.6Q3 channels in the open state and imply that the pyrethroid receptor of resting and open channels occupies different conformations that exhibit distinct structure-activity relationships.     

Modification of sodium channel kinetics by the insecticide tetramethrin in crayfish giant axons

The effects of the pyrethroid insecticide tetramethrin on voltage-dependent sodium channels were studied with internally perfused crayfish giant axons. At low concentrations in the order of 10-8-10-9M, tetramethrin caused an increase in depolarizing after-potential which in turn triggered repetitive after-discharges. Under Voltage clamp conditions, the sodium current was markedly prolonged during a step depolarization, and a large and prolonged sodium tail current appeared upon step repolarization. A population of sodium channels having activation and inactivation kinetics identical to those in control axons was observed in the tetramethrin-poisoned axons, indicating that only a fraction of the channels was modified. The modified channels exhibited remarkably slow kinetics, activating with a time course of 100 msec to 2 sec and inactivating with a time course of 1-5 sec depending on the membrane potential. The voltage dependence of the modified channels was shifted in the direction of hyperpolarization by about 10-20 mV with respect to normal sodium channels. The large inward sodium tail current associated with step repolarization of the membrane decayed with a time course of 20-600 msec. A kinetic hypothesis describing the behavior of sodium channels in a tetramethrin-poisoned axon is presented and discussed in relation of the behavior of the sodium channels modified by other toxins.     

Interactions of the pyrethroid fenvalerate with nerve membrane sodium channels: temperature dependence and mechanism of depolarization

Depolarization of nerve membranes is an important component of the mode of action of pyrethroids, and its negative temperature dependence parallels that of insecticidal activity. We studied the mechanism and temperature dependence of depolarization of crayfish giant axons by pyrethroids, using intracellular microelectrode and voltage clamp techniques. Membrane depolarization caused by tetramethrin and fenvalerate was greater at 10 degrees C than at 21 degrees C, and was reversible upon changing the temperature. Short-duration depolarizing pulses in voltage-clamped fenvalerate-treated axons induced prolonged sodium currents that are typical of other pyrethroids, but the decay of the tail current following repolarization was extremely slow, lasting several minutes at the large negative holding potential of -120 mV. At the normal resting potential, the tail current did not decay completely, and even without stimulation, a steady-state sodium current developed, which could account for the depolarization. The steady-state current induced by fenvalerate at the resting potential was much larger at 8 degrees C than at 21 degrees C, accounting for the negative temperature dependence of the depolarization. The negative temperature dependence of the steady-state current seems to be due ultimately to the great stabilizing effect of low temperature on the open-modified channel. When the steady-state current was induced at the resting potential, hyperpolarization to more negative potentials caused it to decay with exactly the same time course as tail currents induced by short-duration depolarizing pulses, indicating that both types of currents are carried by identically-modified channels. The modified channels were shown to be inactivated very slowly at potentials more positive than - 100 mV, accounting for the limited depolarization observed in micro-electrode experiments. Even when applied directly to the internal face of the membrane, the effect of fenvalerate on the sodium channel developed slowly, taking more than 90 min to reach its final level. Fenvalerate did not significantly affect potassium currents.     

Modulation of nerve membrane sodium channel activation by deltamethrin.

Deltamethrin is a highly potent pyrethroid insecticide that causes hypersensitivity, choreoathetosis, tremors, and paralysis in mammals. It is known to modify the sodium channel in such a way as to prolong the tail current associated with step repolarization following a depolarizing pulse. Using the axial-wire voltage-clamp technique with the giant axon of the squid Loligo pealei, we have demonstrated that deltamethrin also greatly slows the opening of the sodium channel. This was first observed as a decrease, by as much as 80%, in the peak sodium current flowing during a short, 10 ms depolarization. Current flowing through these slowly opening deltamethrin modified sodium channels was observed during the first depolarizing pulse after deltamethrin exposure and developed with a time constant of 320 ms. This supports the idea that deltamethrin can modify sodium channels when they are in the closed or resting state. Further, evidence of this hypothesis was provided by experiments using 0.1 and 10 microM deltamethrin and measuring the tail current amplitude after depolarizing pulses of varying duration (1-1200 ms). The mean time constant for the increase in tail current amplitude was almost concentration independent; 253 ms at 0.1 microM and 193 ms at 10 microM. We conclude that deltamethrin modifies the activation kinetics of sodium channels in such a way as to slow opening and that this modification occurs predominantly when channels are in the closed or resting state.     

Alanine to valine substitutions in the pore helix IIIP1 and linker-helix IIIL45 confer cockroach sodium channel resistance to DDT and pyrethroids

Pyrethroid insecticides exert toxic effects by prolonging the opening of voltage-gated sodium channels. More than 20 sodium channel mutations from arthropod pests and disease vectors have been confirmed to confer pyrethroid resistance. These mutations have been valuable in elucidating the molecular interaction between pyrethroids and sodium channels, including identification of two pyrethroid receptor sites. Previously, two alanine to valine substitutions, one in the pore helix IIIP1 and the other in the linker-helix connecting S4 and S5 in domain III (IIIL45), were found in Drosophila melanogaster mutants that are resistant to DDT and deltamethrin (a type II pyrethroid with an α-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids), but their role in target-site-mediated insecticide resistance has not been functionally confirmed. In this study, we functionally examined the two mutations in cockroach sodium channels expressed in Xenopus laevis oocytes. Both mutations caused depolarizing shifts in the voltage dependence of activation, conferred DDT resistance and also resistance to two Type I pyrethroids by almost abolishing the tail currents induced by Type I pyrethroids. In contrast, neither mutation reduced the amplitude of tail currents induced by the Type II pyrethroids, deltamethrin or cypermethrin. However, both mutations accelerated the decay of Type II pyrethroid-induced tail currents, which normally decay extremely slowly. These results provided new insight into the molecular basis of different actions of Type I and Type II pyrethroids on sodium channels. Computer modeling predicts that both mutations may allosterically affect pyrethroid binding.     

A use-dependent sodium current modification induced by type I pyrethroid insecticides in honeybee antennal olfactory receptor neurons

We studied the mode of action of type I pyrethroids on the voltage-dependent sodium current from honeybee olfactory receptor neurons (ORNs), whose proper function in antenna is crucial for interindividual communication in this species. Under voltage-clamp, tetramethrin and permethrin induce a long lasting TTX-sensitive tail current upon repolarization, which is the hallmark of an abnormal prolongation of the open channel configuration. Permethrin and tetramethrin also slow down the sodium current fast inactivation. Tetramethrin and permethrin both bind to the closed state of the channel as suggested by the presence of an obvious tail current after the first single depolarization applied in the presence of either compounds. Moreover, at first sight, channel opening seems to promote tetramethrin and permethrin binding as evidenced by the progressive tail current summation along with trains of stimulations, tetramethrin being more potent at modifying channels than permethrin. However, a use-dependent increase in the sodium peak current along with stimulations suggests that the tail current accumulation could also be a consequence of progressively unmasked silent channels. Experiments with the sea anemone toxin ATX-II that suppresses sodium channels fast inactivation are consistent with the hypothesis that these silent channels are either in an inactivated state at rest, or that they normally inactivate before they open so that they do not participate to the control sodium current. In honeybee ORNs, three processes lead to a use-dependent pyrethroid-induced tail current accumulation: (i) a recruitment of silent channels that produces an increase in the peak sodium current, (ii) a slowing down of the sodium current inactivation produced by prolongation of channels opening and (iii) a typical deceleration in current deactivation. The use-dependent recruitment of silent sodium channels in honeybee ORNs makes pyrethroids more potent at modifying neuronal excitability.     

Block of deltamethrin-modified sodium current in cultured mouse neuroblastoma cells: local anesthetics as potential antidotes

Effects of local anesthetics and anticonvulsants on the pyrethroid-modified sodium current in cultured mouse neuroblastoma cells have been investigated using the suction pipette voltage clamp technique. In the presence of 10 microM of the pyrethroid deltamethrin the sodium current consists of an enhanced peak current during membrane depolarization and a slowly decaying, deltamethrin-induced tail current remaining after repolarization. At the onset of block the local anesthetics tetracaine, lidocaine and QX 314 reduced the deltamethrin-induced tail current more effectively than the peak current. Lidocaine, but not phenytoin, caused a time-dependent block of tail currents evoked by membrane depolarizations lasting 10-1000 ms. Both lidocaine- and phenytoin-induced blocks were independent of the membrane potential during the tail current. The anticonvulsants phenytoin, phenobarbital and valproate blocked the tail and the peak sodium current to the same extent, but diazepam, mephenesin and urethane blocked the peak current more effectively. Vitamin E, which suppresses pyrethroid-induced paresthesia of the skin, had no effect on the voltage-dependent sodium current. It is concluded that indirect effects of anticonvulsants on pyrethroid-induced toxic symptoms predominate, whereas local anesthetics preferentially block the pyrethroid-induced tail current. Therefore, local anesthetics are potentially useful pyrethroid antidotes.     

Features of the kinetic and stationary characteristics of aconitine-modified sodium channels

Kinetic and steady-state characteristics of aconitine-modified sodium channels were studied in the Ranvier node membrane. Aconitine-modified sodium channels are shown to be inactivated only partially. The voltage dependence of the fraction of noninactivated channels (h infinity) may be described by a three-state model of the channel with closed, open and inactivated states. A reasonable agreement with the data was obtained when parameters of the inactivated state were supposed to be not changed after aconitine modification of the channels. The membrane repolarization to -70 divided by -110 mV, after long (10 ms) depolarizing shift induces firstly fast current decay ("tail") and then its rather slow increase to a steady-state level. Kinetics of this current requires two or more open states to be postulated.     

Biophysical properties of scorpion alpha-toxin-sensitive background sodium channel contributing to the pacemaker activity in insect neurosecretory cells (DUM neurons)

A scorpion alpha-toxin-sensitive background sodium channel was characterized in short-term cultured adult cockroach dorsal unpaired median (DUM) neurons using the cell-attached patch-clamp configuration. Under control conditions, spontaneous sodium currents were recorded at different steady-state holding potentials, including the range of normal resting membrane potential. At -50 mV, the sodium current was observed as unclustered, single openings. For potentials more negative than -70 mV, investigated patches contained large unitary current steps appearing generally in bursts. These background channels were blocked by tetrodotoxin (TTX, 100 nm), and replacing sodium with TMA-Cl led to a complete loss of channel activity. The current-voltage relationship has a slope conductance of 36 pS. At -50 mV, the mean open time constant was 0.22 +/- 0.05 ms (n = 5). The curve of the open probability versus holding potentials was bell-shaped, with its maximum (0.008 +/- 0.004; n = 5) at -50 mV. LqhalphaIT (10-8 m) altered the background channel activity in a time-dependent manner. At -50 mV, the channel activity appeared in bursts. The linear current-voltage relationship of the LqhalphaIT-modified sodium current determined for the first three well-resolved open states gave three conductance levels: 34, 69 and 104 pS, and reversed at the same extrapolated reversal potential (+52 mV). LqhalphaIT increased the open probability but did not affect either the bell-shaped voltage dependence or the open time constant. Mammal toxin AaHII induced very similar effects on background sodium channels but at a concentration 100 x higher than LqhalphaIT. At 10-7 m, LqhalphaIT produced longer silence periods interrupted by bursts of increased channel activity. Whole-cell experiments suggested that background sodium channels can provide the depolarizing drive for DUM neurons essential to maintain beating pacemaker activity, and revealed that 10-7 m LqhalphaIT transformed a beating pacemaker activity into a rhythmic bursting.     

Calcium-dependent depolarizations originating in lizard motor nerve terminals

Action potentials and afterpotentials were recorded via a microelectrode inserted into motor axons innervating the lizard ceratomandibularis muscle. Intra-axonal injection of Lucifer yellow dye indicated that these axons innervate multiple (at least 6-25) motor terminals. In 10 mM tetraethylammonium (TEA), the action potential was followed by a sequence of afterpotentials, whose amplitude and duration increased with increasing proximity to motor nerve terminals. In axons impaled within 1 mm of their most distal terminals, these afterpotentials included a depolarizing plateau (mean amplitude and duration: 32 mV, 24 msec) and a subsequent smaller depolarization that decayed over a time course of several hundred milliseconds. These depolarizing afterpotentials were Ca dependent: They increased with increasing bath [Ca] and were abolished by low [Ca]-high [Mg] solutions, by omega-conotoxin (GVIA, 1 microM), by addition of Cd (1 microM) or Mn (0.3-1 mM) to the bath, and by selective perfusion of Cd over the terminal region. In [Ca]-free solutions the afterpotentials were restored by selective perfusion of Ca over the terminal region but not by Ca applied to the more proximal nerve trunk. When Na influx was eliminated by 1-10 microM tetrodotoxin or by substitution of TEA for bath Na, passage of depolarizing current into the axon evoked prolonged depolarizing afterpotentials that were blocked by Mn. Bay K 8644 (0.1-1 microM), a dihydropyridine that prolongs the opening of certain calcium channels, enhanced mainly the slower component of the depolarizing afterpotential. Nimodipine (0.1-1 microM), a dihydropyridine that favors the closed state of some calcium channels, shortened the plateau phase of the depolarizing afterpotential. Another antagonist dihydropyridine, nitrendipine (0.1-1 microM), had little or no effect on the depolarizing afterpotential but did antagonize the actions of Bay K 8644. These results suggest that the intraaxonally recorded Ca-dependent afterpotentials are caused by electrotonic spread of depolarizations produced by calcium influx into that axon's terminals and that some motor nerve terminal calcium channels are sensitive to certain dihydropyridines.     

ATP modulation of sodium currents in rat dorsal root ganglion neurons

The modulation of tetrodotoxin-sensitive (TTX-S) and slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons by ATP was studied using the whole-cell patch-clamp method. The effects of ATP on two types of sodium currents were either stimulatory or inhibitory depending on the kinetic parameters tested. At a holding potential of -80 mV ATP suppressed TTX-S sodium currents when the depolarizing potential was positive to -30 mV but it increased them when the depolarizing potential was negative to -30 mV. At the same holding potential slow TTX-R sodium currents were always increased by ATP regardless of the depolarizing potential. In both types of sodium currents ATP shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction, and accelerated the time-dependent inactivation. ATP decreased the maximum conductance of TTX-S sodium currents but increased that of slow TTX-R sodium currents. The results suggest that ATP would decrease the excitability of neurons with TTX-S sodium channels but would increase that of neurons with slow TTX-R sodium channels. The effects of ATP on sodium currents were preserved in the presence of a G-protein inhibitor, GDP-beta-S, or purinergic antagonists, suramin and Reactive Blue-2, suggesting that purinergic receptors might not be involved in ATP modulation of sodium currents.     

A model of mouse motor nerve excitability and the effects of polarizing currents

This study was undertaken to develop a theoretical model of mouse motor nerve excitability, and to validate it by application to recordings from mouse axons with membrane potential altered by polarizing currents. Multiple excitability tests were performed on the caudal motor nerves of 12 mice. Membrane potential was artificially modified by injecting polarizing currents through the stimulating electrodes (±20% of threshold for 1 ms pulse). A human motor nerve model was adapted to the unpolarized mouse recordings by adjusting membrane parameters to optimize the fit. To evaluate the usefulness of the model, we then determined the parameter changes which best fitted the recordings from depolarized and hyperpolarized tail nerves. The recordings from the normal axons were well fitted by a model in which axons were hyperpolarized by 4 mV, and nodal sodium and slow potassium conductances were reduced by half, compared with human median nerve motor axons. The changes with polarizing currents were qualitatively similar to those in human axons, and modeling correctly identified a change in current as the best-fitting explanation for the altered excitability. We conclude that this new model should help identify changes in membrane potential and probably in other membrane parameters in mouse models of neurological disease.     

Calcium-activated chloride channels in müller cells acutely isolated from tiger salamander retina

Ca(2+)-activated chloride channels were identified with whole-cell patch-clamp recording techniques in salamander retinal Müller cells. Cl(Ca) channels were activated by membrane depolarizations that elicited Ca2+ influx or the application of the Ca2+ ionophore, ionomycin. The Ca channel blocker, Cd2+, abolished the Cl(Ca) channel tail currents. Increasing the duration of the depolarizing pulse resulted in enhancement of the Cl(Ca) channel tail current. Repetitive depolarizations with rapid pulses to +20 mV produced a buildup of I(Cl(Ca)), which reversed at 0 mV in symmetrical [Cl-] and at -40 mV when intracellular [Cl-] was reduced to 10% of the external concentration. I(Cl(Ca)) was blocked by the Cl channel blocker niflumic acid, while niflumic acid had no effect on voltage-gated Ca channels. These results offer the first demonstration of Cl(Ca) channels in a nonastrocytic glial cell and expand our understanding of the functional capacities of retinal glial cells.     

Modulation of sodium currents in rat dorsal root ganglion neurons by sulfur dioxide derivatives

The effect of sulfur dioxide (SO2) derivatives, a common air pollutant and exists in vivo as an equilibrium between bisulfate and sulfite, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in cultured post-natal dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. SO2 derivatives on two types of sodium currents were either inhibitory or stimulatory depending on the kinetic parameters tested. At a holding potential of -80 mV, SO2 derivatives suppressed TTX-S sodium currents when depolarizing potential was negative to -30 mV and TTX-R sodium currents when negative to -10 mV but they increased them when the depolarizing potential was positive to -30 or -10 mV. SO2 derivatives shifted the conductance-voltage curve for TTX-R sodium currents in the depolarizing direction but had little effect on that for TTX-S sodium currents. The steady-state inactivation curve for TTX-R sodium channel was shifted by SO2 derivatives in the depolarizing direction as that for TTX-S sodium channel. SO2 derivatives changed the reversal potential and increased the maximum conductance of two types of sodium channels. SO2 derivatives postponed the activating time and delayed the inactivation of sodium currents. The results suggest that SO2 derivatives would increase the excitability of neurons and alter the ion selectivity for two types of sodium currents.     

Inhibition of Ca-channels by diazepam compared with that by nicardipine in pheochromocytoma PC12 cells.

The effects of diazepam on voltage-gated Ca channels were studied in PC12 pheochromocytoma cells using whole-cell voltage-clamp techniques. An inward current activated by a depolarizing voltage step to +10 mV from a holding potential of -60 mV in 10.8 mM Ba was larger than that activated in 10.8 mM Ca. The Ba current was completely blocked by a low concentration of Cd (30 microM) and was also sensitive to nicardipine (100 nM to 10 microM). Diazepam (1-100 microM) inhibited the Ba current in a concentration-dependent manner. Neither diazepam nor nicardipine affected the current-voltage relationship or the dependence on holding potentials of the Ba current. Both slightly accelerated the inactivation time course of the Ba current. When diazepam was applied to the cells in combination with nicardipine, the observed inhibition agreed with a value predicted assuming independent blockade by diazepam and by nicardipine. These results suggest that diazepam inhibits Ca channels in a manner similar to nicardipine, but that the binding sites for diazepam are different from those for nicardipine.     

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.     

Activation of neurokinin receptors modulates K and Cl channel activity in cultured astrocytes from rat cortex

Short application of the neurokinin receptor agonist substance P (SP) leads to a biphasic depolarization of astrocytes cultured from rat cortex. The rapid and transient depolarizing event lasted few seconds, the slow one several minutes. In some cells, only the slow depolarizing component was observed. During the slow depolarizing event, the sensitivity of the membrane potential for a change in the K+ gradient decreased, indicating a decrease in the relative K+ permeability of the membrane. The rapid SP-induced depolarization could be reversed, when the membrane potential was depolarized to about 0 mV by elevation of the extracellular K+ concentration, indicating a reversal potential close to the Cl- equilibrium potential. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, SP induced a biphasic inward current with a similar time course as the SP-induced membrane depolarization. Evaluating current-to-voltage curves indicated a conductance decrease during the slow inward current with a reversal potential of the SP-dependent current close to the K+ equilibrium potential. The mean open time of single K+ channels, measured in the cell-attached configuration of the patch-clamp technique, decreased after application of SP. In contrast, the mean open time of single Cl- channels increased. We conclude that activation of neurokinin receptors in astrocytes modulates the activity of K+ and Cl- channels, leading to a complex depolarization of the membrane potential.