Low-threshold Calcium Current Research Articles

In vivo, in vitro, and computational analysis of dendritic calcium currents in thalamic reticular neurons

Thalamic reticular (RE) neurons are involved in the genesis of synchronized thalamocortical oscillations, which depend in part on their complex bursting properties. We have investigated the intrinsic properties of RE cells using computational models based on morphological and electrophysiological data. Simulations of a reconstructed RE cells were compared directly with recordings from the same cell to obtain precise values for the passive parameters. In a first series of experiments, the low-threshold calcium current (I(Ts)) was studied via voltage clamp in acutely dissociated RE cells that lack most of their dendrites. Simulations based on a cell with truncated dendrites and Hodgkin-Huxley kinetics reproduced these recordings with a relatively low density of I(Ts). In a second series of experiments, voltage-clamp recordings obtained in intact RE cells in slices showed a higher amplitude and slower kinetics of I(Ts). These properties could be reproduced from the reconstructed cell model assuming higher densities of I(Ts) in distal dendrites. In a third series of experiments, current-clamp recordings were obtained on RE cells in vivo. The marked differences with in vitro recordings could be reconciled by simulating synaptic bombardment in the dendrites of RE cells, but only if they contained high distal densities of I(Ts). In addition, simpler models with as few as three compartments could reproduce the same behavior assuming dendritic I(Ts). These models and experiments show how intrinsic bursting properties of RE cells, as recorded in vivo and in vitro, may be explained by dendritic calcium currents.

Chapter 3 Neurochemical microcircuitry underlying visual and oculomotor function in the cat superior colliculus

The cat superior colliculus (SC) plays an important role in visual and oculomotor functions, including the initiation of saccadic eye movements. We have studied the organization of neurochemical specific circuits in SC that underly these functions. In this chapter we have reviewed three microcircuits that can be identified by cell type, chemical content, and synaptic input from specific afferents. The first is located within the upper sgl and is related to the W retinal pathway to this region of SC. This circuit includes relay and interneurons that contain the calcium binding protein calbindin (CB), GABA containing presynaptic dendrites, and retinal terminals that have a distribution and size typical of W retinal terminals in the cat SC. This circuit is a typical synaptic triad that mediates feedforward inhibition, possibly to regulate outflow of the W pathway to the lateral geniculate nucleus. CB neurons in SC and other structures may be uniquely related to low threshold calcium currents in these neurons. The second microcircuit consists of neurons that contain parvalbumin (PV), another calcium binding protein. These neurons are located in a dense tier with the deep sgl and upper ol and they receive input from retinal terminals that are likely from 'Y' retinal ganglion cells. Some of these neurons also project to the lateral posterior nucleus and some colocalize glutamate. We speculate that these neurons also receive cortical 'Y' input although we have yet to prove this experimentally. The role of PV in these cells is unknown, but PV has been shown to be contained in fast spiking, non-accomodating neurons in visual cortex which have very rapid spike discharges that are also characteristic of SC neurons innervated by 'Y' input. The third microcircuit consists of a group of clustered neurons within the igl of the cat SC that overlaps the patch-like innervation of afferents to this region that come from the pedunculopontine tegmental and lateral dorsal tegmental nuclie, the substantia nigra, and the cortical frontal eye fields. These clustered neurons project through the tectopontobulbar pathway and terminate within the cuneiform region (CFR) of the midbrain tegmentum. They transiently express NOS during development. Ongoing studies in our laboratory suggest that these cells receive synaptic inputs directly from the PPTN and SN and may represent functional modules involved in the initiation of saccadic eye movements.

Glutamate metabotropic receptor inhibition of voltage-gated calcium currents in visceral sensory neurons

1. Metabotropic glutamate receptors (mGluRs) have been suggested to modulate neurotransmission of glutamatergic pathways via autoreceptive action. Visceral sensory afferents and baroreceptor afferents in particular are thought to utilize L-glutamate (L-glu) as a primary neurotransmitter. The purpose of this study was to investigate whether visceral sensory afferents possess a mGluR and determine the effect of mGluR activation on voltage-gated calcium currents in these neurons. 2. Activation of mGluRs by the selective agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) reversibly suppressed the voltage-gated calcium currents in visceral sensory afferents of the nodose ganglion. Concentrations of t-ACPD ranging from 50 to 1,000 microM consistently decreased the evoked calcium current with a maximum suppression of the peak current of 25-30%. This response was repeatable and reversible within a given cell. 3. Metabotropic GluR activation selectively decreased the high-threshold calcium current evoked from step potentials greater than -30 mV and had no effect on the low-threshold calcium current. The inhibitory effects of t-ACPD on the high-threshold channel was partially blocked by omega-conotoxin (omega-CTx-GVIA) suggesting that at least part of the effects of mGluR inhibition of the voltage-gated calcium current is because of a modulation of the omega-CTx-GVIA sensitive high-threshold current. 4. Finally, the inhibitory effects of quisqualate (quis) on the high-threshold calcium current were blocked by pretreatment of the neurons with pertussis toxin (PTX). These results suggest that visceral sensory afferents do possess a PTX-sensitive mGluR and activation of this receptor results in the inhibition of a omega-CTx-GVIA sensitive high-threshold calcium channel.

Depression of high-threshold calcium currents by activation of human D2 (short) dopamine receptors expressed in differentiated NG108-15 cells.

1. This study examined the regulation of calcium currents in differentiated NG108-15 cells that had been stably transfected with cDNA encoding the short isoform of the human D2 dopamine receptor. Whole cell calcium currents were recorded by nystatin-perforated patch clamp recording. 2. Transient low-threshold calcium currents elicited by depolarizations from -100 mV to -20 mV were reversibly depressed by NiCl2 (84 +/- 8% at 30 microM; n = 3) and by omega-agatoxin IVA (15 +/- 5%; 100 nM, n = 7). These currents were unaffected by hD2 receptor activation. 3. High-threshold calcium currents elicited by depolarizations from -80 mV to 0 mV were partly blocked by omega-conotoxin GVIA (67 +/- 6% at 100 nM, n = 4) and by the subsequent addition of the dihydropyridine, nisoldipine (94 +/- 3% at 1 microM). Consistent with the presence of at least two distinct types of high-threshold calcium channels, nisoldipine alone (38 +/- 15% at 1 microM, n = 6) did not preclude the inhibition caused by omega-conotoxin GVIA (69 +/- 13% at 100 nM, n = 4). The residual current was completely blocked by 100 microM CdCl2 (98.8 +/- 0.4%, n = 7). 4. In hD2-transfected cells, but not untransfected cells, high-threshold currents were depressed by quinpirole (30 +/- 4% at 100 nM; n = 15) with a pEC50 of 8.61 +/- 0.22 (n = 5), as well as by (-)-noradrenaline (28 +/- 5% at 1 microM, n = 9). Responses to both agonists were selectively antagonized by S-(-)sulpiride (100 nM) but not by the alpha-adrenoceptor antagonist, phentolamine (1O microM). The depression caused by (-)-noradrenaline was positively correlated with that of quinpirole for each cell(r2 = 0.91, slope = 0.99).5. hD2-receptor-mediated inhibition of high-threshold calcium currents was abolished by pretreatment of cells with omega-conotoxin GVIA (100 nM; n = 4). However, a component of the high-threshold current was reversibly depressed by omega-conotoxin GVIA (67% to 45% depression after 10 min wash). This current was also depressed by hD2 receptor activation (59 +/- 9% depression in 100 nM quinpirole, n = 3),and was completely blocked by nisoldipine (95 +/- 2% at 1 MicroM).6. These data demonstrate that activation of hD2(short) dopamine receptors can regulate both wconotoxinGVIA, and dihydropyridine-sensitive high-threshold calcium currents in neuroblastoma cells.Morever, the ability of human D2 dopamine receptors to regulate more than one type of calcium current supports the notion that these receptors have a diverse functional role in the central nervous system.

Low-threshold calcium current and resonance in thalamic neurons: a model of frequency preference

1. We constructed a mathematical model of the subthreshold electrical behavior of neurons in the nucleus mediodorsalis thalami (MDT) to elucidate the basis of a Ni(2+)-sensitive low-frequency (2-4 Hz) resonance found previously in these neurons. 2. A model that included the low- and high-threshold Ca2+ currents (IT and IL), a Ca(2+)-activated K+ current (IC), a rapidly inactivating K+ current (IA), a voltage-dependent K+ current which we call IKx, and a voltage-independent leak current (Il), successfully simulated the low-threshold spike observed in MDT neurons. This model (the MDT model) and a minimal version of the model containing only IT and I1 (the minimal MDT model) were used in the analysis. 3. An impedance function was derived for a linearized version of the MDT model. This showed that the model predicts a low-frequency (2-4 Hz) resonance in the voltage response to "small" oscillatory current inputs (producing voltage changes of < 10 mV) when the membrane potential is between -60 and -85 mV. 4. Further examination of the impedances for the MDT and minimal MDT models shows that IT underlies the frequency- and voltage-dependent resonance. The slow inactivation of IT results in an attenuation of voltage responses to low frequencies, resulting in a band-pass behavior. The fast activation of IT amplifies the resonance and modulates the peak frequency but does not, in itself, cause resonance. 5. When voltage responses are small (< 10 mV), the strength and voltage-dependence of resonance of the minimal MDT model are determined by the steady-state window conductance, gw, due to IT. This steady-state conductance arises where the steady-state activation, m(infinity2)(V), and inactivation, h(infinity) (V), curves overlap. Parallel shifts in the inactivation curve can eliminate or enhance resonance with little effect on the IT-dependent low-threshold spike evoked after hyperpolarizing current pulses. When the peak magnitude of gw was large, the minimal MDT model showed spontaneous oscillations at 3 Hz with amplitudes > 30 mV. 6. Large oscillatory current inputs evoked significantly nonlinear voltage responses in the minimal MDT model, but the 2- to 4-Hz frequency selectivity (predicted from the linearized impedance) remained. 7. We conclude that the properties of the low-threshold Ca2+ current, IT, are sufficient to explain the Ni(2+)-sensitive 2- to 4-Hz resonance seen in MDT neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential developmental fates of the two calcium currents in early embryos of the ascidian Ciona intestinalis.

Two voltage-dependent calcium currents have been described in unfertilized eggs of the ascidian Ciona intestinalis: a low threshold, slowly activating current, and a high threshold fast one. According to the classical criteria for classification of calcium currents, they both share some of the features of L-like and T-like currents. We have studied these two calcium currents further by measuring their sensitivity to permeant ions, temperature and inhibitors. Both currents were sensitive to relatively high concentrations of nitrendipine, which was a selective blocker of the low threshold channel. The lanthanide ion gadolinium was a potent blocker of the low threshold current, and cadmium preferentially inhibited the high threshold current. The two calcium currents were regulated in a different manner after fertilization. The density of the high threshold current remained relatively constant, while the low threshold current was lost by the time of first cleavage. This loss following fertilization is similar to the loss of a low threshold sodium current in fertilized eggs of the ascidian Boltenia villosa. Block of the cell cycle with various compounds did not prevent loss of the low threshold calcium current. This observation adds weight to the hypothesis that a loss of excitability is a general property of early development. We conclude that fertilization can differentially modulate channel populations before first cleavage. The mechanism by which this occurs in the ascidian embryo has yet to be discovered.

Low-threshold calcium current in dendrites of the adult rat hippocampus

The patch-clamp technique was employed in hippocampal slices to examine the characteristics of a low-threshold Ca current in adult CA1 pyramidal neurons. We found that adult CA1 pyramidal neurons possess a distinct transient low-threshold Ca current, located predominantly in the distal dendrites. Surgical cuts that separated the dendrites from the soma and left a dendritic length of 150 μm, completely abolished the low-threshold Ca current while a transient K current persisted even in cells with dendrites as short as 50 μm. The transient low-threshold Ca current in dendrites may represent a voltage-dependent Ca entry pathway which contributes to the regulation of cellular excitability, plasticity and pathology.

Antiepileptic drug mechanisms of action.

Clinically used antiepileptic drugs (AEDs) decrease membrane excitability by interacting with ion channels or neurotransmitter receptors. Currently available AEDs appear to act on sodium channels, GABAA receptors, or calcium channels. Phenytoin, carbamazepine, and possibly valproate (VPA) decrease high-frequency repetitive firing of action potentials by enhancing sodium channel inactivation. Benzodiazepines and barbiturates enhance GABAA receptor-mediated inhibition. Ethosuximide and possibly VPA reduce a low-threshold calcium current. The mechanisms of action of AEDs currently under development are less clear. Lamotrigine may decrease sustained high-frequency repetitive firing. The mechanisms of action of felbamate are unknown. Gabapentin (GBP) appears to bind to a specific binding site in the central nervous system with a restricted regional distribution, but the identity of the binding site and the mechanism of action of GBP remain uncertain.

Voltage dependence and activation kinetics of pharmacologically defined components of the high-threshold calcium current in rat neocortical neurons.

1. As a first step toward identification of the functional significance of the spatial distribution of calcium channels we examined the high voltage-activated calcium current (HVA current) in acutely isolated pyramidal neurons from rat sensorimotor cortex using whole-cell voltage clamp. The goals of this study were (1) to determine whether the pharmacologically separable components of the HVA current differed in voltage dependence or activation kinetics and (2) to develop an empirical model that could predict the HVA current evoked by action potentials or other physiological responses. 2. Cells with short dendrites were chosen for study. Input resistance averaged 3.5 +/- 0.4 (SE) G omega. Specific membrane resistance averaged 51.9 +/- 6.8 K omega-cm2 on the basis of estimated membrane surface area. 3. Using 2 mM calcium in the extracellular solution, we evoked the HVA current by depolarizations positive to -45 mV from a holding potential of -60 mV, a potential where the low-threshold calcium current is fully inactivated. Maximum HVA current amplitude (484.9 +/- 42.3 pA) occurred near -15 mV. The evoked current was completely and reversibly blocked by 200 microM cadmium. 4. Tail current amplitude at a fixed potential increased as a sigmoidal function of prepulse potential. A plot of normalized tail current amplitude, taken as the fraction of HVA channels open at each prepulse potential, was best described by a Boltzmann function (maximum slope: e-fold per 11.3 mV; half activation: -24.6 mV) raised to the power of 2. This relation was not altered by extracellular application of 5 microM nifedipine or 10 microM omega-conotoxin, each of which reduced a separate component of the HVA current uniformly at all potentials. We conclude that the pharmacologically separable components of the HVA current do not differ significantly in voltage dependence. 5. The time course of current onset during a step depolarization was best described by second-order activation kinetics. Activation time constants ranged from a maximum of 1.2 ms at -40 mV to 0.3 ms at +25 mV. Neither activation nor tail current time constants were altered by extracellular application of 5 microM nifedipine or 10 microM omega-conotoxin. After application of 1 microM Bay K 8644 tail current decay was best described by a fast time constant similar to control values and a slow time constant. We conclude that the pharmacologically separable components of the HVA current in these neurons do not differ significantly in kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)

Inward rectifier and low-threshold calcium currents contribute to the spontaneous firing mechanism in neurons of the rat suprachiasmatic nucleus

Intracellular and voltage-clamp studies were carried out to clarify the mechanism for spontaneous firing activity in neurons of the suprachiasmatic nucleus (SCN) of rat hypothalamic brain slices in vitro. SCN neurons displayed spontaneously firing action potentials that were preceded by a depolarizing pre-potential and followed by a short spike after-hyperpolarization (AHP). Injection of inward current with a duration longer than 50 ms resulted in a depolarizing voltage "sag" on hyperpolarizing electrotonic potentials. The inward rectification was depressed by bath application of caesium (1 mM) but not by barium (500 microM). SCN neurons also showed a rebound depolarization associated with spike discharge (anodal break) in response to relaxation of hyper polarizing current injection. The rebound depolarization was reduced by nominally zero calcium. Cadmium (500 microM), cobalt (1 mM) or caesium (1 mM) but not nicardipine also depressed the rebound depolarization. Under voltage-clamp conditions, hyperpolarizing steps to membrane potentials negative to approximately -60 mV caused an inward rectifier current, probably H current (IH), which showed no inactivation with time. Bath application of caesium (1-2 mM) suppressed IH. Caesium (2 mM) depressed the slope of the depolarizing spike pre-potential, resulting in a prolongation of the interspike interval of tonic firing neurons. We conclude that both the inward rectifier current, IH, and the low-threshold calcium current contribute to the spike prepotential of spontaneous action potentials in firing neurons of the rat SCN.

Thalamic low threshold calcium current in a genetic model of absence epilepsy.

The low threshold calcium current (IT) in thalamo-cortical neurones contributes to the generation of spike and wave discharges (SWDs) characteristic of generalized, non-convulsive absence epilepsy. The biophysical properties of this current were analysed in dorsal lateral geniculate neurones from the Genetic Absence Epilepsy Rats from Strasbourg (GAERS+). No difference was found in the voltage dependence and kinetics of IT between GAERS+ and rats of the non epileptic control strain (GAERS-). Thus, a dysfunction of IT does not appear to underlie the occurrence of SWDs in absence epilepsy.

Transient enhancement of low-threshold calcium current in thalamic relay neurons after corticectomy

1. The alterations of voltage-sensitive calcium currents produced in thalamic cells by injury were investigated under voltage clamp using patch-clamp recordings in the whole-cell configuration. 2. One day after unilateral cortical ablation in immature rats (postnatal day 7), low-threshold transient calcium (T) currents in acutely isolated thalamic relay neurons (RNs) were increased by 68% compared with contralateral controls (P < 0.001). Three days after the operation, T currents in injured neurons were at 44% of control levels (P < 0.001). On the other hand, high-threshold (L) calcium currents in RNs did not change over the same interval. 3. To investigate the mechanism for the increase of T current, both kinetics and voltage dependency of activation and inactivation were examined. At a test voltage of -40 mV, the activation time constant decreased from 4.1 to 3.2 ms (P < 0.05); however, this small change was insufficient to explain the large increase in T current. Time constants for both fast and slow inactivation did not change significantly, nor did voltage dependence of activation or inactivation of thalamic T currents. 4. Methyl-phenyl-succinimide (MPS, 1 mM), a compound known to block T currents, was used to examine possible alterations in the pharmacological properties of T channels after injury. MPS was more effective in reducing T currents in normal versus injured RNs (24 and 20% reductions, respectively; P < 0.05), suggesting that pharmacological properties of T channels in the injured RNs may be different from those of the normal RNs.(ABSTRACT TRUNCATED AT 250 WORDS)

A variable-threshold motoneuron model that incorporates time- and voltage-dependent potassium and calcium conductances.

1. A "threshold-crossing" motoneuron model was developed to relate recently described biophysical features of cat alpha-motoneurons to motoneuron discharge behavior. This model incorporated three features not included in precedent models: 1) a low-threshold, persistent calcium current; 2) realistic voltage dependencies of the major ionic conductances; and 3) a variable spike threshold. The effects of these additional biophysical features on model behavior were investigated by successively adding them to a fixed threshold model with a single potassium conductance. 2. Fixed-threshold models with either one or two potassium conductances could not produce appropriate discharge behavior. Steady-state frequency-current (F-I) relations were characterized by a continuously increasing slope, unlike the piecewise linear relations observed in real motoneurons. These models also produced unrealistically high discharge rates at the highest levels of "injected" current. 3. The addition of a variable spike threshold, which was made to increase linearly with the magnitude of injected current, could limit maximum discharge rates to more realistic levels. However, steady-state F-I relations still did not exhibit the appropriate shape. 4. The incorporation of a low-threshold calcium current led to a good quantitative agreement between the steady-state F-I relations produced by the model and those obtained in real motoneurons. In addition, the steady-state relation between total membrane current and membrane voltage (I-V relation) of the model was very similar to those measured in real motoneurons. The model's I-V and F-I relations were both very sensitive to the exact form of the steady-state relation between the magnitude of the calcium conductance and membrane voltage. 5. Additional modifications, which included a second calcium conductance and a factor relating spike threshold to membrane voltage, helped to produce more realistic afterhyperpolarizations and first-interval F-I relations. 6. Bistable discharge behavior could be produced by reducing the slow potassium conductance and increasing the time constants governing the activation and deactivation of the low-threshold calcium conductance. 7. The final model thus reproduces a wide range of motoneuron behaviors including subthreshold rectification, piecewise linear first interval and steady-state F-I relations, and, with appropriate modifications, bistable discharge behavior. Nonetheless, by simplifying the representation of fast spike conductances as well as the kinetics of the other ionic conductances, the model remains simple enough to be incorporated into a larger neural network.

Calcium currents in aged rat dorsal root ganglion neurones.

1. The whole-cell voltage clamp technique was used to record calcium currents in the somatic membrane of rat cultured dorsal root ganglion neurones. 2. Neurones were enzymatically isolated from animals of three age groups (neonatal, 2-7 days; adult, 7 months; and old, 30 months) and maintained in primary culture 3-14 days. 3. The neurones isolated from neonatal and old rats showed two distinct types of Ca2+ currents, a low-threshold transient current and a high-threshold sustained current, whereas neurones from old rats showed only a high-threshold calcium current. 4. The density of the high-threshold calcium current was 28.4 +/- 6.3 pA/pF (mean +/- S.E.M., n = 54) in neonatal, 39.1 +/- 7.2 pA/pF (n = 62) in adult and 11.0 +/- 4.6 pA/pF (n = 64) in old dorsal root ganglion neurones. 5. We found no difference in elementary high-threshold Ca2+ current characteristics in neurones from different age groups. The single-channel conductance was (with 60 mM Ca2+ in the recording pipette) 16.0 +/- 2.7 pS (mean +/- S.E.M., n = 9) in neonatal, 16.2 +/- 1.7 pS (n = 11) in adult and 16.4 +/- 1.2 pS (n = 12) in old neurones. 6. Current-voltage relations and kinetics of high-threshold calcium currents showed no detectable age-dependent difference. 7. The run-down of high-threshold calcium currents in dorsal root ganglion neurones from old rats was practically insensitive to intracellular administration of cyclic AMP and ATP. The same intervention caused a significant deceleration of Ca2+ current run-down in the majority of neonatal and in some adult cells. 8. We suggest that the disappearance of the low-threshold calcium current and reduction of high-threshold calcium current with ageing is due to a depression of calcium channel expression during late ontogenesis. The decrease of sensitivity of high-threshold calcium channels to phosphorylation by cyclic AMP-dependent protein kinase in aged neurones could also be a reason for altered turnover between silent and functional pools of calcium channels, which may underlie the age-dependent decline in the density of high-threshold calcium channels.

Different action of ethosuximide on low- and high-threshold calcium currents in rat sensory neurons

The effects of ethosuximide on calcium channels were studied on dorsal root ganglion neurons from one-day-old rats using the patch-clamp technique. Bath application of ethosuximide induced dose-dependent and reversible suppression of calcium currents without affecting their time-course. Substantial differences between the effects of ethosuximide on the low-threshold and high-threshold (T- and L-) currents were observed. Ethosuximide reduced the T-current with greater potency than the L-current (Kd for T-current is 7 μM vs 15 μM for L-current). This relative specificity of its action still remained if applied at concentrations up to 1 mM. These data support the hypothesis according to which the anti-epileptic action of ethosuximide is related to reduction of the low-threshold calcium currents in sensory neurons.

Computer model of ethosuximide's effect on a thalamic neuron.

Ethosuximide appears to have a specific effect on the low-threshold calcium current in thalamic cells. This may be related to its efficacy in the treatment of absence epilepsy. We used a computer model of an individual thalamocortical neuron to better understand the alteration in the low-threshold calcium current under voltage clamp and to predict response to current injection in the presence of ethosuximide. The full model included nine voltage-sensitive ionic channels and a realistic dendritic morphology. The model reproduced the two major responses seen in tissue slices: repetitive spiking with depolarization and the low-threshold calcium spike elicited on release from hyperpolarization. The alteration in low-threshold calcium current with ethosuximide can be explained by a 10-mV depolarizing shift in the steady-state activation curve for this channel with a 10% reduction in maximum channel permeability. Simulations of current injection showed that ethosuximide diminished the low-threshold calcium spike while leaving the tonic firing pattern unaffected. Our results support the hypothesis that ethosuximide's effects on low-threshold calcium current might selectively alter the dynamics of slow bursting in thalamic cells.

Cytoplasmic alkalinization increases high-threshold calcium current in chick dorsal root ganglion neurones

Changes of calcium currents with intracellular pH (pHi) were investigated in chick dorsal root ganglion (DRG) neurones. High-threshold calcium currents decreased after extracellular application of a permeable weak acid, sodium acetate (CH3COONa), and increased when applying a permeable weak base, ammonium chloride (NH4Cl), whereas both compounds were ineffective against the low-threshold calcium current. These weak electrolytes, employed to change pHi, did not alter the kinetic and steady-state parameters of activation and inactivation of the calcium current. Extracellular application of concanavalin A (Con A) and wheat germ agglutinin to elevate pHi increased the high-threshold calcium current. Their effect developed within 2-5 min and was independent of lectin concentration varied from 0.1 to 1 mg/ml. The lectin effects were greatly diminished if Na/H exchange was blocked by amiloride or suppressed by low external sodium. Succinilated Con A and Con A in the presence of D-mannose were less effective. Calcium currents were recorded simultaneously with the pHi, monitored with a proton-sensitive microelectrode. It was found that 50% inhibition of the calcium current occurred at pHi of 6.5. Histidine-specific reagents--diethylpyrocarbonate, Rose Bengal and Methylene Blue--prevented the modulation of the calcium conductance by CH3COONa and NH4Cl. Extracellular baclofen and theophylline or intracellular phorbol esters, staurosporine, calmodulin antagonists R24571 and W-13, and neomycine failed to prevent the modulation of the calcium current by weak electrolytes. These observations are consistent with an interaction between intracellular protons and calcium channels.

Blocking effect of intraperitoneal injection of phenylalanine on high-threshold calcium currents in rat hippocampal neurones

Calcium currents were recorded in cultured (5–7 days) hippocampal neurones isolated from one-day-old rats. The animals obtained intraperitoneal injections of l-phenylalanine which induces in the brain biochemical changes characteristic of phenylketonuria. It has been found that the amplitude of the low-threshold calcium current in l-phenylalanine-affected neurones was not appreciably changed compared with that in neurones from control (non-injected) animals. However, the amplitude of the high-threshold calcium current was essentially decreased. Its relative amplitude at V t = +20 mV became 40 ± 30% as contrasted to 416 ± 130% in neurones from control animals (the amplitude of the calcium currents at V t = −10 mV taken as 100%). The decrease remained during the whole time of culturing. Addition of l-tyrosine to the cultivation medium (50 μM) restored the high-voltage calcium current, its relative amplitude reaching 280 ± 57%. The data are discussed in conjunction with the previously obtained results about antagonistic modulatory action of tyrosine and phenylalanine on the functioning of high-threshold calcium channels and possible mechanisms of brain dysfunction during phenylketonuria.

Contributions of low-threshold calcium current and anomalous rectifier ( Ih) to slow depolarizations underlying burst firing in human neocortical neurons in vitro

The slow depolarization that underlies the voltage-dependent burst-firing behavior of human neocortical neurons is mediated by a low-threshold calcium conductance in concert with the anomalous rectifier current, I h . The slow depolarization could be elicited by depolarization from negative membrane potentials or as a rebound following hyperpolarization. The rebound depolarization was time- and voltage-dependent. Most of the slow depolarization was blocked by inorganic calcium blockers. The remainder of the depolarization and the ‘sag’ in the hyperpolarizing voltage responses were blocked by extracellular Cs +.

High- and low-threshold calcium currents in neurons acutely isolated from rat sensorimotor cortex

Neurons were isolated by papain treatment and trituration of the frontoparietal cortex of 14 to 28-day-old rats. Whole cell voltage clamp revealed a slowly inactivating high-threshold Ca 2+ current, activated positive to −45 mV, and a transient low-threshold Ca 2+ current, activated positive to −65 mV. The high-threshold current was more sensitive to block by Cd 2+ and the low-threshold current was more sensitive to block by Ni 2+. Replacement of Ca 2+ by Ba 2+ increased the high-threshold current and reduced the low-threshold current. The high-threshold current was enhanced by Bay K 8644 and reduced by nimodipine and ω-conotoxin. The low-threshold current was also reduced by nimodipine but was insensitive to Bay K 8644 and ω-conotoxin. The properties of the currents were consistent with different underlying Ca 2+ channel types.