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.     

Modulation of tetrodotoxin-resistant sodium channels by dihydropyrazole insecticide RH-3421 in rat dorsal root ganglion neurons

The effects of the dihydropyrazole insecticide RH-3421 on the retrodotoxin-resistant (TTX-R) voltage-gated sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole-cell patch clamp technique. RH-3421 at 10 nM to 1 microM completely blocked action potentials. The sodium currents were irreversibly suppressed by 1 microM RH-3421 in a time- and a dose-dependent manner and the IC50 value of RH-3421 was estimated to be 0.7 microM after 10 min of application. RH-3421 blocked the sodium currents to the same extent over the entire range of test potentials. The sodium conductance-voltage curve was not shifted along the voltage axis by 1 microM RH-3421 application In contrast, both fast and slow steady-state sodium channel inactivation curves were shifted in the hyperpolarizing direction in the presence of 1 microM RH-3421. It was concluded that RH-3421 bound to the resting and inactivated sodium channels to cause block with a higher affinity for the latter state.     

Kinetic parameters of calcium currents in maturing acutely isolated CA1 cells

Calcium currents were recorded in CA1 hippocampal cells from immature (P(4-10)) and older (P(22-55)) rats, using whole-cell voltage clamp techniques. Parameters defining the voltage-dependence of activation (tau(m)) and inactivation (tau(h)), steady-state inactivation and activation were determined at both stages of maturation. Current density increased with maturation. A transient low voltage activated (l.v.a.) current was found in P(4-10) cells, but not in the older cells. At voltages less negative than -30 mV, current inactivation was best described by two exponentials (tau(hf), tau(hs)); the ratio of the amplitudes of the two components changed with maturation, with a dominance of the faster component (tau(hf)) in the younger cells. The voltage dependence of tau(hf) followed a simple dependence model, decreased with increasing depolarization, in all cells at both stages of maturation. In P(4-10) cells, tau(hs) was voltage insensitive (range -25 to +30 mV); in P(22-55) cells, the voltage dependence of tau(hs) was found to be complex. Two current components were identified from the voltage dependence of the conductance in both groups. The first, more hyperpolarized component, the l.v.a. current found in P(4-10) cells; this was absent in the older cells, in which we found a component with a different voltage dependence. The voltage dependence of the conductance of the second, more depolarized component did not differ in younger and older cells. In the course of maturation, the steady-state inactivation of the second component underwent a hyperpolarizing shift and a decrease in voltage sensitivity.     

Different effects of mexiletine on two mutant sodium channels causing paramyotonia congenita and hyperkalemic periodic paralysis

Effects of the antiarrhythmic and antimyotonic drug mexiletine were studied on two sodium channel mutants causing paramyotonia congenita (R1448H) and an overlap paramyotonic and hyperkalemic paralytic syndrome (M1360V). Channels were expressed in human embryonic kidney cells and studied electrophysiologically, using the whole-cell patch-clamp technique. Compared to the wild-type, channel, both mutants showed alterations of inactivation, i.e. slower inactivation, left shift of steady-state inactivation and faster recovery from inactivation. Mexiletine caused a significantly larger use-dependent block of the R1448H mutant when compared to M1360V and wild-type channels. This can be explained by a prolonged recovery from mexiletine block as observed for R1448H channels, since the affinity of mexiletine for the inactivated state was similar for all three clones. The use-dependent block of sodium channels by mexiletine reduces repetitive series of action potentials and therefore improves muscle stiffness in myotonic patients. The enhanced use-dependent block as seen with R1448H may explain the extraordinary therapeutic efficacy of mexiletine in most patients with paramyotonia congenita.     

Differential properties of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons.

TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) sodium channel currents were analyzed in acutely dissociated dorsal root ganglion (DRG) neurons isolated from 3-12-d-old and adult rats. Currents were recorded using the whole-cell patch-clamp technique. TTX-R current was more likely to be present in younger animals (3-7 d), whereas TTX-S current was more common in older animals (7-10 d), although TTX-R current was recorded from adult rat DRG neurons. The TTX-R and TTX-S currents differed in their steady-state inactivation, with 50% inactivation voltage at -40 +/- 5 mV (n = 10) for TTX-R currents and -70 +/- 4 mV (n = 10) for TTX-S currents. These current types also differed in their activation kinetics, with 50% activation values of -15 +/- 5 mV (n = 5) for TTX-R currents and -26 +/- 6 mV (n = 5) for TTX-S currents. The interactions of TTX-R and TTX-S channels with various pharmacological agents and divalent cations were studied. The Kd values for TTX-S and TTX-R currents were estimated to be 0.3 nM and 100 microM for TTX, 0.5 nM and 10 microM for saxitoxin, and 50 microM and 200 microM for lidocaine, respectively. TTX-S channels did not exhibit a marked use-dependent block by lidocaine, whereas lidocaine significantly decreased TTX-R current in a use-dependent manner at frequencies ranging from 1 to 33.3 Hz. Several external divalent cations exerted different effects on these current types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Block of sodium currents in rat dorsal root ganglion neurons by diphenhydramine

To elucidate the local anesthetic mechanism of diphenhydramine, its effects on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were examined by the whole-cell voltage clamp method. Diphenhydramine blocked TTX-S and TTX-R sodium currents with K(d) values of 48 and 86 microM, respectively, at a holding potential of -80 mV. It shifted the conductance-voltage curve for TTX-S sodium currents in the depolarizing direction but had little effect on that for TTX-R sodium currents. Diphenhydramine caused a shift of the steady-state inactivation curve for both types of sodium currents in the hyperpolarizing direction. The time-dependent inactivation became faster and the recovery from the inactivation was slowed by diphenhydramine in both types of sodium currents. Diphenhydramine produced a profound use-dependent block when the cells were repeatedly stimulated with high-frequency depolarizing pulses. The use-dependent block was more pronounced in TTX-R sodium currents. The results show that diphenhydramine blocks sodium channels of sensory neurons similarly to local anesthetics.     

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.     

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.     

Effects of nordihydroguaiaretic acid on Na+ currents in rat dorsal root ganglion neurons

Nordihydroguaiaretic acid (NDGA) is a lipoxygenase (LO) inhibitor with a strong antioxidant activity. It attenuates nociceptive responses produced by various stimuli, which has been ascribed to its LO inhibition. Primary sensory neurons express multiple Na+ channels that are important in processing normal and abnormal nociception. We examined the effects of NDGA on tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ currents in rat dorsal root ganglion neurons. NDGA inhibited both types of Na+ currents concentration dependently and reversibly. Both activation and inactivation time courses were slowed by NDGA, which were not reversible. NDGA produced a hyperpolarizing shift of the steady-state inactivation curves and reduced the maximal availability of both Na+ currents, indicating that it blocks both inactivated and resting Na+ channels. NDGA shifted the conductance-voltage curves of both Na+ currents toward a depolarizing direction and increased the slope factors of the curves. The recovery of Na+ channels from inactivation was retarded by NDGA. All these effects will reduce the excitability of sensory neurons and should be taken into account when it comes to the antinociceptive effects of NDGA.     

Sodium channel currents in maturing acutely isolated rat hippocampal CA1 neurones.

Sodium channel currents were recorded in excised inside-out patches from immature (P(4-10)) and older (P(20-46)) rat CA1 neurones. Channel conductance was 16.6+/-0.013 pS (P(20-46)) and 19.0+/-0.031 pS (P(4-10)). Opening patterns varied with step voltage and with age. In some patches bursting was apparent at voltages positive to -30 mV. Non-bursting behaviour was more dominant in patches from younger animals. In older animals mean open time (m.o.t.) was best described by two exponentials especially in the older cells; in the immature, there were fewer cases with two exponentials. The time constant of inactivation (tau(h)) estimated in ensemble averages was best described by two exponentials (tau(hf) and tau(hs)) in most patches from older cells. tau(hf) decreased with depolarization; tau(hs) increased in the range -30 to 0 mV. The voltage dependence of tau(hf) in the older cells is identical to that of the single tau(h) found in the younger; the results indicate a dominance of tau(hf) in the younger. Patches from younger cells more often showed one apparent active channel; in such cases, m.o.t. was described by a single exponential. However, in two cases, channels showed bursting behaviour with one of these channels showing a shift between bursting and non-bursting modes. Our findings are consistent with a heterogeneous channel population and with changes in the population in the course of maturation.     

Voltage-dependent block of sodium channels in mammalian neurons by the oxadiazine insecticide indoxacarb and its metabolite DCJW

Indoxacarb is a newly developed insecticide with high insecticidal activity and low toxicity to non-target organisms. Its metabolite, DCJW, is known to block compound action potentials in insect nerves and to inhibit sodium currents in cultured insect neurons. However, little is known about the effects of these compounds on the sodium channels of mammalian neurons. We compared the effects of indoxacarb and DCJW on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons by using the whole-cell patch clamp technique. Indoxacarb and DCJW at 1-10 microM slowly and irreversibly blocked both TTX-S and TTX-R sodium channels in a voltage-dependent manner. The sodium channel activation kinetics were not significantly modified by 1 microM indoxacarb or 1 microM DCJW. The steady-state fast and slow inactivation curves were shifted in the hyperpolarization direction by 1 microM indoxacarb or 1 microM DCJW indicating a higher affinity of the inactivated sodium channels for these insecticides. These shifts resulted in an enhanced block at more depolarized potentials, thus explaining voltage-dependent block, and an apparent difference in the sensitivity of TTX-R and TTX-S channels to indoxacarb and DCJW near the resting potential. Indoxacarb and its metabolite DCJW cause toxicity through their action on the sodium channels.     

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.     

Brevetoxin modulates neuronal sodium channels in two cell lines derived from rat brain

Single Na+ channel currents were recorded from cell-attached membrane patches from two neuronal cell lines derived from rat brain, B50 and B104, and compared before and after exposure of the cells to purified brevetoxin, PbTx-3. B50 and B104 Na+ channels usually exhibited fast activation and inactivation as is typical of TTX-sensitive Na+ channels. PbTx-3 modified channel gating in both cell lines. PbTx-3 caused (1) significant increases in the frequency of channel reopening, indicating a slowing of channel inactivation, (2) a change in the voltage dependence of the channels, promoting channel opening during steady-state voltage clamp of the membrane at voltages throughout the activation range of Na+ currents, but notably near the resting potential of these cells (-60 - -50 mV), and (3) a significant, 6.7 mV hyperpolarized shift in the threshold potential for channel opening. Na+ channel slope conductance did not change in PbTx-3-exposed B50 and B104 neurons. These effects of Pbx-3 may cause hyperexcitability as well as inhibitory effects in intact brain.     

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.     

Effects of arachidonic acid on sodium currents in rat dorsal root ganglion neurons

The effects of arachidonic acid on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons were assessed using the whole-cell patch-clamp method. Both sodium currents were modulated in a similar way by extracellular application of arachidonic acid. Arachidonic acid increased the currents at lower depolarizing potentials, while it suppressed the currents at higher depolarizing potentials and at less negative holding potentials. These effects were due to the shifts of both the conductance–voltage curve and the steady-state inactivation curve in the hyperpolarizing direction. Indomethacin, a cyclooxygenase inhibitor, suppressed the arachidonic acid-induced shift of the conductance–voltage curve but not that of the steady-state inactivation curve. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable arachidonic acid analog, failed to shift the conductance–voltage curve but still produced the shift of the steady-state inactivation curve. Thus it is assumed that the effect of arachidonic acid on the sodium channel activation is caused by the metabolite(s) of arachidonic acid. However, the effect on the steady-state sodium channel inactivation is exerted by arachidonic acid itself. It is suggested that arachidonic acid, by modulating sodium currents, may alter the excitability of sensory neurons depending on the resting membrane potential.     

Activation and inactivation of batrachotoxin-modified sodium channels of nerve fiber membranes in the frog

Currents through batrachotoxin (BTX)-modified sodium channels in frog myelinated nerve were measured under voltage-clamp conditions. Nonlinearity of "instantaneous" current-voltage relations was taken into account when determining steady-state parameters of channel activation. BTX induces the shift of voltage dependence of channel activation towards more negative potentials by 67 mV, without changes in its steepness. Current kinetics and effect of preceding depolarization on current size suggest that BTX-modified channels are capable for partial inactivation. High level of steady-state conductance of BTX-modified channels can be explained by suggestion that open state of the channel is energetically more profitable than inactivated one. It is concluded that effect of BTX on inactivation is different in principle from that of pronase and protein reagents.     

Ethanol inhibits voltage-gated sodium channels in cultured superior cervical ganglion neurons

To determine whether actions on sodium channels contribute to ethanol's depressant effects on the autonomic nervous system, the acute effects of ethanol on Na+ currents in primary cultured superior cervical ganglion were examined by whole-cell patch clamp recordings. Ethanol inhibited Na+ currents concentration dependently, and decreased action potential firing. Ethanol (100 mM) did not affect activation curve, but resulted in a left shift of the inactivation curve and prolonged the recovery from inactivation. This finding indicates that the channels in the inactivated state are more susceptible to ethanol than those in the resting state. For the first time, this study demonstrates acute inhibitory effects of ethanol on sodium channel gating in sympathetic neurons.     

Inhibitory effect of lamotrigine on A-type potassium current in hippocampal neuron-derived H19-7 cells

PURPOSE: We investigated the effects of lamotrigine (LTG) on the rapidly inactivating A-type K+ current (IA) in embryonal hippocampal neurons. METHODS: The whole-cell configuration of the patch-clamp technique was applied to investigate the ion currents in cultured hippocampal neuron-derived H19-7 cells in the presence of LTG. Effects of various related compounds on IA in H19-7 cells were compared. RESULTS: LTG (30 microM-3 mM) caused a reversible reduction in the amplitude of IA. The median inhibitory concentration (IC50) value required for the inhibition of IA by LTG was 160 microM. 4-Aminopyridine (1 mM), quinidine (30 microM), and capsaicin (30 microM) were effective in suppressing the amplitude of IA, whereas tetraethylammonium chloride (1 mM) and gabapentin (100 microM) had no effect on it. The time course for the inactivation of IA was changed to the biexponential process during cell exposure to LTG (100 microM). LTG (300 microM) could shift the steady-state inactivation of IA to a more negative membrane potential by approximately -10 mV, although it had no effect on the slope of the inactivation curve. Moreover, LTG (100 microM) produced a significant prolongation in the recovery of IA inactivation. Therefore in addition to the inhibition of voltage-dependent Na+ channels, LTG could interact with the A-type K+ channels to suppress the amplitude of IA. The blockade of IA by LTG does not simply reduce current magnitude, but alters current kinetics, suggesting a state-dependent blockade. LTG might have a higher affinity to the inactivated state than to the resting state of the IA channel. CONCLUSIONS: This study suggests that in hippocampal neurons, during exposure to LTG, the LTG-mediated inhibition of these K+ channels could be one of the ionic mechanisms underlying the increased neuronal excitability.     

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.     

The role of the divergent amino and carboxyl domains on the inactivation properties of potassium channels derived from the Shaker gene of Drosophila.

Several products generated from the Drosophila Shaker gene by alternative splicing predict a group of similar proteins with an identical central and variable amino and carboxyl domains. We have constructed 9 Sh cDNAs combining 3 different 5' domains with 3 different 3' domains. RNA transcribed from 6 of these cDNAs induce K+ currents in Xenopus oocytes. All currents share similar properties of voltage dependence, potassium selectivity, and block by 4-AP, TEA, and charybdotoxin. These properties presumably result from a channel core formed by the identical central region of the proteins. The currents differ in macroscopic inactivation kinetics. Five RNAs induced K+ currents which inactivate, each at distinct rates, during short depolarizations. The sixth RNA induces a current that essentially does not inactivate unless depolarized for many seconds. This raises the possibility that Sh may encode nontransient as well as transient K+ currents. Analysis of currents produced by the various combinations suggests that the divergent amino domains influence the stability of a first, nonabsorbing, inactivated state. This results in striking differences in the probability of channel reopening, as observed in single-channel recordings, of those channels with identical carboxyl but different amino domains. Furthermore, based on macroscopic analysis of the currents, we suggest that the primary role of the carboxyl domains is to influence the relative stability between the first and a second inactivated state. The second inactivated state is essentially absorbing, and recovery from this state is very slow. The observed differences in the rates of recovery from inactivation of channels containing different carboxyl domains reflect differences in the rates at which they enter this second inactivated state.