Slow Tail Current Research Articles

Switching Time Delay Optimization for “SiC+Si” Hybrid Device in a Phase-Leg Configuration

Compared to SiC MOSFET, the switching loss of Si IGBT is much higher due to its slow switching speed and tail current. Si IGBT/SiC MOSFET hybrid switch device can reach to optimal performance with low static and dynamic loss, which can improve the current capacity of SiC devices and reduce the power loss of Si IGBT based converters. With the separated gate control signals, the switching moments of the two devices can be controlled independently to ensure Si IGBT under zero-voltage switching (ZVS) conditions. This measurement tends to reduce the switching loss of Si IGBT. However, the switching time delay between these two devices has significant impacts on its power loss. In this paper, the switching time delay optimization method is proposed to minimize the power loss of the hybrid switch. The static and dynamic characteristics of Si IGBT/SiC MOSFET hybrid-paralleled switch are studied, and a generalized power loss model for hybrid switch is developed. The influence of switching time delay on the characteristics of hybrid switch is analyzed and verified through double pulse tests in a phase-leg configuration. The experimental results show that the optimal turn-on delay time is that the two devices turn on at the same time and the turn-on loss can be reduced by about 73% compared with the solely Si IGBT and by about 52% compared with the solely SiC MOSFET. While the optimal turn-off sequence is that the Si IGBT turns off ahead of the SiC MOSFET. Under the proposed optimal turn-off delay time of the hybrid switch, the turn-off loss is reduced by about 61.4%. This optimization strategy is used in a Buck converter to verify the superiority of the SiC/Si hybrid switch and the optimal switching sequence. Simulation results show that the optimal switching sequence is consistent with theoretical analysis, and the efficiency is improved by 2.5% compared with Buck converter using solely Si IGBT.

Fractional Ca2+ Currents through TRP and TRPL Channels in Drosophila Photoreceptors

Light responses in Drosophila photoreceptors are mediated by two Ca2+ permeable cation channels, transient receptor potential (TRP) and TRP-like (TRPL). Although Ca2+ influx via these channels is critical for amplification, inactivation, and light adaptation, the fractional contribution of Ca2+ to the currents (Pf) has not been measured. We describe a slow (τ ∼ 350 ms) tail current in voltage-clamped light responses and show that it is mediated by electrogenic Na+/Ca2+ exchange. Assuming a 3Na:1Ca stoichiometry, we derive empirical estimates of Pf by comparing the charge integrals of the exchanger and light-induced currents. For TRPL channels, Pf was ∼17% as predicted by Goldman-Hodgkin-Katz (GHK) theory. Pf for TRP (29%) and wild-type flies (26%) was higher, but lower than the GHK prediction (45% and 42%). As predicted by GHK theory, Pf for both channels increased with extracellular [Ca2+], and was largely independent of voltage between –100 and –30 mV. A model incorporating intra- and extracellular geometry, ion permeation, diffusion, extrusion, and buffering suggested that the deviation from GHK predictions was largely accounted for by extracellular ionic depletion during the light-induced currents, and the time course of the Na+/Ca2+ exchange current could be used to obtain estimates of cellular Ca2+ buffering capacities.

A comparison of the effects of veratridine on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in isolated rat dorsal root ganglion neurons

The effects of veratridine have been compared on tetrodotoxin-sensitive (TTXS) and tetrodotoxin-resistant (TTXR) voltage-gated sodium channels (VGSC) in rat dorsal root ganglion neurons. Veratridine caused a dose-dependent decrease in the peak amplitude of both TTXR and TTXS VGSC currents. When exposed to 25 microM veratridine, TTXS currents but not TTXR currents developed a clear persistent component. The deactivation of both TTXS and TTXR currents was slowed, as evidenced by the appearance of slowly decaying tail currents in voltage clamp records, but the slowing of deactivation was nearly 100 times greater for TTXS than for TTXR currents. Properties of the veratridine-modified VGSCs, derived from an analysis of the slow tail currents, were similar for both TTXS and TTXR in that the V50 for activation and the reversal potential were shifted to more negative potentials than control currents and by a similar amount for each. The relatively fast decay of veratridine-modified TTXR tail currents reflects a faster dissociation of veratridine from TTXR than from TTXS VGSCs. This difference probably underlies the lack of effect of veratridine on TTXR VGSCs in cells that are not voltage-clamped and undermines its value as a chemical activator of putative NaV1.8 TTXR channels.

BK Channels with β3a Subunits Generate Use-Dependent Slow Afterhyperpolarizing Currents by an Inactivation-Coupled Mechanism

Large-conductance, Ca2+- and voltage-activated K+ (BK) channels are broadly expressed proteins that respond to both cellular depolarization and elevations in cytosolic Ca2+. The characteristic functional properties of BK channels among different cells are determined, in part, by tissue-specific expression of auxiliary beta subunits. One important functional property conferred on BK channels by beta subunits is inactivation. Yet, the physiological role of BK channel inactivation remains poorly understood. Here we report that as a consequence of a specific mechanism of inactivation, BK channels containing the beta3a auxiliary subunit exhibit an anomalous slowing of channel closing. This produces a net repolarizing current flux that markedly exceeds that expected if all open channels had simply closed. Because of the time dependence of inactivation, this behavior results in a Ca2+-independent but time-dependent increase in a slow tail current, providing an unexpected mechanism by which use-dependent changes in slow afterhyperpolarizations might regulate electrical firing. The physiological significance of inactivation in BK channels mediated by different beta subunits may therefore arise not from inactivation itself, but from the differences in the amplitude and duration of repolarizing currents arising from the beta-subunit-specific energetics of recovery from inactivation.

Calcium permeable AMPA receptors and autoreceptors in external tufted cells of rat olfactory bulb

Glomeruli are functional units of the olfactory bulb responsible for early processing of odor information encoded by single olfactory receptor genes. Glomerular neural circuitry includes numerous external tufted (ET) cells whose rhythmic burst firing may mediate synchronization of bulbar activity with the inhalation cycle. Bursting is entrained by glutamatergic input from olfactory nerve terminals, so specific properties of ionotropic glutamate receptors on ET cells are likely to be important determinants of olfactory processing. Particularly intriguing is recent evidence that AMPA receptors of juxta-glomerular neurons may permeate calcium. This could provide a novel pathway for regulating ET cell signaling. We tested the hypothesis that ET cells express functional calcium-permeable AMPA receptors. In rat olfactory bulb slices, excitatory postsynaptic currents (EPSCs) in ET cells were evoked by olfactory nerve shock, and by uncaging glutamate. We found attenuation of AMPA/kainate EPSCs by 1-naphthyl acetyl-spermine (NAS), an open-channel blocker specific for calcium permeable AMPA receptors. Cyclothiazide strongly potentiated EPSCs, indicating a major contribution from AMPA receptors. The current–voltage (I–V) relation of uncaging EPSCs showed weak inward rectification which was lost after >∼10 min of whole-cell dialysis, and was absent in NAS. In kainate-stimulated slices, Co 2+ ions permeated cells of the glomerular layer. Large AMPA EPSCs were accompanied by fluorescence signals in fluo-4 loaded cells, suggesting calcium permeation. Depolarizing pulses evoked slow tail currents with pharmacology consistent with involvement of calcium permeable AMPA autoreceptors. Tail currents were abolished by Cd 2+ and (±)-4-(4-aminophenyl)-2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), and were sensitive to NAS block. Glutamate autoreceptors were confirmed by uncaging intracellular calcium to evoke a large inward current. Our results provide evidence that calcium permeable AMPA receptors reside on ET cells, and are divided into at least two functionally distinct pools: postsynaptic receptors at olfactory nerve synaptic terminals, and autoreceptors sensitive to glutamate released from dendrodendritic synapses.

Evidence for the involvement of G-proteins in the generation of the slow poststimulus afterdepolarisation (sADP) induced by muscarinic receptor activation in rat olfactory cortical neurones in vitro

The involvement of G-proteins in generating the slow poststimulus afterdepolarising potential (sADP) induced by muscarinic receptor activation in immature (P10–20) rat olfactory cortical brain slice neurones was investigated under whole-cell patch clamp, using GTP-γ-S (G-protein activator) or GDP-β-S (G-protein blocker)-filled electrodes. In control experiments using K methylsulphate electrodes, cell resting potential ( V m) and spike firing properties were unaffected over 10–15 min recording, although input resistance ( R N) was slightly increased (∼14%). Oxotremorine-M (OXO-M; 10 μM) produced a reversible slow depolarisation, an increase in R N (∼90%) and induction of a slow poststimulus inward tail current ( I ADP) (measured under voltage clamp at −60 mV) that was sustained during drug exposure (up to 15 min); the amplitude of slow inward rectifier ( I h) currents activated from −50 mV were also apparently increased. By contrast, in GTP-γ-S-loaded cells, R N was consistently decreased (∼22%) and spike firing threshold ( V th) was raised (∼5 mV) after 10 min recording. In ∼60% of loaded cells, a persistent muscarinic slow inward current and I ADP were induced by OXO-M; I h relaxation amplitude was also significantly decreased. The effects of GTP-γ-S on R N, V th and I h were partly counteracted by adding Ba 2+ (100 μM) to the bathing medium or mimicked by adding baclofen (GABA B receptor agonist; 100 μM) to normally-recorded cells. Intracellular GDP-β-S (up to 30 min) had no effect on cell membrane properties or I h, but irreversibly blocked the muscarinic slow inward current and I ADP induced by OXO-M. We conclude that both muscarinic responses require G-protein-linked transduction mechanisms for their generation.

Calcium channels: when is a subunit not a subunit?

The textbook view is that a calcium channel contains four subunits: α1, α2-δ, β and γ. This was initially based on biochemical purification of the L-type channel from skeletal muscle, as all four subunits remained associated during the harsh conditions needed for protein purification. The core of the channel is the α1 subunit, which contains the voltage sensors, ion pore, and binding sites for drugs such as dihydropyridines. So what are the roles of the other subunits? In Xenopus oocytes, the α1 subunit alone produces a low level of channel activity, but Tarelius et al. (1997) found that antisense oligonucleotides against β subunits prevented functional expression of α1. Furthermore, coexpression of α1 with additional β both increased current amplitudes and modified channel gating. Thus, β subunits must have two separable effects: they not only facilitate initial channel expression, but also modify the kinetics of existing channels (Tareilus et al. 1997). How can β subunits have two distinct actions? Figure 1 shows two possibilities. Perhaps there is only one β binding site, and β interacts with α1 at two distinct stages of the calcium channel lifecycle (1-site model). Reversible binding of β to α1 facilitates expression of α1 in the plasma membrane, perhaps by masking an endoplasmic reticulum retention signal (Bichet et al. 2000). While in the plasma membrane, α1 can reversibly associate with β, changing a minimally functional α1-only channel to a mature channel. These α1-β interactions are sequential, and thus could involve a single binding site. In the alternative view (2-site model), stoichiometric assembly of the α1-β complex is necessary for expression of α1 in the cell membrane, while occupancy of a second (low affinity?) site modulates channel kinetics. Both models seem consistent with existing data, although there is evidence for a second binding site (Birnbaumer et al. 1998; Gerster et al. 1999). Figure 1 Two models for interactions of calcium channel α1 and β subunits Put bluntly, are β subunits really stoichiometric subunits of the Ca2+ channel, or are they just modulatory proteins that can be associated with Ca2+ channels under some circumstances (like G protein βγ subunits, syntaxin, ryanodine receptors, etc.)? In the 1-site model, the β subunit appears as a modulatory protein, with two distinct effects at different stages. In the 2-site model, β is both a true subunit and a modulatory protein (at distinct binding sites). One key question is how β modulates preexisting α1 subunits in the plasma membrane. How rapid is that action? Is it reversible? Are α1 subunits normally saturated with β? These questions have been difficult to address, in part because most studies have examined effects of β on α1 by coexpression, which does not allow direct determination of whether the interaction is dynamic. Previous evidence for reversible association of β with α1 has mostly been indirect (Canti et al. 2001; Restituito et al. 2001), although injection of purified β subunits into Xenopus oocytes can modulate calcium currents within 1 h (Yamaguchi et al. 1998). Furthermore, a peptide corresponding to the β-binding site in the I-II loop of the α1 subunit can can modify single channel gating (Hohaus et al. 2000), possibly by causing β to dissociate from α1. In a paper in this issue of The Journal of PhysiologyGarcia et al. (2002) now show that acute application of purified β subunits affects currents through calcium channels, using whole cell dialysis of membrane vesicles from adult skeletal muscle. Over ≈20 min (the expected time for delivery of a ≈60 kDa protein from a patch pipette), the current doubled in amplitude. There was no obvious effect on activation kinetics, but a ≈16 mV shift of inactivation towards more negative voltages and an increased slow tail current were seen. Three lines of evidence suggest that the β subunits were modulating preexisting α1 subunits, rather than enhancing membrane expression of α1: (1) the time course was too fast for expression of new α1 subunits; (2) the vesicles appeared to have no sarcoplasmic reticulum or other organelles; and (3) the gating currents did not increase, implying no change in the number of α1 proteins that could move their voltage sensors. One implication of this study is that α1 subunits are not normally fully saturated with β. It seems that β subunits act (at least in part) by rapidly reversibly binding, in stark contrast to the traditional picture of an invariant stoichiometric subunit. Could this also be true for the other calcium channel ‘subunits’, notably the enigmatic γ?

Mechanism of Postinhibitory Rebound in Molluscan Neurons1

Postinhibitory rebound (PIR) is an intrinsic property of many neurons but the underlying mechanism is not well understood. We studied PIR and its relationship to spike adaptation in B-cells isolated from the buccal ganglia of Aplysia. These neurons exhibit PIR following inhibitory synaptic input and following direct membrane hyperpolarization. Hyperpolarizing and depolarizing voltage clamp pulses from the resting potential evoke slow changes in membrane current that persist in the form of tail currents following the pulses. A subtraction method was used to isolate slow tail currents for study. Current-voltage measurements indicate that slow outward tail currents following depolarizing pulses result from increases in membrane conductance, while inward tail currents following hyperpolarizations to −50 and −60 mV result from conductance decreases. The reversal potential of both outward and inward tail current is between −60 and −70 mV. Tail currents activated by pulses more positive than −60 mV are sensitive to the external K concentration and blocked by injection of Cs and TEA. When Ca2 influx is prevented by bathing cells in Ca2 free saline or by adding Co2 or Ni2 , the tail currents are reduced but a significant fraction of the current is insensitive to these treatments. More negative conditioning pulses activate a second component of inward tail current that is weakly sensitive to K but more strongly effected by substitution of N-methyl glucamine or Li for external Na . We conclude that both PIR and adaptation result from slow changes in a voltage dependent, non-inactivating K conductance that is active at voltages near the resting potential and is not tightly coupled to Ca2 influx. In addition, a second inward current is activated by large hyperpolarizing pulses that results from an increase in Na and K conductance. This second process is likely to contribute to PIR under particular circumstances.

Novel action of BAPTA series chelators on intrinsic K+ currents in rat hippocampal neurones.

1. Whole-cell recordings were made from rat CA1 neurones in brain slices. When electrodes contained diazo-2 (2 mM) or dibromo BAPTA (1 mM) a large steady-state outward current (hundreds of picoamps) developed within 5 min of breakthrough at a VH of -60 mV. BAPTA itself (1 mM) caused qualitatively similar but smaller effects. 2. The outward current was accompanied by increased conductance with a null potential close to the calculated K+ equilibrium potential (EK) of -110 mV. Development of outward current occurred concurrently with progressive loss of slow AHP tail current (IsAHP) evoked by brief depolarizations. The peak latency of IsAHP increased during the onset of chelator action. 3. The persistent outward current was reversibly inhibited by noradrenaline (10 microM) or isoprenaline (2-5 microM), and completely prevented by 8-bromoadenosine 3',5' cyclic monophosphate (8-Br cAMP; 100 microM) or QX-314 (10 mM) in recording electrodes. After development of outward current, diazo-2 photolysis caused inward current and decreased conductance. Both flash- and noradrenergic-sensitive responses were inwardly rectifying outward currents with null potentials close to EK. 4. The outward current induced by dibromo BAPTA was not blocked by internal EGTA (10 mM). However, experiments incorporating Ca2+ influx or Ca2+ loading of the buffer indicate that Ca2+ facilitated the outward current. 5. The outward currents induced by dibromo BAPTA or diazo-2 were not associated with significant changes in resting [Ca2+]i. Regions of the cell contributing to the outward current were deduced from measurements of fura-2 diffusion. These were compared with regions of [Ca2+]i elevation during IsAHP. 6. These results are consistent with the hypothesis that the BAPTA series Ca2+ buffers can activate those Ca2+-activated K+ channels that underlie the slow AHP, without the predicted elevation of bulk [Ca2+]i. Therefore these results cannot be interpreted solely in terms of Ca2+ concentration changes, although the observations illustrate a novel, investigative role for these compounds in the study of Ca2+-dependent processes.

Li+ and muscarine cooperatively enhance the cationic tail current in rat cortical pyramidal cells.

Li+ is known to facilitate the onset of status epilepticus induced by cholinergic stimulation, although the underlying mechanisms are not clear. Under whole-cell current clamp conditions with a CsCl-based internal solution, cortical pyramidal cells display a single plateau-spike followed by a slow depolarizing afterpotential (DAP) in response to injection of a short current pulse. However, the same current pulse generated a burst of plateau-spikes after application of Li+ (2 mM) and muscarine (10 microM). As similar bursts of plateau-spikes were generated through an enhancement of the slow DAP when [K+]o was raised (Kang et al. 1998), we have investigated the effects of Li+ and muscarine on the Ca2+-dependent cationic current underlying the slow DAP, measured as the slow tail current evoked after the offset of depolarizing voltage pulses. Muscarine enhanced the amplitudes of both early and late components of the slow tail current. This effect of muscarine was markedly potentiated by Li+, while Li+ by itself affected the slow tail current only slightly. Intracellular application of heparin (0.5-1 mg/mL) suppressed the effect of muscarine in the presence of Li+. These results suggest that inositol-trisphosphate-induced Ca2+ release is involved in the cooperative enhancement of the slow tail current, and this cooperation may be one of the mechanisms underlying facilitation of the onset of epilepsy induced by these agents.

Properties and expression of Ca2+-activated K+ channels in H9c2 cells derived from rat ventricle.

H9c2 is a clonal myogenic cell line derived from embryonic rat ventricle that can serve as a surrogate for cardiac or skeletal muscle in vitro. Using whole cell clamp with H9c2 myotubes, we observed that depolarizing pulses activated slow outward K+ currents and then slow tail currents. The K+ currents were abolished in a Ca2+-free external solution, indicating that they were Ca2+-activated K+ currents. They were blocked by apamin, a small-conductance Ca2+-activated K+ (SK) channel antagonist (IC50 = 6.2 nM), and by d-tubocurarine (IC50 = 49.4 microM). Activation of SK channels exhibited a bell-shaped voltage dependence that paralleled the current-voltage relation for L-type Ca2+ currents (ICa,L). ICa,L exhibited a slow time course similar to skeletal ICa, L, were unaffected by apamin, and were only slightly depressed by d-tubocurarine. RT-PCR analysis of the mRNAs revealed that rSK3, but not rSK1 or rSK2, was expressed in H9c2 myotubes but not in myoblasts. These results suggest that rSK3 channels are expressed in H9c2 myotubes and are primarily activated by ICa,L directly or indirectly via Ca2+-induced Ca2+ release from sarcoplasmic reticulum.

Effects of phosphorylation-related drugs on slow Ca 2+ tail current in guinea-pig detrusor cells

In isolated guinea-pig detrusor cells, large conditioning depolarizations evoke slowly deactivating Ca 2+ tail currents, considered to represent the second open state. The possible involvement of channel phosphorylation in this open state was examined. Application of isoprenaline caused a marginal increase in Ca 2+ channel current evoked by simple depolarization, while forskolin did not. During application of either drug, slow-tail currents were never observed after simple depolarizations. The conditions necessary to induce slow-tail currents were not changed, even when cyclic AMP, ATP-γ-S (adenosine 5′- O-(3-thiotriphosphate)), GDP-β-S (guanosine 5′- O-(2-thiodiphosphate)) (in the pipette) or H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) (to the bathing solution) was applied. The frequent depolarization protocol, known to facilitate Ca 2+ current via Ca 2+ and cyclic AMP-dependent phosphorylation mechanism(s) in cardiac myocytes, did not induce slow-tail currents. These results suggest that the transition of Ca 2+ channels to the second open state during large depolarization is not a result of (voltage-operated) channel phosphorylation itself. Possible underlying mechanisms are discussed.

A phenytoin-sensitive cationic current participates in generating the afterdepolarization and burst afterdischarge in rat neocortical pyramidal cells.

We report here on the ionic mechanisms underlying the depolarizing afterpotential (DAP) in neocortical pyramidal cells, with special interest in those underlying the burst afterdischarge. Injections of short depolarizing current pulses under whole-cell current clamp with a CsCl-based internal medium generated, in most pyramidal cells, a single action potential with a plateau phase (plateau-AP), followed by a slowly decaying DAP both in the absence and presence of TTX. Under voltage-clamp, the same cells displayed a slow tail current (tail-I) at the offset of depolarization. When intracellular free Ca2+ was chelated with 10 mM BAPTA or when extracellular Ca2+ was replaced with equimolar Ba2+, neither the slow DAP nor the slow tail-I was observed. Extracellular application of Co2+ or Cd2+ reduced Ca2+ currents and the slow tail-I. Cation substitution experiments revealed that the channel generating the slow tail-I was permeable to K+ and Cs+ more than to Na+ (PK is approximately equal to PCs > PNa > PNMDG is approximately equal to PTEA). The cationic slow tail-I was not reduced by applying antagonists of the metabotropic glutamate receptor (MCPG, 1 mM) and the muscarinic receptor (atropine, 1-10 microM). Thus, the slow DAP was produced by activation of the cationic channel whose gating is solely dependent on [Ca2+]i. An increase in [K+]o from 3 to 6 or 9 mM enhanced the slow DAP, and resulted in a generation of burst afterdischarges. An anticonvulsant, phenytoin (PT; 1-10 microM) suppressed the slow DAP while enhancing the plateau-AP in the presence of TTX, most likely by blocking the cationic channel.

Effects of veratridine on sodium currents and fluxes.

Veratridine causes Na+ channels to stay open during a sustained membrane depolarization by abolishing inactivation. The consequential Na+ influx, either by itself or by causing a maintained depolarization, leads to many secondary effects such as increasing pump activity, Ca2+ influx, and in turn exocytosis. If the membrane is voltage clamped in the presence of the alkaloid, a lasting depolarizing impulse induces, following the "normal" transient current, another much more slowly developing Na+ current that reaches a constant level after a few seconds. Repolarization then is followed by an inward tail current that slowly subsides. Development of these slow currents is enhanced by additional treatment with agents that inhibit inactivation. Most of these phenomena can be satisfactorily explained by assuming that Na+ channels must open before veratridine binds to them, and that the slow current changes reflect the kinetics of binding and unbinding. It is unclear, however, where the alkaloid stays when it is not bound. Although the effect sets in promptly, once this pool is filled, access to it from outside must be impeded since in most preparations veratridine can only partially be washed out. Cooling acts as if the available concentration is reduced, but this reversible "reduction" takes much longer to develop than the cold-induced changes in kinetics. Several authors assume that the binding site, site 2, is accessed from the lipid phase of the membrane. Considerations of this kind are often based on experiments with batrachotoxin, the widely used site-2 ligand which has a much higher affinity and acts as a full agonist in contrast to the partial agonist veratridine. Batrachotoxin thus lends itself to binding studies using radiolabeled derivatives. Such experiments may eventually lead to the characterization of neurotoxin site 2; the first promising steps have been taken. Modern techniques of molecular biology will almost certainly be successful, and one hopes for point-mutated channels with distinctly different reactions also to veratridine. A considerable amount of research is still required to clarify the structural basis for the numerous allosteric interactions with other sites, the mechanism of the very large potential shift of activation, the reduced single-channel conductance and selectivity, and the chemical nature of the different affinities of the site-2 toxins. Note Added in Proof. A report on point mutations with effects on neurotoxin site 2 (see Sect. 8) has just appeared: Wang S-Y, Wang GK (1988) Point mutations in segment I-S6 render voltage-gated Na+ channels resistant to batrachotoxin. Proc Natl Acad USA 95:2653-2658. In microliter muscle Na+ channels expressed in mammalian cells, mutation Asn434Lys leads to complete, Asn434Ala to partial insensitivity to 5 mM batrachotoxin. (Asn434 corresponds to Asn419 of Trainer et al. 1996). The mutant channel displays almost normal current kinetics and in the presence of veratridine little, if any, slow tail current. However, veratridine inhibits peak Na+ currents in the mutant which may point to a complex structure of site 2.

Calcium current during a single action potential in a large presynaptic terminal of the rat brainstem.

1. The calcium current of a 'giant' synaptic terminal (the calyx of Held) was studied using two-electrode voltage clamp in slices of the rat brainstem. 2. In terminals with a long axon (length > 100 microns), the passive current transient decayed biexponentially following voltage steps. In terminals with a short axon (length < 30 microns), the slow component was reduced or absent. These terminals also had small slow calcium tail currents following long depolarizing voltage steps, suggesting that these are largely due to axonal calcium channels. 3. Terminals were voltage clamped with action potential waveform commands. At both 24 and 36 degrees C the calcium current began shortly after the peak of the action potential and ended before the terminal was fully repolarized. 4. The calcium current during the repolarization phase was 69 +/- 1% (n = 3) of maximal, judged from the increase in this current when a plateau phase was added to the action potential waveform. 5. A Hodgkin-Huxley m2 model, based on the measured activation and deactivation of the calcium current, reproduced both the time course and the amplitude increase of the calcium currents during the different action potential waveforms well. 6. The fast gating of the calcium channels in the terminal ensures that they are effectively opened during the repolarization phase of an action potential. This implies that the distance between open calcium channels is minimized, which is in agreement with the view that multiple calcium channels are needed to release a vesicle in this synapse.

Facilitation of L-type calcium current in thalamic neurons.

We have studied facilitation of the L-type calcium current in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus. Currents were recorded after pretreatment with 1 microM omega-conotoxin GVIA and 5 microM omega-conotoxin MVIIC, to better isolate L-current. Long, strong depolarizations induced slow tail currents at negative voltages, but did not affect currents at voltages where channels were strongly activated. The initial peak tail current was not measurably increased. The time course of recovery from facilitation paralleled the time course of the tail current, indicating that facilitation does not outlast channel closing. The kinase inhibitors staurosporine and H-7 and the phosphatase inhibitor okadaic acid had no significant effect on L-current facilitation compared with control, but facilitation was greater with H-7 than with okadaic acid. The guanosine 5'-triphosphate (GTP) analogs GTP-gamma-S and GDP-beta-S did not affect facilitation. We conclude that L-current facilitation in thalamic neurons does not result from Ser/Thr phosphorylation, although phosphorylation may modulate facilitation. This form of facilitation differs kinetically and pharmacologically from facilitation induced by activation of G protein-coupled receptors.

On the nature of anomalous rectification in thalamocortical neurones of the cat ventrobasal thalamus in vitro.

1. Intracellular sharp electrode current clamp and discontinuous single electrode voltage clamp recordings were made from thalamocortical neurones (n = 57) of the cat ventrobasal thalamus in order to investigate the mechanism underlying anomalous rectification. 2. Under current clamp conditions, voltage-current (V-I) relationships in a potential range of -55 to -110 mV demonstrated anomalous rectification with two components: fast rectification, which controlled the peak of negative voltage deviations, and time-dependent rectification. Time-dependent rectification was apparent as a depolarizing sag generated during the course of negative voltage deviations, was first formed at potentials in the range -60 to -70 mV, and was sensitive to 3 mM Cs+ (n = 6). Similarly, under voltage clamp conditions, instantaneous and steady-state I-V relationships demonstrated anomalous rectification. A slowly activating inward current with an activation threshold in the range of -65 to -70 mV formed time-dependent rectification. This current was sensitive to Cs+ (3 mM) (n = 3) and had properties similar to the slow inward mixed cationic current (Ih). 3. 4-(N-Ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium++ + chloride (ZD 7288) (100-300 microM) irreversibly blocked time-dependent rectification mediated by Ih (n = 23 of 25 neurones), and led to a hyperpolarization of the resting membrane potential (6.8 +/- 0.5 mV). In the presence of ZD 7288, V-I and I-V relationships, exhibited fast anomalous rectification, first activated from potential more negative than -80 mV. 4. Ba2+ (100 microM) (n = 8), in the continuous presence of ZD 7288, reversibly linearized peak V-I and instantaneous I-V relationships over a potential range of -70 to -120 mV, and led to a membrane depolarization (13.3 +/- 4.2 mV) or tonic inward current (192 +/- 36 pA). 5. The co-application of ZD 7288 and Ba2+ revealed a depolarizing sag in negative voltage deviations under current clamp conditions, or a large inward current with kinetics two to three times slower than those of Ih under voltage clamp conditions. This novel form of time-dependent rectification was first apparent at potentials more negative than about -85 mV, was sensitive to 5 mM Cs+ (n = 4), and is termed Ih,slow. Ih,slow tail currents reversed between -65.3 and -56.6 mV (with potassium acetate electrodes, n = 3) or -57.6 and -50.3 mV (with KCl electrodes, n = 3). 6. Computer simulations confirmed that the pattern of anomalous rectification in thalamocortical neurones of the cat ventrobasal thalamus is mediated by the concerted action of Ih and a Ba(2+)-sensitive current with properties similar to an inwardly rectifying K+ current (IKIR).

Mibefradil (Ro 40m5967) blocks multiple types of voltage-gated calcium channels in cultured rat spinal motoneurones

The actions of the novel calcium (Ca 2+) channel antagonist mibefradil (Ro 40–5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca 2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca 2+ current flowed through N-type Ca 2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca 2+ channel currents. Inhibition was nearly complete at 10 μM suggesting mibefradil blockade of all subclasses of Ca 2+ channels. The IC 50 was approximately 1.4 μM on currents measured at 0 mV, from a holding potential of −90 mV. Inhibition of LVA Ca 2+ current occurred over the same concentration range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC 50 = 3.4 μM). These broad inhibitory effects of mibefradil on Ca 2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca 2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca 2+ channels other than type T.

Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway.

With use of the whole cell patch-clamp technique, effects of the potent muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activated Ca2+ channel currents were investigated in acutely dissociated adult rat intracardiac neurons. In all tested neurons oxo-M reversibly inhibited the peak Ba2+ current. Inhibition of the peak Ba2+ current by oxo-M was associated with slowing of activation kinetics and was concentration dependent. The concentration of oxo-M necessary to produce a half-maximal inhibition of current and the maximal inhibition were 40.8 nM and 75.9%, respectively. Inhibitory effect of oxo-M was completely abolished by atropine. Among different muscarinic receptor antagonists, methoctramine (100 and 300 nM) significantly antagonized the current inhibition by oxo-M, with a negative logarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5'-(thio)triphosphate (GTPgammaS, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significantly occlude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS, 2 mM) also significantly reduced the muscarinic inhibition of the peak Ba2+ current by oxo-M. Inhibitory effects of oxo-M were significantly abolished by pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (400 ng/ml). Furthermore, the bath application of N-ethylmaleimide (50 microM) significantly reduced the inhibition of the peak Ba2+ current by oxo-M. The oxo-M shifted the activation curve derived from measurments of tail currents toward more positive potentials. A strong conditioning prepulse to +100 mV significantly relieved the muscarinic inhibition of peak Ba2+ currents by oxo-M and the GTPgammaS-induced current inhibition. In a series of experiments, changes in intracellular concentration of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid and protein kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 microM) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 microM) failed to reduce the slow tail currents induced by the L-type agonist methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176; 2 microM). However, omega-conotoxin (omega-CgTX) GVIA (1 microM) significantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 microM) and nifedipine (10 microM), oxo-M could further inhibit approximately 20% of the total Ca2+ current. After complete removal of N-, Q-, and L-type currents with use of omega-CgTX GVIA, omega-agatoxin IVA, and nifedipine, 70% of the R-type current (approximately 6-7% of the total current) was inhibited by oxo-M (3 microM). In conclusion, the M2 muscarinic receptor activation selectively inhibits N-, Q-, and R-type Ca2+ channel currents, sparing L-type Ca2+ channel currents mainly via a PTX- and voltage-sensitive pathway in adult rat intracardiac neurons.

Metabotropic glutamate receptor subtypes mediating slow inward tail current (IADP) induction and inhibition of synaptic transmission in olfactory cortical neurones

1. The pharmacological features of the pre- and postsynaptic metabotropic glutamate receptors (mGluRs) present in the guinea-pig olfactory cortex, were examined in brain slices in vitro by use of a conventional intracellular current clamp/voltage clamp recording technique. 2. Bath-application of trans-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (50 microM) produced a sustained membrane depolarization, increase in cell excitability and induction of a post-stimulus inward (after depolarizing) tail current (IADP) (measured under 'hybrid' voltage clamp) similar to those evoked by the muscarinic receptor agonist oxotremorine-M (OXO-M, 2 microM). 3. L-Glutamate (0.25 1 mM. in the presence of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 microM-DL-amino-5-phosphono valeric acid (DL-APV)) or the broad spectrum mGluR agonists 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 10 microM), 1S,3S-ACPD (50 microM), ibotenate (Ibo; 25 microM. in the presence of 100 microM DL-APV), the selective mGluR I agonists (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG, 10 microM), (S)-3-hydroxyphenylglycine ((S)-3HPG, 50 microM), or quisqualate (10 microM, in the presence of 20 microM CNQX), but not the mGluR II agonist 2S,1'S,2'S-2-(2'-carboxycyclopropyl)-glycine (L-CCG1,1 microM) or mGluR III agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 1 mM), were all effective in producing membrane depolarization and inducing a post-stimulus IADP. Unexpectedly, the proposed mGluR II-selective agonist (2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)-glycine (DCG-IV, 10 microM, in the presence of 100 microM DL-APV) was also active. 4. The excitatory effects induced by 10 microM 1S,3R-ACPD were reversibly antagonized by the mGluR I/II antagonist (1)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG, 0.5 1 mM), as well as the selective mGluR I antagonists (S)-4-carboxyphenylglycine ((S)-4CPG) and (S)-4-carboxy-3-hydroxyphenyl glycine ((S)-4C3HPG) (both at 1 mM), but not the nonselective mGluR antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP3, 1 mM) or the selective mGluR III antagonist (S)-alpha-methyl-L-AP4 (MAP4, 1 mM). 5. The excitatory postsynaptic potentials (e.p.s.ps), induced by single focal stimulation of cortical excitatory fibre tracts, were markedly reduced by 1S,3R-ACPD or L-AP4 (both at 10 microM), and by the selective mGluR II agonists (mGluR 1 antagonists) (S)-4CPG or (S)-4C3HPG (both at 1 mM) but not (S)-3,5-DHPG or (S)-3HPG (both at 100 microM). 6. The inhibitory effects of 1S-3R-ACPD, but not L-AP4, were reversibly blocked by (+)-MCPG (1 mM), whereas those produced by L-AP4, but not 1S,3R-ACPD, were blocked by the selective mGluR III antagonist MAP4 (1 mM). 7. It is concluded that a group I mGluR is most likely involved in mediating excitatory postsynaptic effects, whereas two distinct mGluRs (e.g. group II and III) might serve as presynaptic inhibitory autoreceptors in the guinea-pig olfactory cortex.