Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells.

Clostridium perfringens epsilon toxin (ET) is a potent pore-forming cytotoxin causing fatal enterotoxemia in livestock. ET accumulates in brain and kidney, particularly in the renal distal-collecting ducts. ET binds and oligomerizes in detergent-resistant membranes (DRMs) microdomains and causes cell death. However, the causal linkage between membrane permeabilization and cell death is not clear. Here, we show that ET binds and forms 220-kDa insoluble complexes in plasma membrane DRMs of renal mpkCCD(cl4) collecting duct cells. Phosphatidylinositol-specific phospholipase C did not impair binding or the formation of ET complexes, suggesting that the receptor for ET is not GPI anchored. ET induced a dose-dependent fall in the transepithelial resistance and potential in confluent cells grown on filters, transiently stimulated Na+ absorption, and induced an inward ionic current and a sustained rise in [Ca2+]i. ET also induced rapid depletion of cellular ATP, and stimulated the AMP-activated protein kinase, a metabolic-sensing Ser/Thr kinase. ET also induced mitochondrial membrane permeabilization and mitochondrial-nuclear translocation of apoptosis-inducing factor, a potent caspase-independent cell death effector. Finally, ET induced cell necrosis characterized by a marked reduction in nucleus size without DNA fragmentation. DRM disruption by methyl-beta-cyclodextrin impaired ET oligomerization, and significantly reduced the influx of Na+ and [Ca2+]i, but did not impair ATP depletion and cell death caused by the toxin. These findings indicate that ET causes rapid necrosis of renal collecting duct cells and establish that ATP depletion-mediated cell death is not strictly correlated with the plasma membrane permeabilization and ion diffusion caused by the toxin.

High sensitivity of mouse neuronal cells to tetanus toxin requires a GPI-anchored protein.

Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis. Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP. Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin.

Identification of living oligodendrocyte developmental stages by fractal analysis of cell morphology.

The Mandelbrot's fractal dimension (D), a measure of shape complexity, has been used to quantify the complex morphology of living cells. Previous studies on glial cells have shown that as cells increase in morphological complexity, their "D" value increases, suggesting that "D" could be used to estimate their stage of differentiation. In the present study the box-counting method was used to calculate the "D" values of rat cerebellar oligodendrocytes during their differentiation in primary culture. These values were correlated with the immunoreactivity of cells to antigenic markers commonly used for assessing their stages of differentiation: A2B5, O4 and anti-galactocerebroside (Gal-C). Our results show that changes of the fractal dimension during differentiation follow the well known pattern of markers expression by these cells. These results demonstrate that A2B5-, O4-, and Gal-C-expressing oligodendrocytes can be confidently estimated from their respective fractal dimension values. Based on this immunocytochemical calibration, the calculation of "D" allows an easy and fast determination of the developmental stage of living (unstained) oligodendrocytes before the study of their physiological characteristics. Using this method we precisely identified living oligodendrocyte progenitors and early pro-oligodendrocytes expressing voltage-activated sodium currents that is a common characteristic of these two immature developmental stages (Sontheimer et al. [1989b] Neuron 2:1135-1145).

Dendro-somatic distribution of calcium-mediated electrogenesis in purkinje cells from rat cerebellar slice cultures.

The role of Ca2+ entry in determining the electrical properties of cerebellar Purkinje cell (PC) dendrites and somata was investigated in cerebellar slice cultures. Immunohistofluorescence demonstrated the presence of at least three distinct types of Ca2+ channel proteins in PCs: the alpha1A subunit (P/Q type Ca2+ channel), the alpha1G subunit (T type) and the alpha1E subunit (R type). In PC dendrites, the response started in 66 % of cases with a slow depolarization (50 +/- 15 ms) triggering one or two fast (approximately 1 ms) action potentials (APs). The slow depolarization was identified as a low-threshold non-P/Q Ca2+ AP initiated, most probably, in the dendrites. In 16 % of cases, this response propagated to the soma to elicit an initial burst of fast APs. Somatic recordings revealed three modes of discharge. In mode 1, PCs display a single or a short burst of fast APs. In contrast, PCs fire repetitively in mode 2 and 3, with a sustained discharge of APs in mode 2, and bursts of APs in mode 3. Removal of external Ca2+ or bath applications of a membrane-permeable Ca2+ chelator abolished repetitive firing. Tetraethylammonium (TEA) prolonged dendritic and somatic fast APs by a depolarizing plateau sensitive to Cd2+ and to omega-conotoxin MVII C or omega-agatoxin TK. Therefore, the role of Ca2+ channels in determining somatic PC firing has been investigated. Cd2+ or P/Q type Ca2+ channel-specific toxins reduced the duration of the discharge and occasionallyinduced the appearance of oscillations in the membrane potential associated with bursts of APs. In summary, we demonstrate that Ca2+ entry through low-voltage gated Ca2+ channels, not yet identified, underlies a dendritic AP rarelyeliciting a somatic burst of APs whereas Ca2+ entry through P/Q type Ca2+ channels allowed a repetitive firing mainly by inducing a Ca2+-dependent hyperpolarization.

G-protein-mediated desensitization of metabotropic glutamatergic and muscarinic responses in CA3 cells in rat hippocampus.

1. Desensitization of a metabotropic response was investigated in CA3 pyramidal neurons in hippocampal slice cultures using the patch-clamp technique. 2. 1S,3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD), an agonist at metabotropic glutamate receptors (mGluRs), and metacholine (MCh), an agonist at muscarinic receptors, induced a cationic current that appears to be activated through a G-protein-independent transduction process, as previously shown. Prolonged or repetitive bath application of agonists led to rapid desensitization of the cationic current with a time constant of approximately 20 s. 3. Complete recovery from desensitization was observed within 6 min. 4. These responses mediated by mGluRs and muscarinic receptors cross-desensitized. 5. Preventing the activation of G-proteins by loading cells with GDP beta S strongly reduced or suppressed desensitization, and resulted in a sustained inward cationic current. When cells were filled with GTP gamma S to irreversibly activate G-proteins, the desensitization process was enhanced such that a first application of agonist caused a markedly reduced response. 6. These results show that a cationic current induced by metabotropic agonists in hippocampal pyramidal cells undergoes apparent desensitization and suggests that this process occurs through a G-protein-mediated inhibition of the underlying membrane conductance.

Low-threshold Ca2+ currents in dendritic recordings from Purkinje cells in rat cerebellar slice cultures.

Voltage-dependent Ca2+ conductances were investigated in Purkinje cells in rat cerebellar slice cultures using the whole-cell and cell-attached configurations of the patch-clamp technique. In the presence of 0.5 mM Ca2+ in the extracellular solution, the inward current activated with a threshold of -55 +/- 1.5 mV and reached a maximal amplitude of 2.3 +/- 0.4 nA at -31 +/- 2 mV. Decay kinetics revealed three distinct components: a fast (24.6 +/- 2 msec time constant), a slow (304 +/- 46 msec time constant), and a nondecaying component. Rundown of the slow and sustained components of the current, or application of antagonists for the P/Q-type Ca2+ channels, allowed isolation of the fast-inactivating Ca2+ current, which had a threshold for activation of -60 mV and reached a maximal amplitude of 0.7 nA at a membrane potential of -33 mV. Both activation and steady-state inactivation of this fast-inactivating Ca2+ current were described with Boltzmann equations, with half-activation and inactivation at -51 mV and -86 mV, respectively. This Ca2+ current was nifedipine-insensitive, but its amplitude was reduced reversibly by bath-application of NiCl2 and amiloride, thus allowing its identification as a T-type Ca2+ current. Channels with a conductance of 7 pS giving rise to a fast T-type ensemble current (insensitive to omega-Aga-IVA) were localized with a high density on the dendritic membrane. Channel activity responsible for the ensemble current sensitive to omega-Aga-IVA was detected with 10 mM Ba2+ as the charge carrier. These channels were distributed with a high density on dendritic membranes and in rare cases were also seen in somatic membrane patches.

Somatic voltage-gated potassium currents of rat hippocampal pyramidal cells in organotypic slice cultures.

1. The dominant voltage-gated K+ currents in the somatic membrane of CA3 pyramidal cells from hippocampal slice cultures were characterized using the cell-attached configuration of the patch-clamp recording method. The kinetics, the voltage dependence of activation and inactivation, and the pharmacological properties of the current were determined from ensemble averages of large numbers of episodes from multichannel patches. 2. Steady-state analysis revealed that this current was half-inactivated at the resting membrane potential (Vr), and fully inactivated when patches were held 40 mV positive to Vr. Inactivation was removed when patches were hyperpolarized by 50 mV from Vr. Inactivation was well described by the Boltzmann equation with a slope factor of 12.6 mV. Removal of inactivation of the peak outward current could be described by a time-dependent monoexponential function with a time constant of the order of 100 ms. In contrast, the time course of inactivation was very slow: a +40 mV depolarization relative to Vr of several seconds was required for complete inactivation of the total outward current. 3. When steady-state inactivation was removed by hyperpolarization, the outward current activated with a threshold 10 mV positive to Vr and was half-activated at a potential 57 mV positive to Vr. The conductance can be described in terms of a single Boltzmann equation with a slope factor of 13.5 mV. Activation and inactivation properties of the somatic conductance produce a small window current between +10 and +20 mV relative to Vr. 4. The outward current activated in a voltage-dependent manner in less than 10 ms with 500 ms depolarizing steps. A kinetic analysis of its decay revealed at least three components, with the following time constants: a fast (17 ms), a slowly (approximately 150 ms), and a very slowly inactivating component (in the range of seconds). 5. External application of 4-aminopyridine (4-AP) induced a dose-dependent block of the peak outward current with an IC50 of 28 microM. The inhibitory effect of 4-AP saturated at a concentration of 200 microM which blocked 80% of the total current. The slowly and very slowly inactivating components of the current were not observed with 20 mM tetraethylammonium (TEA) in the pipette solution. A fast transient ensemble current (mean decay time constant, 24 ms) persisted in the presence of extracellular TEA in 29% of the patches. 6. In summary, at least two distinct voltage-gated K+ currents were present at the somatic level of hippocampal pyramidal cells. The dominant one, which we named IK(AT), is sensitive to micromolar concentrations of 4-AP and millimolar concentrations of TEA, and contributes three kinetic components to the total outward current. The second is TEA insensitive, and contributes only a fast transient component of outward current probably corresponding to the classic A-type K+ current. Intracellular recordings in CA3 pyramidal cells showed that IK(AT) plays an important role in regulating the duration of the action potential.

Distinct modes of channel gating underlie inactivation of somatic K+ current in rat hippocampal pyramidal cells in vitro.

1. We have used the cell-attached configuration of the patch-clamp recording method to characterize the biophysical properties of the voltage-gated K+ channel underlying a 4-aminopyridine (4-AP)- and tetraethylammonium (TEA)-sensitive K+ current (IK(AT)) in pyramidal cells of hippocampal slice cultures. 2. The unitary conductance of channels carrying IK(AT) current (KAT channels) was 19.1 +/- 5.1 pS with a physiological K+ gradient (2.7 mM external K+) and 39.0 +/- 3.6 pS with high external K+ (140 mM). The reversal potential changed with the external K+ concentration as expected for a channel with a dominant K+ selectivity. Channel activity was blocked under both conditions by either external application of 4-AP at 100 microM or by including 20 mM TEA in the pipette solution. 3. An analysis of kinetic behaviour showed that open times were distributed as a single exponential. The mean open time (+/- S.D.) was 4.4 +/- 1.4 ms at a voltage 30 mV positive to resting potential and increased with further depolarization to reach a value of 16.2 +/- 7.4 ms at 70 mV positive to the resting potential. At this depolarized potential, we observed bursts of channel openings with a mean burst duration around 100 ms. 4. With repeated depolarizing pulses, response failures of the KAT channel occurred in a non-random manner and were grouped (referred to as mode 0). This mode was associated with a voltage-dependent inactivation process of the channel and was favoured when the opening probability of the channel was reduced by increasing steady-state inactivation or by bath application of 4-AP. This is consistent with the localization of the binding site for 4-AP at or near the inactivation gate of the channel. 5. When KAT channel openings were elicited by 500 ms depolarizing steps, activity was either transient or it persisted throughout the duration of the pulse. These two modes of activity alternated in a random manner or occurred in groups giving rise to transient (time constant, 20-100 ms) or sustained ensemble currents. In the presence of low concentrations of 4-AP (20-40 microM), the transient pattern of activity was more frequently observed. 6. In addition to mode 0, we propose the existence of at least two further gating modes for KAT channels: mode T (transient current) and mode S (sustained current) that underlie the three decaying components of the IK(AT) ensemble current. These gating modes are probably under the control of intracellular factors that remain to be identified.

Voltage-activated ionic currents in differentiating rat cerebellar granule neurons cultured from the external germinal layer.

The electrical properties of the precursor cells of the external germinal layer of rat cerebellum were assessed during their differentiation in control medium (Dulbecco's modified Eagle's medium) supplemented or not with either basic fibroblast growth factor (bFGF) or 25 mM potassium chloride (KCl). Resting potential was shown to be -10 mV in all three conditions 3 hours after plating [days in vitro (DIV)0]. By DIV 5, it reached -63 mV for cells cultured in 25 mM KCl but only -28 mV in control and bFGF media. The main voltage-sensitive ionic current measured at DIV 0 under all conditions was a composite IK consisting in a sustained K+ current blocked by tetraethylammonium (IK(TEA)), plus a rapidly activating and inactivating TEA-insensitive IK(A). Both currents increased with time in all conditions, but after 5 days IK(A) became dominant in terms of density. IK(TEA) is likely an IK(Ca), since it was blocked by 67% in 1 mM TEA. On DIV 0, INa and ICa were absent or small in amplitude. By DIV 3, 80% of the cells had currents able to generate a spike. Interestingly, ICa mean amplitude and current density measured at -10 mV in control condition on DIV 1 was significantly larger than those recorded in bFGF and 25 mM KCl. The order of appearance of the ionic currents, IK, ICa, and INa, leads directly to fast spike activity allowing for poor calcium entry. Firing rate likely depends on IK(A), which increased during the first 6 days of development but could be differentially regulated by bFGF.

ADP exerts a protective effect against rundown of the Ca2+ current in bovine chromaffin cells.

In isolated chromaffin cells, the high-voltage-activated Ca2+ current, recorded using 5 mM Ca2+ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5'-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5'-triphosphate (ITP) could not replace ATP, whereas guanosine 5'-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl2 and the liberation of a phosphate group since the ATP analogue 5'-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5'-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg2+ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5'-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca2+ concentration (pCa 6 instead of pCa 7.7, where pCa = -log10[Ca2+]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca2+ channels when going to the inside-out patch recording configuration.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP and G proteins affect the runup of the Ca2+ current in bovine chromaffin cells.

The Ca2+ current recorded by the whole-cell technique in chromaffin cells shows, before the often described rundown, a transient facilitation or runup. Initial current amplitude was 570 +/- 165 pA and then it increased by 49 +/- 23% (n = 19, SD) over 2 +/- 1 min in the absence of adenosine 5'-triphosphate (ATP). In the presence of ATP, this process occurred with the same magnitude but it was slowed in a dose-dependent manner, lasting 17 +/- 2 min with 2 mM ATP (n = 8). Since adenosine 5'-diphosphate (ADP) does not reproduce this ATP effect, a complex series of phosphorylations is likely to intervene and we show that, at least, a cAMP-dependent i.e., cyclic adenosine monophosphate) phosphorylation occurs. Pertussis toxin (PTX) pretreatment yielded an already maximal Ca2+ current (around 1000 pA) at the time of the patch rupture, which only slightly increased thereafter (10%, n = 11). Also, guanosine 5'-diphosphate (GDP) and guanosine 5'-O-(2-thiodiphosphate) (GDP[ beta s]), induced a fast runup, which was absent in the presence of GTP. Furthermore, we show that facilitation does not occur in the presence of dihydrophyridine (DHP) antagonists. Globally, our data suggest that an ATP-dependent phosphorylation stabilizes the inhibitory control exerted by a PTX-sensitive G protein and, as a result, slows down the facilitation of L-type Ca2+ channels. The recruitment of L-type channels can also be facilitated by the application of a DHP agonist or a depolarizing prepulse protocol.l We show that these processes are only effective over a period which parallels the runup and are not additive to it.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of a nonselective cationic conductance by metabotropic glutamatergic and muscarinic agonists in CA3 pyramidal neurons of the rat hippocampus.

We have characterized a cationic membrane conductance activated by metabotropic glutamatergic and muscarinic cholinergic agonists in CA3 neurons in hippocampal slice cultures using the patch-clamp technique. When the potassium concentration in the superfusing fluid was raised above 5 mM, a biphasic current was observed in cells held at -60 mV in response to stimulation of postsynaptic metabotropic glutamate receptors (mGluRs) with 1S,3R-ACPD (50 microM) or muscarinic receptors with methacholine (MCh, 5 microM). The initial inward component was due to an increase in a cationic membrane conductance as determined by its reversal potential and its sensitivity to changes in extracellular K+ or Na+. The conductance underlying this current displayed no apparent voltage sensitivity over the range -120 to -50 mV. The response was reduced by extracellular application of Ba2+, Cd2+, Mg2+, or TEA, whereas extracellular Cs+ or loading cells with BAPTA or Cs+ did not affect the current. The effects of 1S,3R-ACPD were reversibly inhibited by bath-applied MCPG, an antagonist at mGluRs. Experiments with atropine and pirenzepine indicated that non-M1 muscarinic receptors mediated the MCh-induced current. A decrease in a resting leak potassium conductance (IK,leak) was responsible for the late component of the 1S,3R-ACPD- and MCh-induced response, seen as an outward current in the bathing solution with high K+ concentration. Loading cells with GDP beta S, GTP gamma S, or GTP did not alter the cationic current, while, in the same cells, the reduction in IKleak was abolished or irreversibly activated. Single-channel recordings of cationic channel activity in the cell-attached configuration provided evidence for the requirement of second messengers in coupling these receptors to the cationic channels. The data indicate that in addition to the previously described reduction of IK,leak, IM, and IAHP, both 1S,3R-ACPD and MCh activate a nonselective cationic conductance that is clearly revealed upon elevating external K+ concentration. This current is mediated by activation of metabotropic receptors, although no evidence could be obtained to show an involvement of G-proteins.

Voltage-gated ionic currents in mature oligodendrocytes isolated from rat cerebellum.

Mature rat cerebellar oligodendrocytes were isolated in culture using a serum free medium and identified using anti-galactocerebroside (GalC) and OL-1 antibodies. The morphology of such oligodendrocytes changes with time in culture from a multipolar shape at about 4 days in vitro (DIV) to a monopolar shape at about 8 DIV, a transition that has been previously described only in situ. Voltage-gated ionic currents were characterized at both oligodendrocyte developmental stages using the whole cell configuration of the patch-clamp technique. No differences between these two stages were detected, both types expressed a large K+ inward rectifying current similar to that described for oligodendrocytes from other vertebrate neuronal structures. This current could play an important role in the control of oligodendrocyte resting membrane potential. Our culture system provides a valuable model, close to the situation encountered in situ, not only to study the process of oligodendrocyte maturation, but also to identify the possible interactions between oligodendrocytes and cerebellar neurons such as granular and Purkinje cells during development.

Facilitatory coupling between a glutamate metabotropic receptor and dihydropyridine-sensitive calcium channels in cultured cerebellar granule cells.

The effect of metabotropic glutamate receptor activation on Ca dihydropyridine (DHP)-sensitive channels recorded in the presence of 1 microM Bay K 8644 was examined on cultured cerebellar granule cells using the patch-clamp technique in the cell-attached configuration. Bath-applied agonist (trans-ACPD, 1S,3R-, and 1R,3S-ACPD isomers, and glutamate or quisqualate in the presence of CPP and CNQX) evoked an increase in Ca channel activity with a variable latency of 8.9 +/- 8.6 sec in 40% of the recorded cells. Neither L-CCG1, L-AP3, L-AP4, nor AMPA or NMDA activated Ca channels. Two dihydropyridine-sensitive channels present in this cell type were activated by trans-ACPD: the classical 24 pS L-type channel and a smaller-conductance 7 pS channel. The effect was shown to be mediated by neither intracellular Ca2+ nor a pertussis toxin (PTX)-sensitive G protein. Interestingly treatment with BAPTA-AM increased the number of responding patches and the activity was more sustained throughout the drug application. After overnight PTX treatment, activation of the Ca channels persisted even after washout of the agonist. These results indicate that mGluR1/mGluR5 probably mediate the facilitation of dihydropyridine-sensitive Ca channels.

Inhibitory effects of dihydropyridines on macroscopic K+ currents and on the large-conductance Ca(2+)-activated K+ channel in cultured cerebellar granule cells.

In cultured cerebellar granule cells, we examined the effects of dihydropyridines (DHPs) on K+ currents, using the whole-cell recording configuration of the patch-clamp technique and on Ca(2+)-activated K+ channels ("maxi K+ channels") using outside-out patches. We found that micromolar concentrations of nicardipine, nifedipine, (+) and (-) BAY K 8644, nitrendipine, nisoldipine and (-) nimodipine block 10-60% of macroscopic K+ currents. The most potent of these DHPs was nicardipine and the least potent, (-) BAY K 8644. (+) Nimodipine had no effect on this current. The inhibitory effects of nifedipine and nicardipine were not additive with those of 1 mM tetraethylammonium (TEA). Outside-out recordings of "maxi K+ channels" showed a main conductance of 200 pS (in 77% of the patches) and two subconductance states (in 23% of the patches). Neither nifedipine nor nicardipine affected the main conductance, but decreased the values of the subconductance levels. In 10% of these patches, nicardipine induced a flickering activity of the channel. These findings show that both Ca2+ and K+ channels have DHP-sensitive sites, suggesting similarity in electrostatic binding properties of these channels. Furthermore, cerebellar granule cells may express different subtypes of "maxi K+ channels" having different sensitivities to DHPs. These drugs may provide new tools for the molecular study of K+ channels.

Evolution of the Ca2+ current during dialysis of isolated bovine chromaffin cells: effect of internal calcium.

We have examined the internal Ca(2+)-dependence of the long-term evolution of whole cell high voltage activated Ca current in chromaffin cells. The evolution of the peak Ca current was characterized by 2 distinct phases: after an initial facilitation, there followed a rundown, which represented a reduction by 70% within some 10 min. The rundown process was shown not to depend on Ca2+ entry nor on membrane depolarization. It resulted from cell dialysis with a saline solution and, once initiated, it proceeded at a rate of 0.28 min-1 at 4 different Ca2+ concentrations (pCa 5-9). The facilitation is also initiated by cell dialysis but this process developed faster at higher internal Ca2+ concentrations. Thus, globally, high-voltage activated Ca2+ current runs down faster when using a recording pipette solution with a higher internal Ca2+ concentration (pCa 5 or 6). Some leupeptin-sensitive proteases may be involved in the initiation of facilitation and rundown processes.

Characteristics of calcium channels responsible for voltage-activated calcium entry in rat cerebellar granule cells.

The properties and characteristics of calcium channel openings in cerebellar granule cells were analysed by the cell-attached patch-clamp technique. At depolarized potentials, with 110 mM Ba2+ as the divalent charge carrier, 36% of the patches displayed activity that consisted of elementary events whose amplitude ranged from -0.3 to -1.75 pA at 0 mV, giving rise to a high threshold current. In this population of events at least four different types of channel openings were identified by their distinct biophysical and pharmacological properties. Two types of channel openings, with conductances around 24 and 7 pS, had similar characteristics in that both opened following two modes of gating characterized by brief (approximately 2 ms) and longer openings (approximately 8 ms) and both were sensitive to dihydropyridines. A further type of channel opening, with a conductance around 11 pS gated mainly with brief openings (approximately 1 ms), was shown to be insensitive to dihydropyridines but was undetectable in recordings from the cells that had been treated with omega-conotoxin. The last type of event was revealed after treatment of the cell with nicardipine or nifedipine and omega-conotoxin. The corresponding channel had a conductance of 19 pS and opened in one dominant mode characterized by brief openings (approximately 1 ms). The data obtained on single-channel activity of cerebellar granule cells are compared with the properties of the total current recorded in whole-cell conditions.

Voltage-dependent calcium entry in confluent bovine capillary endothelial cells.

Confluent bovine capillary endothelial cells display, when examined for voltage-dependent calcium entries using cell-attached channel recordings, two types of Ca2+ channels (4 and 23.5 pS in 110 mM Ba2+) both sensitive to the dihydropyridine Ca agonist BAY K 8644. In contrast to isolated cells, confluent cells display no T-type, low threshold activity, and Ca currents were typically only elicited at very depolarized potentials. In these cells, voltage-dependent calcium entries will only be made operative by substances able to shift their activation towards the resting potential.

Voltage-gated Ca entry in isolated bovine capillary endothelial cells: evidence of a new type of BAY K 8644-sensitive channel.

Isolated bovine capillary endothelial cells have been examined for voltage-dependent Ca entry. All cells displayed a low threshold activity, with the main characteristics of a T-type transient current, when examined using whole-cell recording for activation and inactivation and cell-attached conditions or inside-out patches for the elementary conductance (8 pS). 25% of the cells displayed an additional sustained current in 5 mM CaCl2 above -40 mV, which was enhanced by application of BAY K 8644, but almost insensitive to superfusion with nicardipine. Two types of channels (2.8 and 21 pS, in 110 mM BaCl2) were shown to have a BAY K 8644 sensitivity. The large conductance channels were L-type channels. The smaller events were elicited at more hyperpolarized potentials (by some 30 mV). Their mean open time was 16 ms in control conditions. In presence of BAY K 8644, additional long open times were observed (up to 100 ms as compared to 7.8 ms for the time constants of the slow mode of the L-type channel). We refer to these channels as SB channels: of small conductance and sensitive to BAY K 8644. In the presence of nicardipine, SB channels are not noticeably modified, in contrast to the L-type openings which are abolished. Also, SB open times are close to control values when nicardipine is added after a BAY K 8644 application. We suggest that, at physiological concentrations of divalent ions, an SB-type activity is elicited above -40 mV which generates the low threshold sustained current.