Characterization of maintained voltage-dependent K(+)-channels induced in Xenopus oocytes by rat brain mRNA.

The voltage-dependent K+ currents encoded by rat brain mRNA were studied in Xenopus oocytes after the voltage-dependent Na+ currents and the Ca(2+)-activated Cl- currents were eliminated pharmacologically. This paper describes the maintained K+ currents (IK), defined primarily by resistance to inactivation for 1 s at a holding potential of -40 mV. IK activates at potentials more positive than -60 to -70 mV and consists of both low-threshold and high-threshold components. IK is partially blocked by both tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which appear to be blocking the same component. Long depolarizing pulses result in incomplete inactivation of IK; the inactivating component is inhibited by TEA. Sucrose density gradient fractionation partially resolves the RNA encoding the several components of IK; most IK arises from size classes between 3.8 and 9.5 kb. The study gives further evidence for the existence of numerous distinct RNA populations that encode brain K+ channels different from previously reported cloned K+ channels that have been expressed in Xenopus oocytes.     

Expression of functional sodium channels in stage II-III Xenopus oocytes

We have injected mRNA from rabbit brain into stage II-III Xenopus oocytes to determine whether they will translate exogenous RNA and incorporate functional ion channels into the membrane. Our results show that 48 h after RNA injection, functional voltage-dependent Na channels are present at sufficient densities to allow quantitative electrophysiological recording. The smaller oocytes have at least 2 experimental advantages over the stage V-VI oocytes normally used for electrophysiological experiments: (1) the smaller membrane capacitance (approximately 5-fold) allows a faster settling time following a voltage step and a more detailed kinetic analysis of membrane currents than was previously possible with the 2-microelectrode technique, and (2) roughly 8-fold less RNA is needed for each injection. Thus, the stage II-III Xenopus oocyte is a suitable preparation for the study of ion channels.     

Protein kinase A reduces voltage-dependent Na+ current in Xenopus oocytes.

The voltage-dependent Na+ channel of the brain is a good substrate for phosphorylation by the cAMP-dependent protein kinase (protein kinase A, or PKA), but the physiological effects of PKA on Na+ channels are poorly documented. We studied modulation by PKA of voltage-dependent Na+ channels expressed in Xenopus oocytes injected with RNA coding for the alpha-subunit of the channel protein (rat brain type IIA and its variant VA200), using the two electrode voltage-clamp technique. Intracellularly injected cAMP or catalytic subunit of PKA, or extracellularly applied forskolin, inhibited the Na+ current by 20-30%. The effect of cAMP was attenuated by prior injection of PKA inhibitors. Injection of small doses of protein phosphatase 2A increased the Na+ current by 10%, whereas larger doses of protein phosphatase 1 and alkaline phosphatase were without effect. The inhibition by PKA showed little voltage dependence, being only slightly stronger at holding potentials at which the availability of the channels was reduced. The voltage dependence of activation and inactivation processes was not altered by cAMP. Similar effects were exerted by forskolin and cAMP on the Na+ channels expressed after the injection of heterologous (total) RNA from rat brain. Thus, PKA modulates the Na+ channel by a mechanism that does not involve major changes in the voltage dependency of the current and is exerted on the channel-forming alpha-subunit.     

Properties of endogenous voltage-dependent sodium currents in Xenopus laevis oocytes.

Endogenous voltage-dependent sodium currents were recorded using standard 2-microelectrode techniques in Xenopus laevis oocytes. Maximal inward current occurred at -10 mV with an average amplitude of -279 +/- 17 nA and steady-state inactivation was half-maximal at a voltage of -38 +/- 0.5 mV. Currents were blocked by low concentrations of tetrodotoxin (TTX) with an IC50 value of 6 nM. These properties make the endogenous sodium current in Xenopus oocytes similar to sodium currents expressed following injection of mammalian brain RNA. While endogenous sodium channels have the potential to complicate analysis when using the oocyte expression system, they are only present at significant levels in rare batches of oocytes (less than 5%). Our results do stress the need, however, to reproduce results from exogenous expression studies across several batches of oocytes from different donors.     

Characterization of mRNA responsible for induction of functional sodium channels in Xenopus oocytes

When Xenopus laevis oocytes were microinjected with poly(A)+ mRNA isolated from adult rat brains or electric organs of Electrophorus electricus, the oocytes developed functional sodium channels. Upon application of veratrine, the microinjected oocytes exhibited transient depolarization, resulting in spontaneous repetitive spikes in some occasions, and action potentials. These responses were mediated mainly by external Na ions, prolonged by scorpion toxin, completely blocked by tetrodotoxin, and suppressed by local anesthetics. Thus the mRNA-induced sodium channels exhibited essentially all the functional properties expected for native sodium channels in nerve and muscle membranes. Rat brain mRNA was fractionated into 4 fractions by sucrose gradient centrifugation. Each fraction and various combinations of them were examined for the efficiency in inducing functional sodium channels in Xenopus oocytes. A fraction corresponding to mRNA of approximately 30S to 46S was found to contain all mRNA necessary for the expression of the channels, indicating that mRNA of smaller sizes expected to code for smaller polypeptides may not be required.     

Voltage-gated influx ion channels expressed in Xenopus oocytes after the administration of brain mRNA]

Expression of "fast", TTX-sensitive sodium and high-threshold calcium channels in the membrane of Xenopus oocytes following mRNA injection from the rat brain has been detected using two microelectrode voltage clamp technique. Barium current through expressed calcium channels was blocked by 200 mumol/l Cd2+ and was insensitive to D-600 (20 mumol/l) and nitrendipine (50 mumol/l). Expressed barium current was inhibited within 20-40 min by omega-conotoxin, a peptide neurotoxin known to block high-threshold calcium channels of the neuronal membrane, in 1 mumol/l concentration. A steady-state inactivation curve for this current could be fitted by the Boltzmann relation with V1/2 = -50 mV and k = 14 mV. Voltage-dependent and pharmacological properties of calcium channels which appeared in the oocyte membrane following mRNA injection from the mammalian brain resembled most of all those of high-threshold inactivating (HTI- or N-type) calcium channels of neurons in spite they did not demonstrate prominent time-dependent inactivation. Evidences in favour of expressed calcium channels heterogeneity were not obtained.     

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

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

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.     

The expression of rat brain synaptic plasma membrane Na(+)-Ca2+ exchange activity in Xenopus oocytes

Injection of Xenopus oocytes with mRNA isolated from 1-day-old rat brains leads to expression of Na+ gradient-dependent Ca2+ uptake activity. Size fractionation of the mRNA by sucrose density gradient centrifugation reveals that an mRNA fraction enriched in 14-18 S mRNA is responsible for the transport activity. Plasma membrane proteins isolated from oocytes injected with total or 14-18 S enriched fraction of rat brain mRNA contain proteins of about 70-kDa molecular mass recognized by a polyclonal antibody prepared against the purified 70-kDa rat brain Na(+)-Ca2+ exchanger. In control H2O-injected oocytes, no proteins are recognized by the anti-70-kDa antibody.     

State-dependent block of rat Nav1.4 sodium channels expressed in xenopus oocytes by pyrazoline-type insecticides

Insecticidal pyrazolines inhibit voltage-sensitive sodium channels of both insect and mammalian neurons in a voltage-dependent manner. Studies on the effects of pyrazoline insecticides on mammalian sodium channels have been limited to experimentation on the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channel populations of rat dorsal root ganglion (DRG) neurons. In this study, we examined the effects of the insecticidal pyrazolines indoxacarb, the N-decarbomethoxyllated metabolite of indoxacarb (DCJW), and RH 3421 on rat Na(v)1.4 sodium channels expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique. Both DCJW and RH 3421 were ineffective inhibitors of rat Na(v)1.4 sodium channels at a membrane potential of -120 mV, but depolarization to -60 mV or -30 mV during insecticide exposure resulted in substantial block. Inhibition by pyrazoline insecticides was nearly irreversible with washout, but repolarization of the membrane relieved block. DCJW and RH 3421 also caused hyperpolarizing shifts in the voltage dependence of slow inactivation without affecting the voltage dependence of activation or fast inactivation. These results suggest that DCJW and RH 3421 interact specifically with the slow inactivated state of the sodium channel. Indoxacarb did not cause block at any potential, yet it interfered with the ability of DCJW, but not RH 3421, to inhibit sodium current. Phenytoin, an anticonvulsant, reduced the efficacy of both DCJW and RH 3421. These data imply that the binding site for pyrazoline insecticides overlaps with that for therapeutic sodium channel blockers.     

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.     

Postnatal development of voltage-dependent calcium channels in the mouse brain disclosed by the Xenopus oocyte assay

We assessed the ontogeny of murine voltage-dependent calcium channels by extracting mRNA from brains of mice at different postnatal ages and injecting the mRNA into oocytes of the frog, Xenopus laevis. Voltage-dependent Ca2(+)-activated Cl- channels were measured to assess the presence of Ca2(+) channels. When compared with water-injected oocytes (controls), an increase in Ca2(+) channels was not detected until postnatal day 7. The number of Ca2+ channels peaked between 9 and 18 days and began to decline by 35 days. Bath application of barium, serotonin and the Ca2+ channel antagonist, verapamil, to mRNA-injected oocytes confirmed the presence of Ca2+ channels.     

Expression of the functional D-glucose transport system in Xenopus oocytes injected with mRNA of rat small intestine

mRNA prepared from rat small intestine was injected into Xenopus oocytes. The injected oocytes showed a clear electrical response to D-glucose (Glu) in the form of membrane depolarization and conductance increase, while none was shown to D-fructose. The membrane electrical response of the injected oocytes evoked by Glu followed the Michaelis-Menten type kinetics and was dependent on the membrane potential of the oocyte. Replacing the Na+ of the bathing buffer with choline+ resulted in no response to Glu. Thus, a Glu transport system coupled to a Na+ gradient was expressed in Xenopus oocytes by injecting mRNA from rat small intestine.     

Messenger RNAs coding for receptors and channels in the cerebral cortex of adult and aged rats.

Poly(A)+ mRNAs from the cerebral cortex of aged (24 months) and young adult (3 months) rats were isolated and injected into Xenopus oocytes to express functional neurotransmitter receptors and voltage-operated channels. Electrophysiological recordings of induced membrane currents were used as a measure of the relative amounts of mRNA encoding different receptors and channels, and to study their functional properties. There were no large differences apparent between mRNAs from aged and adult rats, in marked contrast to the dramatic (1000-fold) changes in mRNA expression that occur during embryonic and postnatal development. The membrane currents induced by glutamate or acetylcholine (ACh) application were roughly one third smaller in oocytes injected with mRNA from aged cerebral cortex than in oocytes injected with mRNA from adult cerebral cortex, whereas currents induced by gamma-aminobutyric acid (GABA), kainate or serotonin (5-HT) application, and by activation of voltage-operated Na+ and Ca2+ channels were not significantly different. We did not observe any age-related differences in the properties of the receptors and channels studied.     

Tetrodotoxin-sensitive voltage-dependent Na currents recorded from Xenopus oocytes injected with mammalian cardiac muscle RNA

Voltage-sensitive sodium (Na) channel currents recorded from mammalian cardiac muscle are blocked by tetrodotoxin (TTX) with a Kd of 1-3 microM. We have observed a Kd for TTX of 4-10 nM for Na currents recorded from Xenopus oocytes injected with RNA extracted from rabbit cardiac muscle. This result suggests that the degree of TTX sensitivity of Na channels encoded by cardiac muscle mRNA is in part determined by post-translational modification(s) or of associations with accessory proteins in the membrane.     

Potential-dependent ion currents in the membrane of the striated muscle of the lamprey

Membrane ionic currents were recorded in thin striated muscle bundles of lamprey suction apparatus by means of double sucrose gap method. In response to depolarization fast inward Na+ and delayed outward K+ currents appeared with steady-state characteristics similar to that in frog muscle membrane. The only difference consisted in lower steepness of the inactivation curve for K+ current. This probably suggests a greater density of slow potassium channels. The presence of two fractions in potassium current is suggested from changes both in reversal potential and in speed of the current deactivation during long lasting depolarizing pulses. No functioning voltage-dependent calcium channels were detected in the lamprey muscle membrane.     

Anticonvulsant pharmacology of voltage-gated Na+ channels in hippocampal neurons of control and chronically epileptic rats

Voltage-gated Na+ channels are a main target of many first-line anticonvulsant drugs and their mechanism of action has been extensively investigated in cell lines and native neurons. Nevertheless, it is unknown whether the efficacy of these drugs might be altered following chronic epileptogenesis. We have, therefore, analysed the effects of phenytoin (100 micro m), lamotrigine (100 micro m) and valproate (600 micro m) on Na+ currents in dissociated rat hippocampal granule neurons in the pilocarpine model of chronic epilepsy. In control animals, all three substances exhibited modest tonic blocking effects on Na+ channels in their resting state. These effects of phenytoin and lamotrigine were reduced (by 77 and 64%) in epileptic compared with control animals. Phenytoin and valproate caused a shift in the voltage dependence of fast inactivation in a hyperpolarizing direction, while all three substances shifted the voltage dependence of activation in a depolarizing direction. The anticonvulsant effects on Na+ channel voltage dependence proved to be similar in control and epileptic animals. The time course of fast recovery from inactivation was potently slowed by lamotrigine and phenytoin in control animals, while valproate had no effect. Interestingly, the effects of phenytoin on fast recovery from inactivation were significantly reduced in chronic epilepsy. Taken together, these results reveal that different anticonvulsant drugs may exert a distinct pattern of effects on native Na+ channels. Furthermore, the reduction of phenytoin and, to a less pronounced extent, lamotrigine effects in chronic epilepsy raises the possibility that reduced pharmacosensitivity of Na+ channels may contribute to the development of drug resistance.     

4-aminopyridine, a specific blocker of K(+) channels, inhibited inward Na(+) current in rat cerebellar granule cells

The effects of 4-aminopyridine (4-AP), a specific blocker of outward K(+) current, on voltage-activated transient outward K(+) current (I(K(A))) and inward Na(+) current (I(Na)) were investigated on cultured rat cerebellar granule cells using the whole cell voltage-clamp technique. At the concentration of 1-5 mM, 4-AP inhibited both I(K(A)) and I(Na). It reduced the amplitude of peak Na(+) current without significant alteration of the steady-state activation and inactivation properties. The inhibitory effect was not enhanced by repeated depolarizing pulses (0.5 or 0.1 Hz), suggesting that the binding affinity of 4-AP on Na(+) channels is state-independent. In contrast, the effect of 4-AP on Na(+) channels appeared to be voltage-dependent, the weaker inhibition occurred at more depolarization. Moreover, 4-AP slowed both the activation and inactivation kinetics of Na(+) current. These effects were similar to those induced by alpha-scorpion toxin and sea anemone toxins on Na(+) channels in other cell model. Our data demonstrate for the first time that 4-AP is able to block not only A-type K(+) channels, but also Na(+) channels in rat cerebellar granule cells. It is concluded that the inhibition exerted by 4-AP on Na(+) current likely differs from that provoked by local anesthetics. The possibility that the binding site of neurotoxin receptor 3 may be involved is discussed.     

Carbamazepine effects on Na+ currents in human dentate granule cells from epileptogenic tissue

PURPOSE: Carbamazepine (CBZ) is a well-established drug in the therapy of temporal lobe epilepsy (TLE). The anticonvulsant action of CBZ has been explained mainly by use-dependent effects on voltage-dependent Na+ channels in various nonhuman cell type. However, it is unclear whether Na+ currents in neurons within the focal epileptogenic area of patients with medically intractable TLE show similar characteristics. METHODS: Therefore we used the whole-cell patch-clamp technique to investigate the effects of CBZ on voltage-dependent Na+ currents in 23 acutely isolated dentate granule cells (DGCs) from the resected hippocampus of eight patients with medically intractable TLE. RESULTS: As in findings in animal preparations, CBZ significantly reduced the amplitude of the Na+ current and significantly shifted the current-voltage dependence of the steady-state inactivation in the hyperpolarizing direction. In contrast, the rapid component of the recovery from inactivation of the Na+ currents was not affected by CBZ. In addition, the reduction of the Na+ current amplitude observed during repetitive stimulation with depolarizing pulses was not significantly altered by CBZ. CONCLUSIONS: In summary, CBZ strongly affects the voltage-dependent steady-state inactivation, with no effects on the removal of inactivation in Na+ currents of human DGCs. In spite of the lack of suitable control material, the CBZ insensitivity of the removal of inactivation may be an interesting concept to explain the medically intractable TLE in these patients.