High Voltage-activated Calcium Currents Research Articles

GABAB receptors and G proteins modulate voltage-dependent calcium channels in cultured rat dorsal root ganglion neurons: Relevance to transmitter release and its modulation

Acutely dissociated and cultured chick and rat dorsal root ganglion (DGR) neurons were studied by means of whole-cell patch-clamp technique. The high voltage-activated calcium current in DRG neurons is a result of mixture of N-type (ω-conotoxin-sensitive), P-type (ω-aga-IVA-sensitive), and L-type (dihydropyridine-sensitive) calcium currents. The modulation of these calcium channel currents in DRG neurons involves the GTP binding proteins and GABAB receptors. This kind of modulation occurs at presynaptic terminalsin vivo due to synaptically released GABA resulting in a primary afferent inhibition.

Dephosphorylated oligoadenylates modulate high voltage-activated calcium currents in GH3 cells

Effects of different forms of C2-5A (2′,5′ApApA; 2′,5′ApApepoxyA; and 3′,5′ApApA) on high voltage-activated (HVA) calcium currents in GH3 cells were studied using the whole-cell patch-clamp recording technique. Addition of 10 µM 2′,5′ApApA, a “core” (dephosphorylated) oligoadenylate, to the pipette solution induced an increase in HVA calcium current. Ten minutes after the whole-cell configuration was established, the current magnitude was enhanced about twofold compared with that observed at 2 min. High concentration of Mg2+ (5 µM) in the pipette solution blocked this effect. 2′,5′ApA and 3′,5′ApApA oligoadenylates, and products of 2′,5′A hydrolysis, adenosine and AMP, did not change the value of HVA current. A chemically modified analog of 2′,5′ApApA (2′,5′ApApepoxyA) has been the oligoadenylate most stable under phosphodiesterase action. Addition of 2′,5′ApApepoxyA to the pipette solution under the same conditions caused a smaller effect than 2′,5′ApApA did.

Age-dependent expression of high-voltage activated calcium currents during cerebellar granule cell development in situ.

Ca2+ currents play a crucial role during neuronal growth. In this paper we describe the development of Ca2+ currents using whole-cell patch-clamp recordings in granule cells of cerebellar slices obtained from 7- to 24-day-old rats. Granule cells expressed high-voltage-activated (HVA) Ca2+ currents in different proportions. The percentage of cells with a measurable HVA current, and the size of HVA current increased in parallel with granule cell maturation. At less than 14 days HVA currents consisted of a fast- and slow-inactivating component, while at more than 19 days only the slow-inactivating component remained. The fast-inactivating component had faster activation and inactivation kinetics, a more negative threshold for activation, and steeper steady-state inactivation than the slow-inactivating component. Nifedipine (5 microM) partially blocked both components. omega-Conotoxin (5 microM, omega-CgTx) blocked the slow-inactivating component rather selectively. These results indicate that HVA currents change their gating and pharmacological properties during development. Although the mechanism at the molecular level remains speculative, the developmental changes of the HVA current are relevant to the processes of granule cell maturation and excitability.

The secretion of alpha-MSH from xenopus melanotropes involves calcium influx through omega-conotoxin-sensitive voltage-operated calcium channels.

The secretory activity of endocrine cells largely depends on the concentration of free cytosolic calcium. We have studied the mechanisms that are involved in supplying the calcium necessary for the secretion of alpha-melanophore-stimulating hormone (alpha-MSH) from melanotrope cells in the pituitary intermediate lobe of the amphibian Xenopus laevis. Using whole-cell voltage clamp, high-voltage activated calcium currents were observed, with a peak current between 0 and +20 mV. Two types of Ca(2+)-currents appeared, depending on the experimental setup. An inactivating current, which was observed after a 10 msec depolarizing prepulse, resembled currents through N-type channels as it was clearly inhibited by 1 microM omega-conotoxin. The second type was a non-inactivating current, which was blocked up to 50% by 1 microM nifedipine, indicating its L-type nature. Only a small component of this inactivating current could be blocked by omega-conotoxin. No evidence was found for the presence of transient, low-voltage activated currents. The spontaneous secretion of alpha-MSH from superfused neurointermediate lobes was dependent on extracellular calcium, as low calcium conditions (10(-4)-10(-8) M) rapidly inhibited this process. Under these conditions, secretion was not affected by depolarizing concentrations of potassium chloride. The calcium ionophore A23187 increased secretion under low calcium conditions, but had no effect on spontaneous alpha-MSH release. These results suggest that spontaneous alpha-MSH release depends on influx of calcium through voltage-operated calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in calcium currents during ethanol withdrawal in a genetic mouse model

Changes in voltage dependent calcium currents in the dentate gyrus of the hippocampal slice during ethanol withdrawal were studied using an alcohol withdrawal seizure prone mouse strain (WSP) and compared to a withdrawal-resistant strain (WSR). There was a significant increase in the high voltage activated calcium currents during the withdrawal period in the WSP strain, while those of the resistant strain showed no significant enhancement. These results suggest that an increase in calcium currents is one factor involved in the alcohol withdrawal hyperexcitability of the prone strain observed in vivo.

Adenosine acting at an A1 receptor decreases N-type calcium current in mouse motoneurons

The neuromodulator adenosine is known to decrease neurotransmitter release at the neuromuscular junction by activation of an A1 adenosine receptor coupled to a pertussis toxin-sensitive G protein. Among the mechanisms that could contribute to the depression of neurotransmitter release is reduced entry of calcium through channels located in the presynaptic terminal. In the present study, we have examined the effects of adenosine on high-voltage-activated (HVA) calcium currents in motoneurons, the presynaptic cells of the neuromuscular junction. The motoneurons were isolated from embryonic mice, placed in primary tissue culture for 16 hr, and analyzed by means of the whole-cell patch-clamp technique. Adenosine (40 microM) reduced both transient and sustained components of HVA calcium current. This effect was blocked by the A1 antagonist 8-cyclopentyltheophylline (CPT; 100 nM) and was mimicked by the A1 agonist N6-cyclohexyladenosine (CHA; 50 nM to 10 microM) but not by the A2a agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine (CGS-21680; 1 micron). Pretreatment with pertussis toxin (200 ng/ml, > 16 hr) abolished the depression of HVA calcium current by adenosine receptor activation. Brief (3 min) exposure of the cells to 10 microM omega-conotoxin GVIA irreversibly blocked a part of the HVA current, which can therefore be attributed to N-type channels; the remaining current was unaffected by adenosine receptor activation. Hence, it appears that adenosine decreases only the N-current portion of HVA current and that this inhibition occurs via an A1 receptor linked to a pertussis toxin-sensitive G protein. Other investigators have shown that N-type channels do not play a primary role in eliciting transmitter release at the mammalian neuromuscular junction. Thus, it is uncertain what motoneuronal functions are influenced by adenosine modulation of N-type channels.

Mechanosensitivity of voltage-gated calcium currents in rat anterior pituitary cells.

Sensitivity of voltage-activated calcium currents to flow-induced mechanical stress was examined in enriched populations of rat anterior pituitary somatotrophs. Voltage-activated calcium currents were recorded with the whole-cell configuration of the patch-clamp technique. Pituitary cells were exposed to flow (from pipettes) which was produced by a hydrostatic pressure of about 3 cmH2O. In 92% of the cells studied (n = 87 cells) flow reduced the amplitude of both low voltage-activated (LVA) and high voltage-activated (HVA) calcium currents. These effects of flow on calcium currents did not result from changes in either seal resistance or leak conductance of the cell and were dependent on the magnitude of flow. The effect of flow is selective. We found that LVA calcium currents were substantially more sensitive to flow than HVA calcium currents. Under constant flow conditions, LVA calcium currents were reduced by 57.6 +/- 29.6% (S.D.), whereas HVA currents (recorded from the same cells) were reduced by only 17.8 +/- 15.9% (S.D.). The effects of flow on calcium currents were associated with effects on their related calcium tail currents. Slowly deactivating calcium tail currents were reduced by 75.3 +/- 25.6% (S.D.), whereas rapidly deactivating calcium tail currents were reduced by 29.1 +/- 14.4% (S.D.). The effect of flow on calcium currents was not associated with any significant shift in the activation curves of the calcium currents (voltage range -60 to +30 mV), suggesting that the effect of flow is not voltage dependent. The effect of flow is not dependent on activation of calcium currents during the exposure to flow. Calcium currents which were evoked immediately after cessation of the exposure to flow were reduced in amplitude and recovered to control values. Possible mechanisms underlying the flow effect and possible physiological relevance of the effect on pituitary cells are discussed.

Opioids decrease high-voltage activated calcium currents in acutely dissociated neostriatal neurons

Although the distribution of opioid receptors is central to the patch-matrix model of neostriatal organization, it has been unclear whether these receptors are located post-synaptically. Moreover, it has not yet been clarified whether opioid receptor activation in neostriatum results in the modulation of calcium and/or potassium conductances. To test this, neostriatal neurons were acutely isolated and their sensitivity to opioid receptor agonists determined. At nanomolar. At nanomolar concentrations, both the μ-agonist [ d-Ala 2, MePhe 4, Gly-ol 5]-enkephalin (DAMGO) and the δ-agonist [ d-Pen 2, d-Pen 5]-enkephalin (DPEPE) reversibly decreased whole-cell calcium currents in medium-sized neurons. These effects were blocked by the opiate antagonist naloxone. These findings argue that activation of post-synaptic, opioid receptors is capable of modulating the excitability of neostriatal neurons.

Fluspirilene block of N‐type calcium current in NGF‐differentiated PC 12 cells

1. High voltage-activated calcium currents were recorded in nerve growth factor (NGF)-differentiated PC12 cells with the whole-cell patch clamp technique. After exposure to NGF for 3-10 days the PC12 cells developed neurone-like processes and calcium currents which were pharmacologically separable into L- and N-types (defined by sensitivity to nifedipine and omega-conotoxin GVIA respectively). 2. After blocking the L-type calcium channels with nifedipine (10 microM), omega-conotoxin GVIA blocked approximately 85% of the remaining calcium current with an IC50 of 3 nM and a Hill coefficient of 1. The block by conotoxin GVIA was irreversible on the time scale of these experiments. These results suggested that the majority of the nifedipine-insensitive calcium current was N-type. 3. Fluspirilene, a substituted diphenylbutylpiperidine with potent neuroleptic properties, reversibly inhibited the N-type component in a dose-dependent manner with an IC50 of 30 nM. The Hill coefficient of the block was 0.25. The fraction of current blocked was the same at all test potentials examined (-30 to +40 mV). 4. These data indicate that the neuroleptic properties of fluspirilene may be due, at least in part, to an inhibition of neuronal N-type calcium channels. This finding raises the possibility that modulation of N-type calcium channel activity by drugs derived from substituted diphenylbutylpiperidines may provide a novel way of altering neurotransmitter release and hence brain function.

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)

Modulation of a sustained calcium current by intracellular pH in horizontal cells of fish retina.

A sustained high voltage-activated (HVA), nifedipine- and cadmium-sensitive calcium current and a sustained calcium action potential (AP) were recorded from horizontal cells isolated from catfish retina. pH indicator dyes showed that superfusion with NH4Cl alkalinized these cells and that washout of NH4Cl or superfusion with Na-acetate acidified them. HVA current was slightly enhanced during superfusion of NH4Cl but was suppressed upon NH4Cl washout or application of Na-acetate. When 25 mM HEPES was added to the patch pipette to increase intracellular pH buffering, the effects of NH4Cl and Na-acetate on HVA current were reduced. These results indicated that intracellular acidification reduces HVA calcium current and alkalinization increases it. Sustained APs, recorded with high resistance, small diameter microelectrodes, were blocked by cobalt and cadmium and their magnitude varied with extracellular calcium concentration. These results provide confirmatory evidence that the HVA current is a major component of the AP and indicate that the AP can be used as a measure of how the HVA current can be modified in intact, undialyzed cells. The duration of APs was increased by superfusion with NH4Cl and reduced by washout of NH4Cl or superfusion with Na-acetate. The Na-acetate and NH4Cl washout-dependent shortening of the APs was observed in the presence of intracellular BAPTA, a calcium chelator, IBMX, a phosphodiesterase inhibitor, and in Na-free or TEA-enriched saline. These findings provide supportive evidence that intracellular acidification may directly suppress the HVA calcium current in intact cells. Intracellular pH changes would thereby be expected to modulate not only the resting membrane potential of these cells in darkness, but calcium-dependent release of neurotransmitter from these cells as well. Furthermore, this acidification-dependent suppression of calcium current could serve a protective role by reducing calcium entry during retinal ischemia, which is usually thought to be accompanied by intracellular acidosis.

Inhibition of high voltage-activated calcium currents by l-glutamate receptor-mediated calcium influx

The modulation of high voltage-activated (HVA) Ca2+ currents by L-glutamate and its agonists was investigated in cultured rat hypothalamic neurons. L-Glutamate and agonists selective for NMDA or non-NMDA receptors reversibly inhibited HVA Ca2+ currents. The putative presynaptic glutamate receptor agonist L-2-amino-4-phosphonobutyric acid and the selective metabotropic agonist trans-ACPD were ineffective. Inhibition was dependent on the presence of extracellular Ca2+ and blocked by internal perfusion of the cells with BAPTA. The calmodulin antagonists trifluoperazine and calmidazolium completely prevented the inhibition. Increases in the intracellular Ca2+ concentration due to Ca2+ influx through non-NMDA receptor channels were visualized using fura-2. These results indicate that not only NMDA but also non-NMDA receptor channels in these neurons are permeable for Ca2+ and that Ca2+ influx through these channels activates a calmodulin-dependent mechanism, which leads to HVA Ca2+ current inhibition.

Calcium Currents and Internal Calcium in Caudo-Dorsal Neuroendocrine Cells in the Snail Lymnaea Stagnalis

Voltage-activated calcium currents and changes in internal calcium in relation to electrical activity were studied in the neuroendocrine Caudo-Dorsal Cells (CDCs) of the pond snail Lymnaea stagnalis. The CDCs control egg-laying via the release of a number of peptides during a prolonged period of spiking activity called afterdischarge. Under whole-cell voltage-clamp conditions two distinct dihydropyridine-sensitive high-voltage-activated (HVA) calcium currents were demonstrated in isolated CDCs, which differed in voltage-dependence of activation and in kinetics and voltage-dependence of inactivation. Stimulation of several second messenger routes (cAMP, cGMP and PKC) increased the amplitudes of both HVA calcium currents. Phosphorylation by protein kinases appeared to be a critical step in the modulation of the HVA calcium channels. Fluctuations in the intracellular calcium concentration ([Ca2+]i) during an afterdischarge were measured in CDCs in the intact central nervous system, using the fluorescent dye fura-2. During an electrically-induced discharge, the intracellular calcium level increased. However, maximal calcium levels were only reached at the final phase of the discharge or several minutes after the cessation of firing. This suggests that calcium rises during the discharge require action potential driven influx of extracellular calcium through calcium channels, whereas the prolonged high level of calcium following the discharge is not directly dependent on action potentials.

Suppression of a calcium current by CNQX and kynurenate

During our investigation of a sustained high voltage-activated (HVA) calcium current in retinal horizontal cells, we found that the glutamate receptor antagonists CNQX and kynurenate but not AP7 reversibly reduced the peak amplitude of the HVA current. Changes in the HVA current kinetics or activation voltage were not apparent; there was only a reduction in the peak current. The novel effects of these antagonists on HVA calcium currents reported here could have an impact on many studies involving glutamatergic synaptic transmission.

Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro

We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively. Inhibitory postsynaptic responses were evoked less frequently and were blocked by the GABAA receptor antagonist (-)-bicuculline methochloride. The pharmacological profiles of whole-cell calcium currents and evoked EPSCs were compared. With 50 microM cadmium chloride (Cd), whole-cell low voltage-activated (LVA) calcium currents were reduced in amplitude and high voltage-activated (HVA) calcium currents and excitatory synaptic transmission were completely blocked. This suggests that the residual calcium influx through LVA channels into the presynaptic terminal does not suffice to trigger transmitter release. A saturating concentration of omega-conotoxin GVIA (omega-CgTx) (2.5 microM) blocked one-third of whole-cell HVA calcium currents and evoked EPSCs. The dihydropyridine nifedipine (50 microM) reversibly reduced whole-cell HVA calcium currents in a voltage-dependent manner but not excitatory synaptic transmission. Cd and omega-CgTx did not alter amplitude distributions of miniature EPSCs, demonstrating that the inhibition of synaptic transmission was due to block of presynaptic calcium channels. We conclude that excitatory glutamatergic transmission in thalamic neurons in vitro was mediated mainly by HVA calcium currents, which were insensitive to omega-CgTx and nifedipine.

Intra and extracellular surface charges near Ca2+ channels in neurons and neuroblastoma cells.

The properties of low (LVA) and high (HVA) voltage-activated calcium currents were investigated in rat sensory neurons and a murine neuroblastoma cell line exposed to various concentrations of intra- or extracellular monovalent ([c+]i/o) and trivalent ([c3+]i/o) cations. In neurons, when [c+]i was changed from 150 to 20 mM, positive shifts of 18-28 mV were observed in activation curves of both LVA and HVA currents, as well as in LVA inactivation curves. Extracellularly, in divalent-free solutions, [c+]o of 20-50 mM produced medium (12-22 mV) negative shifts of the LVA channel properties. These data were used to estimate, by a "screening" model, a negative surface charge density around neuron's calcium channels of 1/1,000 and 1/1,325 eA-2 at the outside or inside face, respectively. In the presence of physiological concentrations of divalent cations, [c+]o of 20-60 mM caused smaller (4-11 mV) negative shifts of the activation and inactivation curves, which can be explained by assuming a partial neutralization of negative charges by divalent cations. By applying the above procedure to LVA channels of neuroblastoma cells, the ratio of extra- to intracellular surface charge density turned out to be more than tenfold higher than in neurons. Effects produced by [c3+]i/o were not in agreement with expectations based on screening or binding models.

Voltage-dependent modulation of calcium current by GTPγS and dopamine in cultured frog pituitary melanotrophs

Dopamine (1 μM) reversibly scaled down barium current through high-voltage activated (HVA) calcium channels but had little effect on the time course of current activation in cultured frog melanotrophs. Intracellular perfusion with guanosine-5′- O-(3-thiotriphosphate) (GTPγS; 100 μM) sustained the effect of dopamine. Moreover, GTPγS drastically slowed down the current activation kinetics. The latter effect was in part reversed by dopamine. A conditioning prepulse to +70 mV facilitated the current in GTPγS-dialyzed cells but not in cells exposed to dopamine. These reults suggest the existence of a dual G protein-mediated mechanism for reducing HVA calcium current.

Pharmacological discrimination of N-type from L-type calcium current and its selective modulation by transmitters

GABA and norepinephrine inhibit high voltage-activated calcium current in chick sensory neurons. Using specific pharmacological tools, we have dissected this current into two components: the major one is omega-conotoxin sensitive and dihydropyridine resistant (N-type) while the minor one is dihydropyridine sensitively and omega-conotoxin resistant (L-type). The ability to selectively eliminate these two components has allowed us to determine whether the transmitters target the same or different channel types. Both GABA and norepinephrine modulate the N-type component as evidenced by their lack of effect on (1) omega-conotoxin-resistant current and (2) pure L-type tail current, prolonged by a dihydropyridine calcium channel agonist. This simple pharmacological profile will allow future tests of the significance of the two channel types in regulating sensory neuron functions.

Calmodulin antagonists and protein phosphatase inhibitor okadaic acid fasten the ‘run-up’ of high-voltage activated calcium current in rat hippocampal neurones

Voltage-activated Ca 2+ channel currents were recorded from cultured rat hippocampal neurones using the whole-cell clamp technique with Ba 2+ as a charge carrier. After breaking into the cell the amplitude of low-voltage activated Ca 2+ channel current increased to a new steady value within 1 min whereas several minutes were required for a full development of the high-voltage activated current ( I HVA). Pretreatment of cells with calmodulin antagonists (trifluoperazine or W-13) or protein phosphatase inhibitor, okadaic acid, fastened the development of I HVA. Trifluoperazine (6–40 μM) also increased I HVA when applied after breaking into the cell in standard external solution. Incubation of cells in the presence of permeable precursor of Ca 2+ chelator, BAPTA, was without effect. The effects of all inhibitors studied allow to suggest that I HVA in intact cells is largely masked due to activity of calmodulin-activated protein phosphatase.

Two high voltage-activated calcium currents are present in isolation in adult rat spinal neurons

In neurons enzymatically isolated from adult rat dorsal root ganglia and used during the following 24 hours, the Ca 2+ currents were investigated with the whole-cell patch-clamp technique. In contrast to the neonatal neurons, the salient feature of these adult neurons is the well separated (in the voltage-range) activation and inactivation properties of each recorded current. The low-threshold T-, the high-threshold inactivating N-, and the long-lasting L-currents have a threshold for activation at −60, −45 and −10 mV, and a 50% inactivation at −75, −45 and −5 mV respectively. The N and L currents were poorly affected by 100 μM Ni, a known blocker of T channels and completely blocked by 100 μM Cd 2+. Frequently we could find neurons with only one type of current present. We conclude that adult sensory neurons are a better preparation for studying, in isolation, the physiological relevance of the three types of Ca 2+ channels.