Activation of Nucleotide Receptors Inhibits High-threshold Calcium Currents in NG108-15 Neuronal Hybrid Cells

A P_2U (UTP-sensitive) nucleotide receptor has previously been cloned from NG108-15 neuroblastoma × glioma hybrid cells and it has been shown that activation of this receptor inhibits the M-type K⁺-current. We now report that UTP also inhibits Ca²⁺-currents in differentiated NG 108-15 cells, but probably through a different nucleotide receptor. UTP (100 μM) inhibited the peak of the high-threshold current by 28.4 ± 3.1% ( n = 38) with no effect on the low-threshold current. Two components of high-threshold current were identified: one inhibited by 100 nM ω-conotoxin (CgTx) and one inhibited by 2 μM nifedipine and enhanced by 1 μM BAY K8644. UTP inhibited the former by 31.0 ± 3.1%, with an IC₅₀ of 2.8 ± 1.1 μM, and the latter by 34.2 ± 6.1% with an IC₅₀ of 1.7 ± 1.3 μM. Pertussis toxin pretreatment prevented inhibition of the CgTx-sensitive, nifedipine-resistant but not CgTx-resistant current. Inhibition was not prevented by intracellular BAPTA (20 mM) or CAMP (1 mM). Effects of UTP on both currents were imitated by UDP, ATP, ADP, AP₄A and ATPγS but weakly or not at all by 2-MeSATP, GTP, AMP-CPP or ITP. Since the receptors which inhibit Ca²⁺-currents are activated by ATP, it is suggested that they might mediate auto-inhibition of transmitter release by ATP if present on purinergic nerve terminals.     

Blockade of nicotinic responses in rat retinal ganglion cells by neuronal bungarotoxin

The effects of neuronal bungarotoxin (NBT) on nicotinic acetylcholine responses recorded from rat retinal ganglion cells in culture were studied with patch electrodes. We observed that the concentration of this toxin needed to induce a total blockade of nicotinic currents varied according to the method of toxin application utilized. Rapid addition of 20 microM NBT by pressure ejection from micropipettes produced total blockade of 50-microM acetylcholine-induced currents. In contrast, when added slowly via the physiological solution which continuously superfused the cells, NBT was able to produce a complete blockade of the response at 200 nM. The IC50 determined for NBT by the superfusion method was 55 nM. Recovery from block was slow and incomplete with both drug application methods, although some differences were found. The results are discussed with reference to a scheme in which NBT binds with both low and high affinity to functional nicotinic receptors in rat retinal ganglion cells in culture.     

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

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

Alteration of Sodium Currents by New Peptide Toxins From the Venom of a Molluscivorous Conus Snail

TxIA and TxIB, peptides with 27-amino acid residues recently isolated from the molluscivorous marine snail Conus textile neovicarius , exhibit strong paralytic activity in molluscs, with no paralytic effects on arthropods and vertebrates. At concentrations of 0.25 – 0.5 μM the toxins cause spontaneous repetitive firing and dramatic broadening of the action potential of cultured Aplysia neurons. The action potential duration partially recovers within 30 min in the presence of the toxins. Under these conditions a second toxin application does not change the spike duration. TxI-induced spike broadening occurs when potassium and calcium conductances are blocked. Voltage-clamp experiments revealed that the toxins alter the kinetics of the sodium current either by slowing down the rate of sodium current inactivation or by recruiting silent sodium channels with slower activation and inactivation kinetics. The toxins shift the voltage-dependent steady-state Na⁺ current inactivation curve to more positive values by 6 mV. These changes are not associated with alteration in the rate of sodium current activation, in the peak sodium current, or the sodium current reversal potential. TxI apparently represents a new class of conotoxins with an unusual phylogenic specificity and may therefore be useful as a probe for the study of molluscan neuronal sodium channels.     

Associative somatodendritic interaction in layer V pyramidal neurons is not affected by the antiepileptic drug lamotrigine

The antiepileptic drug lamotrigine was described to exert its effects on neuronal excitability via voltage-gated sodium and calcium, as well as hyperpolarization-activated conductances. In order to define the effects of lamotrigine on the excitability of layer V pyramidal cells of the rat somatosensory cortex we performed patch-clamp recordings from the soma and dendrite of this major cortical output cell type in acute slices. Voltage-clamp experiments revealed the blockade of the persistent sodium current by 50-100 micro m lamotrigine as well as by 50 micro m of the anticonvulsant drug phenytoin. In somatic current-clamp studies lamotrigine, in a therapeutic concentration range, depolarizes the membrane potential reflecting the activation of the hyperpolarization-activated current. This depolarization reduces the rheobase and increases the spiking frequency at the onset of the spike train. For long depolarizing current pulses under lamotrigine, however, a use-dependent block of sodium channels reduces spiking frequency and spike amplitude. The depolarization due to 50-100 micro m lamotrigine reduces additionally the critical frequency of back-propagating spikes necessary to elicit a dendritic calcium action potential. Ten to thirty micromolar lamotrigine, in contrast, did not change the critical frequency. Lamotrigine blocks long-lasting, high frequent spiking activity due to its use-dependent sodium channel block, while burst activity is not impaired due to a depolarizing shift of the membrane potential. This drug therefore dampens epileptic activity while leaving the somatodendritic association in layer V pyramidal cells intact.     

Voltage-gated Currents in Rabbit Retinal Astrocytes

The voltage-gated currents of the astrocytes associated with the retinal capillaries of the rabbit retina were studied using whole-cell patch clamp recording. The resting potential of these cells was −70 ± 4.8 mV (mean ± SEM; n = 54), and the input resistance and cell capacitance were 558 ± 3.6 MΩ and 19.5 ± 1.8 pF respectively. Depolarization to potentials positive to −50 mV evoked rapidly activating inward and outward currents. The inward current was transient, eliminated by substitution of choline for Na⁺ in the bathing solution, and reduced by 50% in the presence of 1 μM tetrodotoxin. The time-to-peak of the Na⁺ current was more than twice that for the Na⁺ current found in retinal neurons. The glial Na⁺ current was half-inactivated at −55 mV. A transient component of the outward K⁺ current was blocked by external 4-aminopyridine while a more sustained component was blocked by external tetraethylammonium. At potentials between −150 and −50 mV the membrane behaved Ohmically. Voltage-gated currents in retinal astrocytes recorded in situ appear qualitatively similar to those described for some glial cells in vitro.     

Electrophysiological characterization of sodium channel types in the HCN-1A human cortical cell line

Electrically evoked sodium currents were recorded under whole-cell patch clamp from undifferentiated HCN-1A cells. Peak sodium currents had a half-maximal activation, V_m0.5, of −22.6 ± 1.0 mV with a voltage dependence, K_m, of 7.28 ± 0.39 mV⁻¹. Steady-state inactivation indicated the presence of two types of sodium channel. One type inactivated with V_h0.5 = −93.8 ± 1.2 mV and k_h = −6.8 ± 0.4 mV⁻¹. The second type of sodium channel inactivated w V_h0.5 = −44.6 ± 1.5 mV and k_h = −7.3 ± 0.4 mV⁻¹. The occurrence of each channel type varied from cell to cell and ranged from 0 to 100% of the total sodium current. No variation in the rate of inactivation was seen when the holding potential was adjusted to eliminate the more negative of the two inactivation components. Application of tetrodotoxin (TTX) or saxitoxin (STX) revealed channel types with two different affinities for each toxin. TTX blocked peak sodium conductance with apparent IC50s of 22 nM and 5.3 μM. STX was more potent, with apparent IC₅₀s of 1.6 nM and 1.2 μM. There was no statistical correlation between toxin sensitivity and steady-state inactivation voltage, suggesting that these properties varied independently among sodium channel types.     

Hyperosmolar D-mannitol reverses the increased membrane excitability and the nodal swelling caused by Caribbean ciguatoxin-1 in single frog myelinated axons

The effects of hyperosmolar D-mannitol were studied on single frog myelinated nerve fibres previously poisoned with Caribbean ciguatoxin-1 (C-CTX-1), a new toxin isolated from the pelagic fish Caranx latus inhabiting the Caribbean region. In current-clamped myelinated axons, C-CTX-1 (50-120 nM) caused spontaneous and repetitive action potential discharges after a short delay. In addition, the toxin produced a marked swelling of nodes of Ranvier of myelinated axons that reached a steady state within about 90 min, as revealed by using confocal laser scanning microscopy. The increased excitability and the nodal swelling caused by C-CTX-1 were prevented or reversed by an external hyperosmotic solution containing 100 mM D-mannitol. Moreover, the C-CTX-1-induced nodal swelling was completely prevented by the blockade of voltage-sensitive sodium channels by tetrodotoxin (TTX). It is suggested that C-CTX-1, by increasing nerve membrane excitability, enhances Na(+) entry into nodes of Ranvier through TTX-sensitive sodium channels, which directly or indirectly disturb the osmotic equilibrium between intra- and extra-axonal media resulting in an influx of water that was responsible for the long-lasting nodal swelling. The fact, that hyperosmolar D-mannitol either reversed or prevented the neurocellular actions of C-CTX-1, is of particular interest since it provides the rational basis for its use to treat the neurological symptoms of ciguatera fish poisoning in the Caribbean area.     

A new conotoxin isolated from Conus consors venom acting selectively on axons and motor nerve terminals through a Na+-dependent mechanism

A novel conotoxin was isolated and characterized from the venom of the fish-hunting marine snail Conus consors. The peptide was identified by screening chromatography fractions of the crude venom that produced a marked contraction and extension of the caudal and dorsal fins in fish, and noticeable spontaneous contractions of isolated frog neuromuscular preparations. The peptide, named CcTX, had 30 amino acids and the following scaffold: X11CCX7CX2CXCX3C. At the frog neuromuscular junction, CcTx at nanomolar concentrations selectively increased nerve terminal excitability so that a single nerve stimulation triggered trains of repetitive or spontaneous synaptic potentials and action potentials. In contrast, CcTx had no noticeable effect on muscle excitability even at concentrations 100 x higher than those that affected motor nerve terminals, as revealed by direct muscle stimulation. In addition, CcTx increased miniature endplate potential (MEPP) frequency in a Ca2+-free medium supplemented with ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA). Blockade of voltage-dependent sodium channels with tetrodotoxin (TTX) either prevented or suppressed the increase of MEPP frequency induced by the toxin. CcTx also produced a TTX-sensitive depolarization of the nodal membrane in single myelinated axons giving rise, in some cases, to repetitive and/or spontaneous action potential discharges. In addition, CcTx increased the nodal volume of myelinated axons, as determined using confocal laser scanning microscopy. This increase was reversed by external hyperosmolar solutions and was prevented by pretreatment of axons with TTX. It is suggested that CcTx, by specifically activating neuronal voltage-gated sodium channels at the resting membrane potential, produced Na+ entry into nerve terminals and axons without directly affecting skeletal muscle fibres. CcTx belongs to a novel family of conotoxins that targets neuronal voltage-gated sodium channels.     

Indirect activation of neuronal noncapacitative Ca2+ entry is the final step involved in the neurotoxic effect of Tityus serrulatus scorpion beta-toxin

Interweaving strategies of electrophysiology, calcium imaging and immunocytochemistry bring new insights into the mode of action of the Brazilian scorpion Tityus serrulatusbeta-toxin VII. Pacemaker dorsal unpaired median neurons isolated from the cockroach central nervous system were used to study the effects of toxin VII. In current-clamp, 50 nm toxin VII produced a membrane depolarization and reduced spiking. At 200 nM, depolarization associated with multiphasic effects was seen. After artificial hyperpolarization, plateau potentials on which spontaneous electrical activity appeared were observed. In voltage clamp, toxin VII induced a negative shift of the voltage dependence of sodium current activation without significant effect on steady-state inactivation. In addition, toxin VII produced a permanent TTX-sensitive holding inward current, indicating that background sodium channels were targeted by beta-toxin. Cell-attached patch recordings indicated that these channels were switched from unclustered single openings to current fluctuating between distinct subconductance levels exhibiting increased open probability and open-time distribution. Toxin VII also produced a TTX-sensitive [Ca2+]i rise. Immunostaining with Cav2.2(alpha1b) antibodies and calcium imaging data obtained with omega-CgTx GVIA indicated that N-type high-voltage-activated calcium channels initiated calcium influx and were an essential intermediate in the pathway linking toxin VII-modified sodium channels to the activation of an additional route for calcium entry. By using inhibitors of (i) noncapacitative calcium entry (inhibitor LOE-908), (ii) NO-sensitive guanylyl cyclase (ODQ) and (iii) phosphodiesterase 2 (EHNA), together with cGMP antibodies, we demonstrated that noncapacitative calcium entry was the final step in a complex combination of events that was initiated by toxin VII-alteration of sodium channels and then involved successive activation of other membrane ion channels.     

Regulation of bradykinin- and ATP-activated Ca(2+)-permeable channels in rat pheochromocytoma (PC12) cells.

Membrane currents activated by bradykinin (500 nM) and by extracellular ATP (50 microM) were studied in voltage-clamped, NGF-treated rat pheochromocytoma (PC12) cells. Under quasiphysiological ionic conditions, both substances caused an outward current due to opening of Ca(2+)-activated K+ channels. Bradykinin caused an additional inward current that could be studied after blockade by internal Cs+ of the initial transient outward current. The inward current became larger when the extracellular Ca2+ concentration was increased. Neither inositol-1,4,5-trisphosphate, dioctanoylglycerol, phorbol 12-myristat 13-acetate, forskolin, GTP, GTP-gamma-S, or pretreatment with pertussis toxin affected this current component. Increasing the internal Ca buffer concentration [EGTA or bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid] from 1 to 10 mM had no effect on the inward current as long as the free [Ca2+]i was kept constant. However, it was modulated by the resting free [Ca2+]i. Elevation of [Ca2+]i from nominally 0 to 60 or to 180 nM increased the bradykinin-induced average peak current density from 0.14 to 1.04 or to 2.29 pA/pF, respectively. This regulation may depend on a calmodulin-dependent pathway, since CGS 9343B, a calmodulin inhibitor, blocked the effect of elevated [Ca2+]i. With ATP as an agonist, outward current was preceded by a large inward current that was partially blocked by extracellular Ca2+ in the millimolar range. Extracellular Ca2+ was also found to reduce the single-channel conductance estimated from outside-out patches treated with ATP.     

The neuroleptic drug, fluphenazine, blocks neuronal voltage-gated sodium channels

Fluphenazine (Prolixin(R)) is a potent phenothiazine-based dopamine receptor antagonist, first introduced into clinical practice in the late 1950s as a novel antipsychotic. The drug emerged as a 'hit' during a routine ion channel screening assay, the present studies describe our electrophysiological examination of fluphenazine at tetrodotoxin-sensitive (TTX-S) and resistant (TTX-R) voltage-gated sodium channel variants expressed in three different cell populations. Constitutively expressed TTX-S conductances were studied in ND7/23 cells (a dorsal root ganglion-derived clonal cell line) and rat primary cerebrocortical neurons. Recombinant rat Na(V)1.8 currents were studied using ND7/23 cells as a host line for heterologous expression. Sodium currents were examined using standard whole-cell voltage-clamp electrophysiology. Current-voltage relationships for either ND7/23 cell or Na(V)1.8 currents revealed a prominent fluphenazine block of sodium channel activity. Steady-state inactivation curves were shifted by approximately 10 mV in the hyperpolarizing direction by fluphenazine (3 microM for ND7/23 currents and 10 microM for Na(V)1.8), suggesting that the drug stabilizes the inactivated channel state. Fluphenazine's apparent potency for blocking either ND7/23 or Na(V)1.8 sodium channels was increased by membrane depolarization, corresponding IC(50) values for the ND7/23 cell conductances were 18 microM and 960 nM at holding potentials of -120 mV and -50 mV, respectively. Frequency-dependent channel block was evident for each of the cell/channel variants, again suggesting a preferential binding to inactivated channel state(s). These experiments show fluphenazine to be capable of blocking neuronal sodium channels. Several unusual pharmacokinetic features of this drug suggest that sodium channel block may contribute to the overall clinical profile of this classical neuroleptic agent.     

Dihydropyridine actions on calcium currents of frog sympathetic neurons.

Dihydropyridines (DHPs) generally have little effect on whole-cell calcium currents of neurons, even at concentrations far higher than those effective on muscle. Either neuronal calcium currents are much less sensitive to DHPs, or only a small proportion of the current is DHP-sensitive. We find that DHP agonists and antagonists act at low concentration on calcium currents in frog sympathetic neurons but that the effects are small even at optimal concentrations. The half-maximal dose (EC50) of the agonist Bay K 8644 is approximately 50 nM, and the effect of Bay K 8644 is blocked by 50% at approximately 300 nM nifedipine, from a holding potential of -80 mV. Nifedipine is more effective from a holding potential of -50 mV. These results suggest the presence of an L-type calcium current, with DHP sensitivity similar to L-currents in cardiac muscle. The predominant (greater than 90%) calcium current in frog sympathetic neurons is a DHP-resistant N-type current. However, high concentrations of DHPs (10 microM) partially block N-type calcium current, as well as voltage-dependent sodium and potassium currents.     

The roles of sodium channels in nociception: implications for mechanisms of neuropathic pain

Animal models have provided useful insights into the development and treatment of neuropathic pain. New genetic data from both human studies and transgenic mouse models suggest that specific voltage-gated sodium channel subtypes are associated with specific types of pain and, as such, may be useful analgesic drug targets for a variety of pain types including neuropathic pain. Global voltage-gated sodium channel blockers such as lidocaine have proven efficacy in treating pain but can be limited by adverse effects when administered systemically. Selective sodium channel blockers targeting channels at the periphery (Nav1.7, Nav1.8, and Nav1.9) could potentially reduce the side effect profile. Individual isoforms of voltage-gated sodium channels have been linked to particular types of pain. Nav1.7 is a useful target for ameliorating acute mechanical pain and inflammatory pain, and strong evidence also suggests that Nav1.9 could be targeted for treating inflammatory pain. Selective blockers of Nav1.8 could also have clinical benefit for visceral pain. Although there is no association between a single sodium channel isoform and neuropathic pain, combined blockade of peripherally expressed isoforms Nav1.7, Nav1.8, and Nav1.9 may prove useful.     

Effect of a neuronal sodium channel blocker on magnetic resonance derived indices of brain water content during global cerebral ischemia

Diffusion-weighted magnetic resonance imaging (DWI) with calculation of the apparent diffusion coefficient (ADC) of water is a widely used noninvasive method to measure movement of water from the extracellular to the intracellular compartment during cerebral ischemia. Lamotrigine, a neuronal Na(+) channel blocker, has been shown to attenuate the increase in extracellular concentrations of excitatory amino acids (EAA) during ischemia and to improve neurological and histological outcome. Because of its proven ability to reduce EAA levels during ischemia, lamotrigine should also minimize excitotoxic-induced increases in intracellular water content and therefore attenuate changes in the ADC. In this study, we sought to determine the effect of lamotrigine on intra- and extracellular water shifts during transient global cerebral ischemia. Fifteen New Zealand white rabbits were anesthetized and randomized to one of three groups: a control group, a lamotrigine-treated group, or a sham group. After being positioned in the bore of the magnet, a 12-min 50-s period of global cerebral ischemia was induced by inflating a neck tourniquet. During ischemia and early reperfusion there was a similar and significant decrease of the ADC in both the lamotrigine and control group. The ADC in the sham ischemia group remained at baseline throughout the experiment. Lamotrigine-mediated blockade of voltage-gated sodium channels did not prevent the intracellular movement of water during 12 min 50 s of global ischemia, as measured by the ADC, suggesting that the ADC decline may not be mediated by voltage-gated sodium influx and glutamate release.     

The role of dihydropyridine-sensitive voltage-gated calcium channels in potassium-mediated neuronal survival

The survival of isolated neurons from chick embryo ciliary, sympathetic, and dorsal root ganglia is greatly enhanced by concentrations of extracellular potassium that significantly depolarize the neurons (ED50 = 20-25 mM). The survival-promoting effect of elevated potassium on each of these 3 types of neurons appears to be the result of the opening of voltage-gated calcium channels. The dihydropyridine, Bay K 8644, which increases calcium influx through L-type voltage-gated calcium channels in neurons, strongly potentiated the survival-promoting action of elevated potassium (ED50 = 10.8 +/- 7.0 nM). In contrast, chemically closely related dihydropyridines, PN200-110 (ED50 = 0.33 +/- 0.15 nM) and nitrendipine (ED50 = 1.3 +/- 0.3 nM), which block calcium influx through the same voltage-gated channels, completely inhibited potassium-mediated neuronal survival. Chemically different agents that also block calcium influx through voltage-gated channels also inhibited potassium-mediated neuronal survival: the phenylalkylamine verapamil (ED50 = 0.78 +/- 0.38 microM), the benzothiazepine diltiazem (ED50 = 1.7 microM), and the inorganic ion cadmium (ED50 = 5.8 microM). These calcium-channel blockers are not simply toxic to neurons, since they did not inhibit neuronal survival mediated by the neurotrophic proteins, nerve growth factor, basic fibroblast growth factor, or ciliary neurotrophic factor, also suggesting that voltage-gated calcium channels are not involved in the action of these factors. These results suggest that neuronal survival in elevated potassium in ciliary, sympathetic, and dorsal root ganglion neurons is the result of calcium influx through dihydropyridine-sensitive, L-type voltage-gated calcium channels. These findings are discussed in relation to the neuronal toxicity of excitatory amino acids which is also thought to occur through increased calcium influx.     

CAP-1A is a novel linker that binds clathrin and the voltage-gated sodium channel Na(v)1.8

The voltage-gated sodium channel Na(v)1.8 produces a tetrodotoxin-resistant current and plays a key role in nociception. Annexin II/p11 binds to Na(v)1.8 and facilitates insertion of the channel within the cell membrane. However, the mechanisms responsible for removal of specific channels from the cell membrane have not been studied. We have identified a novel protein, clathrin-associated protein-1A (CAP-1A), which contains distinct domains that bind Na(v)1.8 and clathrin. CAP-1A is abundantly expressed in DRG neurons and colocalizes with Na(v)1.8 and can form a multiprotein complex with Na(v)1.8 and clathrin. Coexpression of CAP-1A and Na(v)1.8 in DRG neurons reduces Na(v)1.8 current density by approximately 50% without affecting the endogenous or recombinant tetrodotoxin-sensitive currents. This effect of CAP-1A is blocked by bafilomycin A1 treatment of transfected DRG neurons. CAP-1A thus is the first example of an adapter protein that links clathrin and a sodium channel and may regulate Na(v)1.8 channel density at the cell surface.     

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.     

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.