High-threshold Ca Currents Research Articles

A model of on/off transitions in neurons of the deep cerebellar nuclei: deciphering the underlying ionic mechanisms

The neurons of the deep cerebellar nuclei (DCNn) represent the main functional link between the cerebellar cortex and the rest of the central nervous system. Therefore, understanding the electrophysiological properties of DCNn is of fundamental importance to understand the overall functioning of the cerebellum. Experimental data suggest that DCNn can reversibly switch between two states: the firing of spikes (F state) and a stable depolarized state (SD state). We introduce a new biophysical model of the DCNn membrane electro-responsiveness to investigate how the interplay between the documented conductances identified in DCNn give rise to these states. In the model, the F state emerges as an isola of limit cycles, i.e. a closed loop of periodic solutions disconnected from the branch of SD fixed points. This bifurcation structure endows the model with the ability to reproduce the text{F}to text{SD} transition triggered by hyperpolarizing current pulses. The model also reproduces the text{F}to text{SD} transition induced by blocking Ca currents and ascribes this transition to the blocking of the high-threshold Ca current. The model suggests that intracellular current injections can trigger fully reversible text{F}leftrightarrow text{SD} transitions. Investigation of low-dimension reduced models suggests that the voltage-dependent Na current is prominent for these dynamical features. Finally, simulations of the model suggest that physiological synaptic inputs may trigger text{F}leftrightarrow text{SD} transitions. These transitions could explain the puzzling observation of positively correlated activities of connected Purkinje cells and DCNn despite the former inhibit the latter.

Central Nervous System–Toxic Lidocaine Concentrations Unmask L-Type Ca2+ Current–Mediated Action Potentials in Rat Thalamocortical Neurons: An In Vitro Mechanism of Action Study

High systemic lidocaine concentrations exert well-known toxic effects on the central nervous system (CNS), including seizures, coma, and death. The underlying mechanisms are still largely obscure, and the actions of lidocaine on supraspinal neurons have received comparatively little study. We recently found that lidocaine at clinically neurotoxic concentrations increases excitability mediated by Na-independent, high-threshold (HT) action potential spikes in rat thalamocortical neurons. Our goal in this study was to characterize these spikes and test the hypothesis that they are generated by HT Ca currents, previously implicated in neurotoxicity. We also sought to identify and isolate the specific underlying subtype of Ca current. We investigated the actions of lidocaine in the CNS-toxic concentration range (100 μM-1 mM) on ventrobasal thalamocortical neurons in rat brain slices in vitro, using whole-cell patch-clamp recordings aided by differential interference contrast infrared videomicroscopy. Drugs were bath applied; action potentials were generated using current clamp protocols, and underlying currents were identified and isolated with ion channel blockers and electrolyte substitution. Lidocaine (100 μM-1 mM) abolished Na-dependent tonic firing in all neurons tested (n = 46). However, in 39 of 46 (85%) neurons, lidocaine unmasked evoked HT action potentials with lower amplitudes and rates of de-/repolarization compared with control. These HT action potentials remained during the application of tetrodotoxin (600 nM), were blocked by Cd (50 μM), and disappeared after superfusion with an extracellular solution deprived of Ca. These features implied that the unmasked potentials were generated by high-voltage-activated Ca channels and not by Na channels. Application of the L-type Ca channel blocker, nifedipine (5 μM), completely blocked the HT potentials, whereas the N-type Ca channel blocker, ω-conotoxin GVIA (1 μM), had little effect. At clinically CNS-toxic concentrations, lidocaine unmasked in thalamocortical neurons evoked HT action potentials mediated by the L-type Ca current while substantially suppressing Na-dependent excitability. On the basis of the known role of an increase in intracellular Ca in the pathogenesis of local anesthetic neurotoxicity, this novel action represents a plausible contributing candidate mechanism for lidocaine's CNS toxicity in vivo.

Ca(2+)-dependent inactivation of high-threshold Ca(2+) currents in hippocampal granule cells of patients with chronic temporal lobe epilepsy.

Intracellular Ca(2+) represents an important trigger for various second-messenger mediated effects. Therefore a stringent control of the intracellular Ca(2+) concentration is necessary to avoid excessive activation of Ca(2+)-dependent processes. Ca(2+)-dependent inactivation of voltage-dependent calcium currents (VCCs) represents an important negative feedback mechanism to limit the influx of Ca(2+) that has been shown to be altered in the kindling model of epilepsy. We therefore investigated the Ca(2+)-dependent inactivation of high-threshold VCCs in dentate granule cells (DGCs) isolated from the hippocampus of patients with drug-refractory temporal lobe epilepsy (TLE) using the patch-clamp method. Ca(2+) currents showed pronounced time-dependent inactivation when no extrinsic Ca(2+) buffer was present in the patch pipette. In addition, in double-pulse experiments, Ca(2+) entry during conditioning prepulses caused a reduction of VCC amplitudes elicited during a subsequent test pulse. Recovery from Ca(2+)-dependent inactivation was slow and only complete after 1 s. Ca(2+)-dependent inactivation could be blocked either by using Ba(2+) as a charge carrier or by including bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) or EGTA in the intracellular solution. The influence of the cytoskeleton on Ca(2+)-dependent inactivation was investigated with agents that stabilize and destabilize microfilaments or microtubules, respectively. From these experiments, we conclude that Ca(2+)-dependent inactivation in human DGCs involves Ca(2+)-dependent destabilization of both microfilaments and microtubules. In addition, the microtubule-dependent pathway is modulated by the intracellular concentration of GTP, with lower concentrations of guanosine triphosphate (GTP) causing increased Ca(2+)-dependent inactivation. Under low-GTP conditions, the amount of Ca(2+)-dependent inactivation was similar to that observed in the kindling model. In summary, Ca(2+)-dependent inactivation was present in patients with TLE and Ammon's horn sclerosis (AHS) and is mediated by the cytoskeleton similar to rat pyramidal neurons. The similarity to the kindling model of epilepsy may suggest the possibility of altered Ca(2+)-dependent inactivation in patients with AHS.

Effects of adrenalectomy on Ca2 currents and Ca2 channel subunit mRNA expression in hippocampal CA1 neurons of young rats

Previously it was found that the amplitude of Ca currents in CA1 hippocampal neurons increases after adrenalectomy (ADX) of young adult rats. Preliminary data suggested that this effect of ADX is age-dependent. In the present study we therefore investigated the effect of ADX on Ca currents in three age groups: rats that were 1, 3, or 6 months old. It appeared that ADX of the youngest age group resulted in a selective enhancement of sustained, high threshold Ca currents. By contrast, ADX of the oldest rats enhanced only the low threshold, transient Ca current amplitude. The age dependency of the ADX-induced effects can be explained by developmental changes in Ca current properties in the adrenally intact rats with which ADX animals were compared. Data from acutely dissociated cells, which lack most of their dendrites, suggest that the ADX-induced changes of Ca current properties are at least partly targeted at currents generated in the distal dendrites. No significant changes were observed with age or after ADX for the mRNA expression of the α1D Ca channel subunit, which forms the ionpore of the sustained high threshold L-type Ca channel. We can therefore presently not exclude that the altered amplitude of Ca current with age and ADX is due to changes in ion channel function rather than in the total number of these channels. Synapse 26:155–164, 1997. © 1997 Wiley-Liss, Inc.

Effects of adrenalectomy on Ca2+ currents and Ca2+ channel subunit mRNA expression in hippocampal CA1 neurons of young rats.

Previously it was found that the amplitude of Ca currents in CA1 hippocampal neurons increases after adrenalectomy (ADX) of young adult rats. Preliminary data suggested that this effect of ADX is age-dependent. In the present study we therefore investigated the effect of ADX on Ca currents in three age groups: rats that were 1, 3, or 6 months old. It appeared that ADX of the youngest age group resulted in a selective enhancement of sustained, high threshold Ca currents. By contrast, ADX of the oldest rats enhanced only the low threshold, transient Ca current amplitude. The age dependency of the ADX-induced effects can be explained by developmental changes in Ca current properties in the adrenally intact rats with which ADX animals were compared. Data from acutely dissociated cells, which lack most of their dendrites, suggest that the ADX-induced changes of Ca current properties are at least partly targeted at currents generated in the distal dendrites. No significant changes were observed with age or after ADX for the mRNA expression of the alpha 1D Ca channel subunit, which forms the ionpore of the sustained high threshold L-type Ca channel. We can therefore presently not exclude that the altered amplitude of Ca current with age and ADX is due to changes in ion channel function rather than in the total number of these channels.

Comparison of ionic currents expressed in immature and mature muscle cells of an ascidian larva

We have compared the voltage-gated ion channels present in larval ascidian muscle at two developmental stages: muscle precursor cells just after the terminal cell division and mature contractile muscle, 7-11 hr later. All precursor cells express a high-threshold transient Ca current and a slowly activating delayed K current, and about half the cells express a low-threshold transient Ca current. An inwardly rectifying K current, which had been present from fertilization until just before the terminal cell division, is absent. Mature muscle retains two of the tailbud currents: the low-threshold transient Ca current and the slow delayed K current, although at larger densities, and also expresses a high-threshold Ca current that is similar in most respects to the precursor cell current but that lacks inactivation. In addition, mature muscle expresses two rapidly activating outward K currents, one voltage and one Ca dependent, that generate a composite outward K current that is eight times larger and activates eight times faster than the tailbud K current. Mature muscle also reexpresses the inward rectifier. We propose that the transient absence of the inward rectifier and the slow activation of the delayed K current early in development create a window of developmental time when spontaneous electrical activity is likely.

Block of Ca channels in rat central neurons by the spider toxin omega- Aga-IIIA

The effects of the spider toxin omega-Aga-IIIA were studied on Ca channel currents in rat central neurons. In hippocampal CA1 pyramidal neurons, omega-Aga-IIIA blocked approximately 70% of the high-threshold Ca currents and had no effect on low-threshold T-type current. Occlusion experiments with blockers of L-, N-, and P-type Ca currents showed that omega-Aga-IIIA abolished dihydropyridine-sensitive L-type current and blocked a substantial fraction of the omega-conotoxin (CgTX)-sensitive N-type and omega-Aga-IVA-sensitive P-type Ca currents. The high-threshold current remaining with saturating concentrations of nimodipine, CgTX, and omega-Aga-IVA was also partially blocked by omega-Aga-IIIA in a variety of central neurons. Block of P-type current by omega-Aga-IIIA was investigated in more detail in cerebellar Purkinje neurons. Block was potent (Kd approximately 0.5 nM), but incomplete and voltage dependent. Tail current activation curves showed that channel gating is shifted in the depolarizing direction by approximately 7 mV. The instantaneous current-voltage curve for P-type current was also altered; the toxin reduced Ba-carried inward currents by approximately 40% and had little effect on Cs-carried outward currents. The partial, voltage-dependent reduction of P-type Ca current can be accounted for by a combination of toxin effects on channel permeation and gating.

Modulation of voltage‐activated Ca currents by pain‐inducing agents in a dorsal root ganglion neuronal line, F‐11

Whole cell currents evoked by pain-inducing agents--bradykinin (Bk), capsaicin (Cap), and reciniferatoxin (RTX), and their modulation of voltage-activated Ca currents were examined in F-11 cells using a patch electrode voltage clamp technique. Most F-11 cells generated action potentials under current clamp if their membrane potentials were held sufficiently negative. Average peak inward Na current (INa) was 100 microA/cm2 and the INa was abolished by 10(-6) M tetrodotoxin. At least two types of Ca currents could be clearly distinguished on the basis of voltage dependency and kinetics; a low threshold transient ICa(t) and a high threshold sustained ICa(l). In addition, another high threshold transient Ca current, presumably ICa(n), was observed. About 30% of the cells produced inward current for these pain-inducing agents, when activated at the membrane holding potential of -70 mV. In some F-11 cells, the amplitude of action potential was observed to increase during 10(-6) M Cap-induced depolarization. Both low and high threshold Ca currents were reduced by 10(-6) M Bk in the majority of the cells. Similarly, both 10(-6) M Cap and 10(-9) M RTX reduced these Ca currents. However, a considerable number of cells showed an initial enhancement followed by reduction in the amplitude of these Ca currents. With higher concentrations of these ligands, all Ca currents were suppressed. Such modulation of voltage-activated Ca currents by pain-inducing agents occurred in both the presence and absence of apparent receptor-activated current flows in the cells. In pertussis toxin (PTX)-treated cells, the inhibitory modulation of Ca currents by pain-inducing agents was suppressed. In contrast, in cholera toxin (CTX)-treated cells, this inhibitory modulation appeared to be enhanced. These data indicate that the inhibitory modulation of Ca channel currents by Cap and RTX, similarly to that of Bk, involves a PTX-sensitive inhibitory G protein (Gi).

A study of the mechanism of Ca2+ current inhibition produced by serotonin in rat dorsal raphe neurons

Calcium currents and their modulation by 5-HT were studied using both whole-cell and single-channel patch-clamp techniques in acutely isolated adult rat dorsal raphe neurons. Evidence for three types of Ca channels (T, N, L) was obtained in both whole-cell and single-channel experiments. Approximately 4% of the total high-threshold Ca current (L-type) was sensitive to dihydropyridines (DHPs) while approximately 40% of the Ca current (N-type) was sensitive to omega-conotoxin (omega-CgTx). About 56% of the whole-cell current was insensitive to either DHPs or omega-CgTx and may thus represent a different kind of Ca current. 5-HT reduced raphe neuron Ca currents by approximately 50%, while slowing activation. 5-HT inhibited both omega-CgTx-sensitive and -insensitive Ca current. Inhibition by 5-HT was voltage dependent; prepulses to +80 mV lasting for 20 msec almost completely abolished the 5-HT-mediated inhibition. The voltage dependence of the response to 5-HT suggested that trains of action potentials might overcome the inhibition due to 5-HT. Trains of brief depolarizations were used to simulate action potentials; only about 5% of the 5-HT-induced inhibition was relieved by the trains. These results suggest that while large depolarizations could restore the Ca current inhibited by 5-HT, physiological stimuli, such as trains of action potentials, could not. The action of 5-HT was made irreversible by inclusion of GTP-gamma-S in the patch pipette, suggesting a G-protein mediation of the response to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual action of prajmalium on the Ca currents in frog isolated cardiomyocytes

The effects of N-n-propylajmaline (prajmalium) on the Na and Ca currents of single frog atrial and ventricular cells were studied by means of the whole-cell patch-clamp technique. Prajmalium (10 −9 to 10 −6 m) depressed the Na current ( I Na) in a dose- and use-dependent manner. In the same range of concentrations, prajmalium induced a dual effect on the high ( I CaL) and low ( I CaT) threshold Ca currents (the latter being only present in atrial cells). At a low concentration (10 −9 m), prajmalium increased both Ca currents while at high concentrations (10 −6 m) it depressed them. Prajmalium appeared very potent on I CaT although this current is generally reported to be barely sensitive to agonists and drugs. The action of the drug was also accompanied by a shortening in the half-time of inactivation of the Ca currents and a slight hyperpolarizing shift of their availability curves. The increase in I CaL by prajmalium was not prevented by prazosin (10 −7 m) nor by propranolol (10 −6 m), and it was also observed after I CaL had been fully stimulated by isoproterenol (10 −7 m). Nifedipine (10 −6 m), however, was able to prevent or block the prajmalium-induced increase in I CaL. Some similarities between the actions of prajmalium and dihydropyridine agonists on Ca currents are discussed.

Stimulation‐dependent facilitation of the high threshold calcium current in guinea‐pig ventricular myocytes.

1. Stimulation-dependent modulation of Ca currents was examined in guinea-pig ventricular myocytes at room temperature. Whole-cell recordings of Ca currents were made under conditions which minimized ionic fluxes through other channels. 2. Stimulation from rest at a rate of 2 Hz resulted in a decrease of the low threshold Ca current within one pulse and facilitation of the high threshold Ca current within five pulses. Facilitation was associated with a reduction in the rate of inactivation. 3. Pulse durations as short as 10 ms facilitated the high threshold Ca current in subsequent pulses. Facilitation produced by a single pulse decayed with a half-time of several seconds. 4. Substitution of Ba2+ or Sr2+ for external Ca2+ reduced the rate of inactivation of the high threshold Ca current and abolished facilitation of the current. 5. Facilitation persisted with 40 microM-Ruthenium Red added to the internal solution or 0.2-2 microM-ryanodine added to the bath solution to reduce Ca2+ release from the sarcoplasmic reticulum. 6. Facilitation was modulated by isoprenaline. Low concentrations of isoprenaline (5-10 nM) increased the amount of facilitation. Isoprenaline (1 microM) increased the Ca current approximately 3-fold, however, facilitation was nearly abolished. 7. Caffeine (0.5 and 1 mM) affected the Ca current and facilitation in a manner similar to 1 microM-isoprenaline. It increased the Ca currents approximately 2.5-fold and facilitation was not observed. 8. We conclude that stimulation-dependent facilitation of the high threshold Ca current is mediated by calcium and hypothesize that calcium affects a site near the Ca channel that modifies the rate of inactivation. The common actions of caffeine and high concentrations of isoprenaline suggest that calcium modulates a phosphorylation step.

Ca currents in human neuroblastoma IMR32 cells: kinetics, permeability and pharmacology

We have investigated the kinetics, permeability and pharmacological properties of Ca channels in in vitro differentiated IMR32 human neuroblastoma cells. The low-threshold (LVA, T) Ca current activated positive to -50 mV and inactivated fully within 100 ms in a voltage-dependent manner. This current persisted in the presence of 3.2 microM omega-conotoxin (omega-CgTx) or 40 microM Cd and showed a weaker sensitivity to Ni and amiloride than in other neurons. The high-threshold Ca currents (HVA,L and N) turned on positive to -30 mV, and inactivated slowly and incompletely during pulses of 200 ms duration. The amplitude of the HVA currents and the number of 125I-omega-CgTx binding sites increased markedly during cell differentiation. In agreement with recent reports, 6.4 microM omega-CgTx blocked only about 85% of the Ba currents through HVA channels in 50% of the cells. Residual omega-CgTx-resistant currents proved to be more sensitive to dihydropyridines (DHP) than total HVA currents. Bay K 8644 (1 microM) had a clear agonistic action on omega-CgTx-resistant currents and was preferred to other Ca antagonists for identifying HVA DHP-sensitive channels. Compared to the omega-CgTx-sensitive, the DHP-sensitive currents turned on at slightly more negative potentials and showed a weaker sensitivity to voltage. The two HVA currents were otherwise hardly distinguishable in terms of activation/inactivation kinetics, Ca/Ba permeability and sensitivity to holding potentials. This suggests that currently used criteria for identifying multiple types of neuronal Ca channels (T;L,N) may be widely misleading if not supported by pharmacological assays.

T-type calcium channels: heterogeneous expression in rat sensory neurons and selective modulation by phorbol esters

We report selective inhibition of low-threshold, T-type calcium channels by a phorbol ester in rat sensory neurons. Cells were exposed, either acutely or with 15-60 min preincubations, to low concentrations of phorbol 12-myristate 13-acetate, (PMA), an activator of protein kinase C; if the temperature was 29 degrees C or higher, T-type Ca current was diminished without effect on high-threshold Ca current. In untreated cells, the amplitude of T-type Ca current varies widely among neighboring sensory neurons. T channels are absent in about 25% of cells, provide a small current near threshold for the majority of cells, and are a dominant pathway for calcium entry in a small subset of neurons. The results are of interest because, by selectively inhibiting a calcium channel expressed differently among subpopulations of sensory neurons, activation of protein kinase C might selectively suppress particular sensations.

Temperature sensitivity of Ca currents in chick sensory neurones

We have investigated the effects of temperature on the Ca currents of chick sensory neurones. Raising the temperature from 17 to 37 degrees C, caused low-threshold (LVA, T) and high-threshold (HVA, L and N) Ca currents to show a marked amplitude increase and a drastic acceleration of their activation-inactivation gatings. Compared to HVA channels, the LVA type showed a weaker temperature sensitivity. Its average Q10 values were closer to those of other voltage-operated ion channels: 1.7 (permeability), 1.9 (activation) and 2.2 (inactivation). Alternatively, the activation kinetics and peak permeability of HVA Ca channels showed maximal Q10 values of about 5 and 2.8, respectively. HVA channel deactivation was less sensitive to temperature (Q10 1.8). Inactivation of these channels was slow and monoexponential between 17 and 22 degrees C, but faster and double exponential above 30 degrees C, uncovering a fast temperature-sensitive decaying phase. The size and rate of decay of this component decreased with increasing membrane depolarizations and persisted at holding potentials positive to -80 mV, suggesting the involvement of temperature-sensitive Ca-mediated processes in the mechanism of HVA channel inactivation. Our data are consistent with the view that heating from 17 to 37 degrees C causes both an increased probability of Ca channels to open and a drastic acceleration of their activation-inactivation kinetics.

The low-threshold Ca current in isolated amygdaloid neurons in the rat

Two types of voltage-dependent Ca currents were recorded from isolated rat amygdaloid neurons under single-electrode voltage-clamp. A low-threshold Ca current was elicited at −60 mV or more positive potentials and inactivated rapidly. At −30 mV or more positive potentials, a high-threshold Ca current was also activated. In the steady-state inactivation curve of the low-threshold Ca current, the half-inhibition value (h 0.5) was −71 mV. The low-threshold Ca current was inhibited by organic and inorganic Ca blockers in a dose-dependent manner. The inhibitory effect of these Ca blockers was completely reversible while that of flunarizine was partly so. It is concluded that the membrane and pharmacological properties of the low-threshold Ca channel in the rat amygdaloid neurons are quite similar to those in the hypothalamic neurons.

Effects of BAY K 8644 on Ca-channel currents and electrical activity in mouse melanotrophs

The effects of BAY K 8644 on both evoked and spontaneous electrical activity were studied in mouse melanotrophs. The action potential in these cells consisted of both a Na and a Ca component. BAY K 8644 greatly increased the duration of the evoked action potential apparently by enhancing the Ca component since this prolongation remained when the Na component was blocked by tetrodotoxin but was diminished by the Ca-channel antagonists Co 2+ or nimodipine. In addition to its effects on the evoked action potential, BAY K 8644 sometimes caused action potentials of long duration to occur in otherwise quiescent melanotrophs, even in the presence of tetrodotoxin. These action potentials appeared to be triggered by small, depolarizing prepotentials. Voltage-clamp experiments suggested the existence of 3 types of Ca-channel currents in these cells: one low-threshold current and two high-threshold currents, one of which rapidly inactivated and another which did not. BAY K 8644 caused a shift in the I–V relation for Ca-channel current to more negative values and reduced or abolished the hump in the initial part of the I–V curve produced by the low-threshold current suggesting that BAY K 8644 selectively affects high-threshold Ca current. The action of BAY K 8644 to initiate trains of long-lasting Ca spikes and thereby increase Ca influx, might explain its secretagogue effect on the unstimulated melanotroph.

Voltage-activated calcium channels in rat Purkinje cells maintained in culture

Cell-attached patch recordings were used to study calcium channels on the dendritic membrane of rat cerebellar Purkinje cells maintained in culture. Experiments were performed with isotonic BaCl2 (110 mM) in the pipette and isotonic potassium gluconate in the bath to zero the cell membrane potential. Two distinct types of voltage-activated calcium channels were identified. The first one had a small conductance (9 pS), was activated at a low threshold (congruent to -50 mV) and could be inactivated by holding the membrane potential at -30 mV. This channel had the same characteristics as the T channel described in other neuronal preparations. The second type of Ca channel activated at a high threshold (-30 or +10 mV depending on whether BAY K 8644 was added or not to the pipette solution) and was still activatable even when the membrane was held at -40 mV. In the presence of BAY K 8644 this channel had a conductance of 21 pS with long openings. All these characteristics are similar to those of the S (L) Ca channel described in many preparations. The present study is in agreement with our previous experiments on Purkinje dendrites, where we identified low and high threshold Ca currents using the whole-cell configuration. Up to now, no channel corresponding to the N current has been observed but we cannot exclude its presence.

Decay kinetics of calcium currents in rat sensory neurones: analysis at two internal free calcium concentrations

The patch-clamp technique in whole-cell configuration was used to investigate the kinetics of decay of calcium currents in rat sensory neurones. Whole-cell recording permitted control of the internal medium, particularly of the internal free calcium concentration, which was maintained at either 10(-9) M or 10(-6) M using a high concentration of Ca buffer. The inactivation decay of the total Ca current elicited above -10 mV was found to be faster at pCa 6 than at pCa 9. The total current contained three exponential components which were tentatively identified as the three types of Ca currents (IcaT, IcaN and IcaS). Kinetic analyses indicated that the control of the inactivation process by internal Ca results from an effect on both high-threshold Ca currents, IcaN and IcaS. The inactivation kinetics reported in the literature presents a large variability depending on the cell type. We propose that this variability may result from differences in the capacity of those cells to control their internal Ca.

Calcium currents in rat cerebellar purkinje cells maintained in culture

Calcium permeabilities were examined in large cerebellar neurons maintained in culture, and morphologically identified as Purkinje cells. When cells were supplied with a Dulbecco Minimum Eagle's Medium with 10% horse serum added (5–10 days), somatic recordings revealed complex spikes and these were shown to be generated by Na and Ca components, the Na one being tetrodotoxin-sensitive. At the dendritic level, Ca currents were better resolved than at the soma. In dendrites, Ca entry was shown to occur through at least two distinct currents. The first was a low-threshold transient current (elicited above −60 mV from a holding potential of −80 mV) which was reduced by almost 30% by 50μ M cadmium. The second was a high-threshold current (above −20 mV) which gave rise to (1) a transient component exhibiting a steady-state inactivation and so requiring holding potentials at −80 mV, and (2) a sustained component. Both components were suppressed by 50 μm cadmium. We measured a total Ca current at the dendritic level reaching values of up to 1 nA. In another culture medium (Leibovitz medium) known to allow expression of three types of calcium currents in nodose cells we observed the development of the dendritic tree of Purkinje cells but with no simultaneous expression of the high-threshold Ca current.

Omega-conotoxin blockade distinguishes Ca from Na permeable states in neuronal calcium channels.

The blocking properties of the neurotoxic peptide omega-conotoxin GVIA (omega-CgTX), on neuronal Ca channels were investigated. In line with previous reports (Feldman et al. 1987; McCleskey et al. 1987), we found that micromolar concentrations of the toxin block selectively and persistently the high-threshold Ca channels of chick sensory neurons. The block by omega-CgTX could be partially relieved in low [Ca2+]o (less than 1 microM) toxin-free solutions, allowing Na ions to flow through open high-threshold Ca channels. Ca currents through these channels, however, remained permanently blocked on returning to normal Ca2+ toxin-free solutions. Also neurons which were preincubated with omega-CgTX in low Ca2+ (5 mM EGTA) failed to show high-threshold Ca currents during washing with normal Ca2+. Thus, appearance of Na currents through Ca channels in CgTX-pretreated cells was neither a consequence of unbinding of the toxin from its receptor site nor due to an interaction of EGTA with bound omega-CgTX. Na currents in CgTX-pretreated cells could be reversibly suppressed by bath applications of verapamil or by further addition of the toxin. At variance with Ca currents, block of Na currents by omega-CgTX was faster and reversible (KD 0.7 microM). Our data are consistent with the idea that neuronal Ca channels are in different conformational states when permeable to Ca2+ or Na+ ions and that omega-CgTX depresses persistently ion permeation primarily in the Ca-permeable state.