Half-maximal Blocking Concentration Research Articles

Automatic modeling of dynamic drug-hERG channel interactions using three voltage protocols and machine learning techniques: A simulation study

BackgroundAssessment of drug cardiac safety is critical in the development of new compounds and is commonly addressed by evaluating the half-maximal blocking concentration of the potassium human ether-à-go-go related gene (hERG) channels. However, recent works have evidenced that the modelling of drug-binding dynamics to hERG can help to improve early cardiac safety assessment. Our goal is to develop a methodology to automatically generate Markovian models of the drug-hERG channel interactions. MethodsThe training and the test sets consisted of 20800 and 5200 virtual drugs, respectively, distributed into 104 groups with different affinities and kinetics to the conformational states of the channel. In our system, drugs may bind to any state (individually or simultaneously), with different degrees of preference for a conformational state and the change of the conformational state of the drug bound channels may be restricted or allowed. To model such a wide range of possibilities, 12 Markovian chains are considered. Our approach uses the response of the drugs to our three previously developed voltage clamp protocols, which enhance the differences in the probabilities of occupying a certain conformational state of the channel (open, closed and inactivated). The computing tool is comprised of a classifier and a parameter optimizer and uses linear interpolation, support vector machines and a simplex method for function minimization. ResultsWe propose a novel methodology that automatically generates dynamic drug models using Markov model formulations and that elucidates the states where the drug binds and unbinds and the preferential binding state using data obtained from simple voltage clamp protocols that captures the preferential state-dependent binding properties, the relative affinities, trapping and non-trapping dynamics and the onset of IKr block. Overall, the tool correctly predicted the class of 92.04% of the drugs and the model provided by the tool accurately fitted the response of the target compound, the mean accuracy being 97.53%. Moreover, generation of the dynamic model of an IKr blocker from its response to our voltage clamp protocols usually takes less than an hour on a common desktop computer. ConclusionOur methodology could be very useful to model and simulate dynamic drug–hERG channel interactions. It would contribute to the improvement of the preclinical assessment of the proarrhythmic risk of drugs that inhibit IKr and the efficacy of antiarrhythmic IKr blockers.

Monovalent cation permeability and Ca(2+) block of the store-operated Ca(2+) current I(CRAC )in rat basophilic leukemia cells.

Like voltage-operated Ca(2+) channels, store-operated CRAC channels become permeable to monovalent cations in the absence of external divalent cations. Using the whole-cell patch-clamp technique, we have characterized the permeation and selectivity properties of store-operated channels in the rat basophilic leukemia (RBL-1) cell line. Store depletion by dialysis with InsP(3) and 10 mM EGTA resulted in the rapid development of large inward currents in Na(+)- and Li(+)-based divalent-free solutions. Cs(+) permeated the channels poorly (P(Cs)/ P(Na)=0.01). Trimethylamine (TMA(+)), tetramethylammonium (TeMA(+)), tetraethylammonium (TEA(+)), N-methyl- D-glucamine (NMDG(+)) and TRIS(+) were not measurably permeant. NH(4)(+) was conducted well. We estimated the minimum pore diameter under divalent-free conditions to be between 0.32 nm and 0.55 nm. When cells were dialysed with buffered Ca(2+) solution and I(CRAC) activated by application of thapsigargin, P(Cs)/ P(Na) was still low (0.08). Outward currents through CRAC channels were carried by intracellular Na(+), K(+) and, to a much lesser extent, by Cs(+). Currents were unaffected by dialysis with Mg(2+)-free solution. The Na(+) current was inhibited by external Ca(2+) (half-maximal blocking concentration of 10 microM). This Ca(2+)-dependent block could be alleviated by hyperpolarization. The monovalent Na(+) current was voltage dependent, increasing as the holding potential depolarized above 0 mV. Our results suggest that CRAC channels in RBL-1 cells have a smaller pore diameter than voltage-operated Ca(2+) channels, discriminate between Group I cations, and differ markedly in their selectivity from CRAC channels reported in lymphocytes.

Effect of drugs used for neuropathic pain management on tetrodotoxin-resistant Na(+) currents in rat sensory neurons.

Tetrodotoxin-resistant Na(+) channels play an important role in generation and conduction of nociceptive discharges in peripheral endings of small-diameter axons of the peripheral nervous system. Pathophysiologically, these channels may produce ectopic discharges in damaged nociceptive fibers, leading to neuropathic pain syndromes. Systemically applied Na(+) channel--blocking drugs can alleviate pain, the mechanism of which is rather unresolved. The authors investigated the effects of some commonly used drugs, i.e., lidocaine, mexiletine, carbamazepine, amitriptyline, memantine, and gabapentin, on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglia. Tetrodotoxin-resistant Na(+) currents were recorded in the whole-cell configuration of the patch-clamp method in enzymatically dissociated dorsal root ganglion neurons of adult rats. Half-maximal blocking concentrations were derived from concentration-inhibition curves at different holding potentials (-90, -70, and -60 mV). Lidocaine, mexiletine, and amitriptyline reversibly blocked tetrodotoxin-resistant Na(+) currents in a concentration- and use-dependent manner. Block by carbamazepine and memantine was not use-dependent at 2 Hz. Gabapentin had no effect at concentrations of up to 3 mm. Depolarizing the membrane potential from -90 mV to -60 mV reduced the available Na(+) current only by 23% but increased the sensitivity of the channels to the use-dependent blockers approximately fivefold. The availability curve of the current was shifted by 5.3 mV to the left in 300 microm lidocaine. Less negative membrane potential and repetitive firing have little effect on tetrodotoxin-resistant Na(+) current amplitude but increase their sensitivity to lidocaine, mexiletine, and amitriptyline so that concentrations after intravenous administration of these drugs can impair channel function. This may explain alleviation from pain by reducing firing frequency in ectopic sites without depressing central nervous or cardiac excitability.

Block of neuronal tetrodotoxin-resistant Na+ currents by stereoisomers of piperidine local anesthetics.

Tetrodotoxin (TTX)-sensitive Na(+) channels in the peripheral nervous system are the major targets for local anesthetics. In the peripheral nociceptive system, a Na(+) channel subtype resistant to TTX and with distinct electrophysiological properties seems to be of importance for impulse generation and conduction. A current through TTX-resistant Na(+) channels displays slower activation and inactivation kinetics and has an increased activation threshold compared with TTX-sensitive Na(+) currents and may have different pharmacological properties. We studied the effects of stereoisomers of piperidine local anesthetics on neuronal TTX-resistant Na(+) currents recorded with the whole-cell configuration of the patch clamp method in enzymatically dissociated dorsal root ganglion neurons of adult rats. Stereoisomers of mepivacaine, ropivacaine, and bupivacaine reversibly inhibited TTX-resistant Na(+) currents in a concentration and use-dependent manner. All drugs accelerated time course of inactivation. Half-maximal blocking concentrations were determined from concentration-inhibition relationships. Potencies for tonic and for use-dependent block increased with rising lipid solubilities of the drugs. Stereoselective action was not observed. We conclude that block of TTX-resistant Na(+) currents may lead to blockade of TTX-resistant action potentials in nociceptive fibers and consequently may be responsible for pain suppression during local anesthesia.

Pharmacological properties of Ca(V)3.2, a low voltage-activated Ca2+ channel cloned from human heart.

Three genes encoding T-type Ca2+ channels have been described but their correspondence to the various native T-type Ca2+ currents remains uncertain. In particular, Ca(V)3.2 (or alpha1H) was cloned from a human heart library, its message was found abundantly in cardiac tissue, and expressed Ca(V)3.2 was shown to conduct low voltage-activated currents, which inactivate rapidly and are sensitive to Ni2+ and mibefradil. These observations suggested that Ca(V)3.2 might encode native cardiac T-type Ca2+ channels but more information on the pharmacology of Ca(V)3.2 was needed to confirm this hypothesis. In the present study, we compare the pharmacology of Ca(V)3.2 expressed in HEK293 cells and of native T-type Ca2+ channels in guinea pig atrial myocytes ("native-T"). (1) Ca(V)3.2 and native-T are insensitive to TTX and to toxins selective for N-, P-, or Q-type Ca2+ channels (omega-CTx-GVIA, omega-Aga-IVA, omega-CTx-MVIIC). (2) The half-maximal blocking concentration (IC50) of mibefradil on Ca(V)3.2 is near that on native-T and the block is similarly voltage-dependent. (3) Ca(V)3.2 is five- to sixfold less sensitive than native-T to the 1,4-dihydropyridine (DHP) amlodipine, suggesting a difference in the DHP binding site. (4) Both channels display similar (but not identical) sensitivities to the inorganic blockers Ni2+ and Cd2+ and the IC50s are in the range of values found for T-type Ca2+ currents in other cell types. (5) Ni2+ shifts the voltage dependence of Ca(V)3.2 activation but not that of native-T. The many similarities between the two channels support the contention that Ca(V)3.2 encodes cardiac T-type Ca2+ channels. The slight differences may be due to species variations and/or to the choice of splice variant.

Blocking effects of the anaesthetic etomidate on human brain sodium channels

Sodium channels from human brain tissue were incorporated into voltage-clamped planar lipid bilayers in presence of batrachotoxin and exposed to increasing concentrations of the intravenous anaesthetic drug etomidate (0.03–1.02 mM). Etomidate interacted with the sodium-conducting pathway of the channel causing a concentration-dependent block of the time-averaged sodium conductance (computer fit of the concentration–response curve: half-maximal blocking concentration, EC 50, 0.19 mM; maximal block, block max, 38%). This block of sodium-conductance resulted from two distinct effects (I) major effect: reduction of the sodium-channel amplitude and (II) minor effect: reduction of the fractional channel open-time. These results were observed at concentrations above clinically-relevant serum concentrations (up to 0.01 mM), suggesting only a limited role for human brain sodium channels in the mechanism of action of etomidate during clinical anaesthesia.

Local anaesthetic effects on tetrodotoxin-resistant Na+ currents in rat dorsal root ganglion neurones.

Besides the fast tetrodotoxin-sensitive Na+ current, small dorsal root ganglion neurones of rats also possess a slower tetrodotoxin-resistant Na+ current. The blocking effect of commonly used local anaesthetics upon the tetrodotoxin-resistant Na+ current was investigated in the present paper. Dorsal root ganglia were dissected from adult rats and cells were enzymatically isolated. The whole-cell patch clamp technique was then used to measure inward Na+ currents of small dorsal root ganglion neurones. Externally applied local anaesthetics reversibly blocked the tetrodotoxin-resistant Na+ current in a dose-dependent manner. Half-maximal blocking concentrations for tonic block were: lignocaine, 326 microM; prilocaine, 253 microM; mepivacaine, 166 microM; etidocaine, 196 microM bupivacaine, 57 microM procaine, 518 microM benzocaine, 489 microM; tetracaine, 21 microM; and dibucaine, 23 microM. Blocking of the current by lignocaine was independent of temperature. The quaternary lignocaine derivative OX-314 did not have any effect upon the tetrodotoxin-resistant Na+ current when applied externally. High concentrations of tetrodotoxin also blocked the tetrodotoxin-resistant Na+ current with a half-maximal blocking concentration of 115 microM. The block by high tetrodotoxin concentrations did not compete with the lignocaine block, suggesting that there were two independent blocking mechanisms for the two substances. The tetrodotoxin-resistant Na+ currents also showed a marked sensitivity to phasic (use-dependent) block by local anaesthetics.

Diltiazem blocks the PH-induced excitation of rat nociceptors together with their mechanical and electrical excitability in vitro.

1. The effect of the calcium channel antagonist diltiazem on pH-induced sustained nociceptor excitation was investigated in a rat skin-saphenous nerve preparation, in vitro, where receptive fields of identified and isolated single fibers were superfused at the corium side with controlled solutions to assess their chemosensitivity. 2. Unmyelinated mechano-heat sensitive ("polymodal") C fiber terminals (n = 78) were superfused with a CO2-saturated synthetic interstitial fluid (CO2-SIF, pH 6.1). Fibers responding to this acid pH condition (n = 60; 77%) were further stimulated for > or = 30 min and additionally treated with diltiazem in various concentrations (10(-6)-10(-3) M) during the middle 10-min period. Usually only one concentration of diltiazem was applied per fiber, although in some cases diltiazem was applied repeatedly and in increasing concentrations. 3. Diltiazem dose-dependently and reversibly reduced the pH-induced sustained nociceptor discharge to a significant degree or completely abolished it. With higher concentrations, both the relative number of units affected and the average amount of suppression were enhanced. The half-maximal blocking concentration (IC50) was estimated to 1.1.10(-4) M diltiazem, the half-maximal concentration for gradual suppression of the pH response was 2.10(-5) M diltiazem. 4. Also, the delay of onset of the suppressive effect decreased with higher diltiazem concentrations. After diltiazem, a partial recovery of the pH-induced discharge was achieved within 10 min depending on the degree of suppression. 5. Before, after, and sometimes during the superfusion, the mechanical (von Frey) thresholds were determined and found to be significantly increased after partial wash out of diltiazem (10(-4) and 10(-3) M; P < 0.006 and P < 0.008, respectively). After 10(-3) M diltiazem superfusion (and 10 min of wash-out), the responsiveness to mechanical stimulation of the majority of the fibers was still totally lost. 6. Heat thresholds were still found to be significantly increased after treatment with diltiazem at 10(-3) and 10(-4) M concentrations (and 10 min of wash out), but appeared unchanged after wash out of lower concentrations. 7. In five mechano-heat-sensitive C fibers, electrical stimulation via a microelectrode placed in the receptive field was used to demonstrate a diltiazem-dose-dependent (10(-5), 10(-4), 10(-3) M) progressive retardation of the nerve conduction velocity and an increase of the electrical threshold. Superfusion for 6 min of diltiazem 10(-5) M was sufficient to block axonal conduction as well as mechanosensitivity, which both recovered synchronously during wash out. 8. It can be concluded from the results that the suppressive effect of diltiazem on pH-induced nociceptor excitation can be explained by a use-dependent axonal block, comparable with the action of local anesthetics and affecting all modalities of sensory responsiveness. 9. The findings provide no indication that a transformed calcium channel specifically sensitive to diltiazem is involved in pH-induced sustained nociceptor excitation.

Activation of a Cl- conductance by SCN- in single proximal tubule cells isolated from Rana temporaria.

1. This study investigated the effect of the anion, SCN-, on the conductive properties of single proximal tubule cells isolated from frog kidney. Ionic currents were measured using the conventional whole-cell patch clamp technique. 2. Addition of SCN- to the bath solution alone had a biphasic effect; there was an initial rapid rise, followed by a slower secondary increase, in both outward and inward conductances. However, when SCN- was added to the bath in the presence of pipette SCN-, such that the concentration gradient for movement of SCN- into the cell was abolished, only the fast changes in conductance were observed. 3. Cells did not discriminate between cations and anions under nominally K(+)-free control conditions. However, in the presence of intracellular SCN- cells became anion selective. Taken together with the increased conductance, this suggests activation of a Cl- channel (gSCN). 4. Niflumic acid, a Cl- channel blocker, inhibited gSCN in a dose-dependent manner, with a half-maximal blocking concentration (Ki) of 28 +/- 2.8 microM and a Hill coefficient of 1.2 +/- 0.3 (n = 6). 5. The activation of gSCN by intracellular SCN- was dose dependent and showed positive co-operativity, with a Ki of 62.3 microM and Hill coefficient of 4.0. 6. Plasma and urine levels of SCN- range between 10 and 70 microM, thus the conductance described here may play a role in the regulation of Cl- handling by the kidney.

Block of native Ca(2+)-permeable AMPA receptors in rat brain by intracellular polyamines generates double rectification.

1. The influence of intracellular factors on current rectification of different subtypes of native alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptors (AMPARs) was studied in rat brain slices by combining fast application of glutamate with patch pipette perfusion. 2. The peak current-voltage (I-V) relation of the AMPARs expressed in Bergmann glial cells of cerebellum and dentate gyrus (DG) basket cells of hippocampus was weakly rectifying in outside-out patches and nystatin-perforated vesicles, but showed a doubly rectifying shape with a region of reduced slope between 0 and +40 mV in nucleated patches. The I-V relation of AMPARs expressed in hippocampal CA3 pyramidal neurones was linear in all recording configurations. 3. Intracellular application of 25 microM spermine, a naturally occurring polyamine, blocked outward currents in outside-out patches from Bergmann glial cells and DG basket cells in a voltage-dependent manner, generating I-V relations with a doubly rectifying shape which were similar to those recorded in nucleated patches. AMPARs in CA3 pyramidal cell patches were unaffected by 25 microM spermine. 4. The half-maximal blocking concentration of spermine at +40 mV was 0.3 microM in Bergmann glial cell patches and 1.5 microM in DG basket cell patches, whereas it was much higher (>> 100 microM) for CA3 pyramidal cell patches. Spermidine also affected current rectification, but with lower affinity. The block of outward current by polyamines following voltage jumps developed within < 0.5 ms. 5. We conclude that current rectification, rather than being an intrinsic property of the Ca(2+)-permeable AMPAR channel, is generated by polyamine block.

Large conductance calcium-activated potassium channels in cultured retinal pericytes under normal and high-glucose conditions

Pericytes are considered to contribute to the regulation of retinal microcirculation which is impaired in diabetic retinopathy. Single, large-conductance, Ca(2+)-dependent K+ channels (BK) were studied in cultured bovine retinal capillary pericytes using the patch-clamp method. In excised patches with symmetrical 135-mmol/l K+ solutions a single channel conductance of 238 +/- 9.9 pS was measured. With a K+ gradient of 4/135 mmol/l (extracellular/intracellular) the slope conductance averaged 148 +/- 2.9 pS at 0 mV. The mean permeability was 4.2 X 10(-13) cm3/s. The channel was highly selective for K+ with a permeability ratio for K+ over Na+ of 1/0.02. The mean open time and the open probability (Po) of the BK channel increased with depolarization and with increasing internal [Ca2+] showing a maximal sensitivity to Ca2+ between 10(-4) and 10(-5) mol/l Ca2+. Ba2+ (5 mmol/l), quinine (5 mmol/l), and verapamil (Michaelis constant 1.5 X 10(-5) mol/l) blocked from the intracellular side. Tetraethylammonium induced a dose-dependent block from the outside only with a half-maximal blocking concentration of 2.5 X 10(-4) mol/l. Charybdotoxin (10(-8) mol/l) blocked completely from the extracellular side. The channel activity was not changed by either internal adenosine triphosphate (ATP, 10(-4) mol/l) or the putative opener of ATP-sensitive K+ channels Hoe 234 (10(-6) mol/l). In cell-attached patches channel Po was less than 3%. After a 3-day incubation in culture medium containing an elevated glucose concentration (22.5 mmol/l) the channel activity in attached patches was markedly increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantal components of unitary EPSCs at the mossy fibre synapse on CA3 pyramidal cells of rat hippocampus.

1. Excitatory postsynaptic currents (EPSCs) were recorded in CA3 pyramidal cells of hippocampal slices of 15- to 24-day-old rats (22 degrees C) using the whole-cell configuration of the patch clamp technique. 2. Composite EPSCs were evoked by extracellular stimulation of the mossy fibre tract. Using the selective blockers 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonopentanoic acid (APV), a major alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor-mediated component and a minor NMDA receptor-mediated component with slower time course were distinguished. For the AMPA/kainate receptor-mediated component, the peak current-voltage (I-V) relation was linear, with a reversal potential close to 0 mV. The half-maximal blocking concentration of CNQX was 353 nM. 3. Unitary EPSCs of the mossy fibre terminal (MF)-CA3 pyramidal cell synapse were evoked at membrane potentials of -70 to -90 mV by low-intensity extracellular stimulation of granule cell somata using fine-tipped pipettes. The EPSC peak amplitude as a function of stimulus intensity showed all-or-none behaviour. The region of low threshold was restricted to a few micrometres. This suggests that extracellular stimulation was focal, and that the stimulus-evoked EPSCs were unitary. 4. Latency and rise time histograms of EPSCs evoked by granule cell stimulation showed narrow unimodal distributions within each experiment. The mean latency was 4.2 +/- 1.0 ms, and the mean 20-80% rise time was 0.6 +/- 0.1 ms (23 cells). When fitted within the range 0.7 ms to 20 ms after the peak, the decay of the EPSCs with the fastest rise (rise time 0.5 ms or less) could be described by a single exponential function; the mean time constant was in the range 3.0-6.6 ms with a mean of 4.8 ms (8 cells). 5. Peak amplitudes of the EPSCs evoked by suprathreshold granule cell stimulation fluctuated between trials. The apparent EPSC peak conductance in normal extracellular solution (2 mM Ca2+, 1 mM Mg2+), excluding failures, was 1 nS. Reducing the Ca2+ concentration and increasing the Mg2+ concentration reduced the mean peak amplitude in a concentration-dependent manner. 6. Peaks in EPSC peak amplitude distributions were apparent in low Ca2+ and high Mg2+. Using the criteria of equidistance and the presence of peaks and dips in the autocorrelation function, five of nine EPSC peak amplitude distributions were judged to be quantal.(ABSTRACT TRUNCATED AT 400 WORDS)

Block of current through T-type calcium channels by trivalent metal cations and nickel in neural rat and human cells.

1. The effects of the trivalent cations yttrium (Y3+), lanthanum (La3+), cerium (Ce3+), neodymium (Nd3+), gadolinium (Gd3+), holmium (Ho3+), erbium (Er3+), ytterbium (Yb3+) and the divalent cation nickel (Ni2+) on the T-type voltage gated calcium channel (VGCC) were characterized by the whole-cell patch clamp technique using rat and human thyroid C cell lines. 2. All the metal cations (M3+) studied, blocked current through T-type VGCC (IT) in a concentration-dependent manner. Smaller trivalents were the best T-channel antagonists and potency varied inversely with ionic radii for the larger M3+ ions. Estimation of half-maximal blocking concentrations (IC50s) for IT carried by 10 mM Ca2+ resulted in the following potency sequence: Ho3+ (IC50 = 0.107 microM) approximately Y3+ (0.117) approximately Yb3+ (0.124) > or = Er3+ (0.153) > Gd3+ (0.267) > Nd3+ (0.429) > Ce3+ (0.728) > La3+ (1.015) >> Ni2+ (5.65). 3. Tail current measurements and conditioning protocols were used to study the influence of membrane voltage on the potency of these antagonists. Block of IT by Ni2+, Y3+, La3+ and the lanthanides was voltage independent in the range from -200 to +80 mV. In addition, the antagonists did not affect macroscopic inactivation and deactivation of T-type VGCC. 4. Increasing the extracellular Ca2+ concentration reduced the potency of IT block by Ho3+, indicative of competitive antagonism between this blocker and the permeant ion for a binding site. 5. The results suggest that the mechanism of metal cation block of T-type VGCC is occlusion of the channel pore by the antagonist binding to a Ca2+/M3+ binding site, located out of the membrane electric field. 6. Block of T-type VGCC by Y3+, lanthanides and La3+ differ from the inhibition of high voltage-activated VGCC block in several respects: smaller cations are more potent IT antagonists; block is voltage independent and the antagonists do not permeate T-type channels. These differences suggest corresponding structural dissimilarities in the permeation pathways of low and high voltage-activated Ca2+ channels.

GABA responses and their partial occlusion by glycine in cultured rat medullary neurons

Whole-cell current responses to bath application of GABA and glycine were studied in medullary neurons cultured from embryonic rats. Two current components were seen in the responses to bath application of GABA, one component which desensitized and another which did not. These two current components have different dose-response characteristics for GABA, with the nondesensitizing component being activated more effectively and reaching its peak amplitude at lower agonist concentrations than the desensitizing one. The agonist concentrations producing half of the maximum responses are 2.8 ± 0.3 (±S.E.M., n = 9) and 14.7 ± 2.7 ( n = 5) μM for the nondesensitizing and desensitizing components, respectively. The two current components for GABA are differentially affected by the antagonists, picrotoxin and bicuculline. The antagonist concentrations which block 50% of the control desensitizing and nondesensitizing responses to GABA are 33 and 320 μM for picrotoxin, and 3 and 50 μM for bicuculline, respectively. Thus, the characteristics of the GABA responses are analagous to those described previously for glycine in that there are two components which are differentially sensitive to agonist concentration [Lewis et al. (1991) J. Neurophysiol. 40, 1178–1187]. We now find there is occlusion between the responses to GABA and glycine, indicating that they share a population of receptors or channels. The occlusion was incomplete (<80%) in half of the cells, suggesting that both agonists also activate unique receptors. Furthermore, the current responses to 35 μM GABA are blocked by the glycinergic antagonist, strychnine, with half-maximal blocking concentrations equal to 2 and 30 μM for the desensitizing and nondesensitizing components, respectively. This strychnine sensitivity is less than that for the glycine receptor. At the same time, the current responses to 100 μM glycine are sensitive to the GABAergic antagonists, picrotoxin and bicuculline. The half-maximal blocking concentrations are 36 and 120 μM picrotoxin, and 120 and 500 μM bicuculline, for the desensitizing and nondesensitizing components of the glycine response, respectively. Consequently, these results suggest that these cultured cells have at least three types of inhibitory receptors: glycine receptors, GABA receptors and GABA/glycine receptors, with all three receptors sensitive to block by strychnine, bicuculline and picrotoxin. The GABA/glycine receptor may be an immature form of the inhibitory receptor. Alternatively, some GABA and glycine receptors may have common ionophores.

Human brain sodium channels as one of the molecular target sites for the new intravenous anaesthetic propofol (2,6-diisopropylphenol)

Single sodium channels from human brain cortex tissue were incorporated into voltage-clamped planar lipid bilayers in the presence of batrachotoxin and studied with various doses of the new anaesthetic compound propofol (2,6-diisopropylphenol). Propofol was found to depress two major sodium channel functions, leading to a reduction of the time-averaged fractional channel open-time (half-maximal blocking concentration, ED 50, 20 μM; maximal block 28%) and an interaction with the steady-state activation. These effects occurred at clinically relevant serum concentrations, suggesting the human brain sodium channel as one of the molecular target sites of action of propofol.

Calcium currents in embryonic and neonatal mammalian skeletal muscle.

The whole-cell patch-clamp technique was used to study the properties of inward ionic currents found in primary cultures of rat and mouse skeletal myotubes and in freshly dissociated fibers of the flexor digitorum brevis muscle of rats. In each of these cell types, test depolarizations from the holding potential (-80 or -90 mV) elicited three distinct inward currents: a sodium current (INa) and two calcium currents. INa was the dominant inward current: under physiological conditions, the maximum inward INa was estimated to be at least 30-fold larger than either of the calcium currents. The two calcium currents have been termed Ifast and Islow, corresponding to their relative rates of activation. Ifast was activated by test depolarizations to around -40 mV and above, peaked in 10-20 ms, and decayed to baseline in 50-100 ms. Islow was activated by depolarizations to approximately 0 mV and above, peaked in 50-150 ms, and decayed little during a 200-ms test pulse. Ifast was inactivated by brief, moderate depolarizations; for a 1-s change in holding potential, half-inactivation occurred at -55 to -45 mV and complete inactivation occurred at -40 to -30 mV. Similar changes in holding potential had no effect on Islow. Islow was, however, inactivated by brief, strong depolarizations (e.g., 0 mV for 2 s) or maintained, moderate depolarizations (e.g., -40 mV for 60 s). Substitution of barium for calcium had little effect on the magnitude or time course of either Ifast or Islow. The same substitution shifted the activation curve for Islow approximately 10 mV in the hyperpolarizing direction without affecting the activation of Ifast. At low concentrations (50 microM), cadmium preferentially blocked Islow compared with Ifast, while at high concentrations (1 mM), it blocked both Ifast and Islow completely. The dihydropyridine calcium channel antagonist (+)-PN 200-110 (1 microM) caused a nearly complete block of Islow without affecting Ifast. At a holding potential of -80 mV, the half-maximal blocking concentration (K0.5) for the block of Islow by (+)-PN 200-110 was 182 nM. At depolarized holding potentials that inactivated Islow by 35-65%, K0.5 decreased to 5.5 nM.

Bepridil and valproate retard Na + reactivation in Myxicola

Two drugs were examined, each causing a similar specific modification of Na + channel inactivation gating when internally applied to voltage-clamped Myxicola giant axons. Bepridil is an antianginal-antiarrhythmic agent with vasodilator and direct cardiac inotropic effects. Sodium valproate has anticonvulsant activity and causes use-dependent inhibition of repetitive firing in CNS neurons. Bepridil and sodium valproate caused a dose-dependent decrease in maximum Na + conductance (K D = 25 μM for bepridil; K D = 0.5 mM for valproate). More importantly, at half-maximal blocking concentrations both drugs shifted steady state Na + inactivation in the hyperpolarizing direction] (by 30 mV for bepridil; 15 mV for valproate) and slowed the recovery of Na + channels from inactivation (by 300% for bepridil; 60% for valproate). There was no effect on the K + conductance, voltage-dependence of Na + activation, or the time-dependence of inactivation of conducting channels. Neither produced non-inactivating Na + current during long depolarizing steps.