Expression and kinetic properties of Na(+) currents in rat cardiac dorsal root ganglion neurons

The expression and properties of voltage-gated Na(+) currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na(+) currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 microM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 microM of TTX) Na(+) currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na(+) currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (-34 mV) than for TTX-S (-40.4 mV) currents. V(1/2) of activation for TTX-S current (-19.6 mV) was significantly more negative than for TTX-R current (-9.2 mV), but k factors did not differ significantly. V(1/2) and the k constant for inactivation for TTX-S currents were -35.6 and -5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V(1/2) and k were -62.3 and -7.7 mV, respectively. The action potential threshold was lower, the 10-90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na(+) current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na(+) currents were present, exhibited both TTX-S and TTX-R Na(+) currents with remarkably similar expression and kinetic properties.     

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)     

An epilepsy mutation in the beta1 subunit of the voltage-gated sodium channel results in reduced channel sensitivity to phenytoin

The antiepileptic drug phenytoin inhibits voltage-gated sodium channels. Phenytoin block is enhanced at depolarized membrane potentials and during high frequency channel activation. These properties, which are important for the clinical efficacy of the drug, depend on voltage-dependent channel gating. In this study, we examined the action of phenytoin on sodium channels, comprising a mutant auxiliary beta1 subunit (mutation C121Wbeta1), which causes the inherited epilepsy syndrome, generalized epilepsy with febrile seizures plus (GEFS+). Whole cell sodium currents in Chinese hamster ovary (CHO) cells coexpressing human Na(v)1.3 sodium channels and C121Wbeta1 exhibited altered gating properties, compared to currents in cells coexpressing Na(v)1.3 and wild type beta1. In addition mutant channels were less sensitive to inhibition by phenytoin, showing reduced tonic block at -70mV (EC(50)=26microM for C121Wbeta1 versus 11microM for wild type beta1) and less frequency-dependent inhibition in response to a 20Hz pulse train ( approximately 40% inhibition for C121Wbeta1 versus approximately 70% inhibition for wild type beta1, with 50microM phenytoin). Mutant and wild type channels did not differ in inactivated state affinity for phenytoin, suggesting that their pharmacological differences were secondary to their differences in voltage-dependent gating, rather than being caused by direct effects of the mutation on the drug receptor. Together, these data show that a sodium channel mutation responsible for epilepsy can also alter channel response to antiepileptic drugs.     

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.     

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.     

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 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.     

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

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

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

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

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.     

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.     

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)     

Effects of ApC, a sea anemone toxin, on sodium currents of mammalian neurons

We have characterized the actions of ApC, a sea anemone polypeptide toxin isolated from Anthopleura elegantissima, on neuronal sodium currents (I(Na)) using current and voltage-clamp techniques. Neurons of the dorsal root ganglia of Wistar rats (P5-9) in primary culture were used for this study. These cells express tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) I(Na). In current-clamp experiments, application of ApC increased the average duration of the action potential. Under voltage-clamp conditions, the main effect of ApC was a concentration-dependent increase in the TTX-S I(Na) inactivation time course. No significant effects were observed on the activation time course or on the current peak-amplitude. ApC also produced a hyperpolarizing shift in the voltage at which 50% of the channels are inactivated and caused a significant decrease in the voltage dependence of Na+ channel inactivation. No effects were observed on TTX-R I(Na). Our results suggest that ApC slows the conformational changes required for fast inactivation of the mammalian Na+ channels in a form similar to other site-3 toxins, although with a greater potency than ATX-II, a highly homologous anemone toxin.     

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.     

Calcium currents in cultured rat cortical neurons

Rat neocortical neurons grown in dissociated cell culture for 4-12 weeks were studied with whole-cell patch-clamp techniques in order to characterize the calcium currents present in these cells. When voltage-dependent Na and K currents were inhibited, depolarizations from negative holding potentials induced inward currents which had 3 components: a low threshold activated, small, relatively persistent component, which was completely inactivated at holding potentials more positive then -60 mV; a higher threshold, relatively persistent component (which was not inactivated at VH = -50 mV); and a higher threshold, larger, transient component. All 3 components were reduced by removal of Ca, and blocked by Cd and Ni at appropriate concentrations. The components were differentially affected by low concentrations of Ni (500 microM), nifedipine (500 microM) and Ba (1.8 mM). Only the first two components were present in very young neurons.     

Interaction of the novel anticonvulsant, BIA 2-093, with voltage-gated sodium channels: comparison with carbamazepine

PURPOSE: BIA 2-093 [(S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz/b,f/azepine-5-carboxamide] is endowed with an anticonvulsant potency similar to that of carbamazepine (CBZ), but produces less cognitive and motor impairment. This study evaluated whether voltage-gated sodium channels (VGSCs) are a primary locus for the action of BIA 2-093. METHODS: We used the whole-cell voltage-clamp technique in the mouse neuroblastoma cell line N1E-115 to investigate the effects of BIA 2-093 and CBZ on VGSCs, displacement of [3H]-batrachotoxinin A 20-alpha-benzoate ([3H]-BTX), and [3H]-saxitoxin to define their relative potency to bind to rat brain sodium channels, and inhibition of uptake of 22Na by rat brain cortical synaptosomes stimulated by veratridine as a measure of sodium entry. RESULTS: The inhibitory potencies of BIA 2-093 and CBZ increased as the holding potential was made less negative (-100, -90, -80, and -70 mV) with median inhibitory concentration (IC50) values (in microM) of, respectively, 4,337, 618, 238, and 139 for BIA 2-093, and 1,506, 594, 194, and 101 for CBZ. BIA 2-093 displayed a similar potency in displacing [3H]-BTX (IC50 values, 222 vs. 361 microM; p > 0.05) and inhibiting the uptake of 22Na (IC50 values, 36 vs. 138 microM; p > 0.05). Both drugs failed to displace [3H]-saxitoxin in concentrations up to 300 microM. CONCLUSIONS: BIA 2-093, like CBZ, inhibits sodium currents in a voltage-dependent way by an interaction predominantly with the inactivated state of the channel and interacts with neurotoxin receptor site 2, but not with receptor site 1. BIA 2-093 displayed a potency blocking VGSCs similar to that of CBZ.     

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

Lysophosphatidic acid (LPA), a simple phospholipid, induces pain. To elucidate an involvement of ion channel mechanism in the LPA-induced pain, its effects on sodium currents in rat dorsal root ganglion (DRG) neurons were investigated. LPA suppressed tetrodotoxin-sensitive (TTX-S) sodium current, but increased tetrodotoxin-resistant (TTX-R) sodium current, when currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV. In both types of currents, LPA produced a hyperpolarizing shift of both activation and inactivation voltages. LPA had a negligible effect on the maximal conductance of TTX-S current, but increased that of TTX-R current. The results suggest that the enhancement of TTX-R current may contribute to the LPA-induced pain.     

Gating of myotonic Na channel mutants defines the response to mexiletine and a potent derivative.

BACKGROUND: Myotonia and periodic paralysis caused by sodium channel mutations show variable responses to the anti-myotonic drug mexiletine. OBJECTIVE: To investigate whether variability among sodium channel mutants results from differences in drug binding affinity or in channel gating. METHODS: Whole-cell sodium currents (I(Na)) were recorded in tsA201 cells expressing human wild-type (WT) and mutant skeletal muscle sodium channels (A1156T, hyperkalemic periodic paralysis; R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). RESULTS: At a holding potential (hp) of -120 mV, mexiletine produced a tonic (TB, 0.33 Hz) and a use-dependent (UDB, 10 Hz) block of peak I(Na) with a potency following the order rank R1448C > WT approximately equal A1156T > G1306E. Yet, when assayed from an hp of -180 mV, TB and UDB by mexiletine were similar for the four channels. The different midpoints of channel availability curves found for the four channels track the half-maximum inhibitory value (IC50) measured at -120 mV. Thus differences in the partitioning of channels between the closed and fast-inactivated states underlie the different IC50 measured at a given potential. The mexiletine-derivative, Me7 (alpha-[(2-methylphenoxy)methyl]-benzenemethanamine), behaved similarly but was approximately 5 times more potent than mexiletine. Interestingly, the higher drug concentrations ameliorated the abnormally slower decay rate of myotonic I(Na). CONCLUSIONS: These results explain the basis of the apparent difference in block of mutant sodium channels by mexiletine and Me7, opening the way to a more rationale drug use and to design more potent drugs able to correct specifically the biophysical defect of the mutation in individual myotonic patients.     

Anticonvulsant phenytoin affects voltage-gated potassium currents in cerebellar granule cells

The anticonvulsant drug phenytoin (diphenylhydantoin, DPH) was examined for its action on potassium currents in cerebellar granule cells using the whole-cell patch-clamp technique. Granular cells expressed two main types of voltage-dependent potassium currents: the first, sensitive to Tetraethylammonium ion (TEA), resembles a delayed rectifier K(+) channel (I(d)); the second shows biophysical and pharmacological properties similar to an I(A)-type potassium current. Phenytoin blocks the I(A) current in a dose-dependent manner, with an apparent dissociation constant K(d) of (73+/-7) microM. The drug shifts the steady-state inactivation curves towards a more negative potential, stabilizing the inactivated state, while the activation kinetics remain unaffected. The estimated K(d) when the cell is held to -100 mV (closed state of the channel) is 145+/-8 microM which decreases to 35+/-10 microM at -80 mV holding potential (partial inactivation of the channel). Phenytoin shows a discriminant behaviour between the two different types of potassium channels because at high concentration the effect of the drug on the delayed rectifier K(+) channel is negligible.