The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium

The SV channel encoded by the TPC1 gene represents a Ca²⁺- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1 , named fou2 for fatty acid oxygenation upregulated 2 , results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca²⁺ signals and cytosolic Ca²⁺ is required for SV channel function, we here studied the Ca²⁺-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca²⁺ sensitivity and Ca²⁺ impermeability. K⁺ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca²⁺ reached the 0.1-m m level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca²⁺ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca²⁺. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca²⁺ before SV channel activity vanishes and K⁺ homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca²⁺ signals resulting in overproduction of jasmonate.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Barium-Evoked Glutamate Release from Guinea-Pig Cerebrocortical Synaptosomes

Abstract: Ba²⁺ has multiple effects on presynaptic terminals. The ion inhibits the K⁺ channels responsible for stabilizing the plasma membrane potential in the same way as previously reported for dendrotoxin and 4-aminopyridine. Secondly, the ion can substitute fully for Ca²⁺ in supporting KCl-evoked release of glutamate from guinea-pig cerebrocortical synaptosomes. In the latter case, the kinetics of glutamate release in the presence of saturating Ca²⁺ or Ba²⁺ are essentially identical. Substantially lower external concentrations of Ba²⁺ are required to achieve the same release kinetics as with Ca²⁺. The average internal free Ba²⁺ concentration attained during KCl depolarization is some 10-fold higher than that for Ca²⁺. However, because the fura-2 signal reflects predominantly the overflow of divalent cation after dissociation from the release trigger, it is not the valid parameter to compare effectiveness of the cations in triggering glutamate exocytosis. In view of the established inability of Ba²⁺ to interact with calmodulin, these results are discussed in relation to theories in which Ca²⁺/calmodulin-dependent protein kinase-mediated phosphorylation is a prerequisite for synaptic vesicle exocytosis.     

Potassium ion channels in the plasmalemma

The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K⁺ transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K⁺ equilibrium potential (E_K) – membrane potential (V_m), or secondary active by coupling through a carrier to the inward driving force of H⁺ or Na⁺. Known K⁺ channels are permeable to monovalent cations, a permeability order being K⁺ > Rb⁺ > NH₄⁺ > Na⁺≥ Li⁺ > Cs⁺. The macroscopic K⁺ currents across a cell or protoplast surface commonly show rectification, i.e. a V^m-dependent conductance which in turn, may be controlled by the cytosolic activity of Ca²⁺, of K⁺, of H⁺, or by the K⁺ driving force. Analysis by the patch clamp technique reveals that plant K⁺ channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K⁺ channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.     

Protein kinase inhibitor K-252a and fusicoccin induce similar initial changes in ion transport of parsley suspension cells

The protein kinase inhibitor K-252a induces a rapid, transient decrease of extracellular pH and [K⁺], and a concomitant increase in extracellular [Ca²⁺] in suspensions of cultured parsley cells. These effects are subsequently reversed. As with K-252a, fusicoccin also induces similar changes in pH and extracellular [Ca²⁺], but reversion does not occur. Acidification by HCI also leads to an increase in external [Ca²⁺], suggesting that the changes in extracellular [Ca²⁺] are mainly due to a pH-dependent displacement of Ca²⁺ ionically bound to the cell wall. The artificial acidification by HCI is rapidly followed by cell-mediated alkalinization, a process associated with K² release and rebinding of Ca²⁺. Any change in external pH or [K⁺] induced by K-252a, fusicoccin, or HCI is followed by an uptake of ⁴⁵Ca²⁺ into cellular pools. The results show that K-252a may be a valuable tool for studying the complex regulation of ion transport which may involve changes in the phosphorylation of unknown proteins.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

NaCl-induced changes in cytosolic free Ca2+ in Arabidopsis thaliana are heterogeneous and modified by external ionic composition

Increases in cytosolic free Ca²⁺ ([Ca²⁺]_cyt) are common to many stress-activated signalling pathways, including the response to saline environments. We have investigated the nature of NaCl-induced [Ca²⁺]_cyt signals in whole Arabidopsis thaliana seedlings using aequorin. We found that NaCl-induced increases in [Ca²⁺]_cyt are heterogeneous and mainly restricted to the root. Both the concentration of NaCl and the composition of the solution bathing the root have profound effects on the magnitude and dynamics of NaCl-induced increases in [Ca²⁺]_cyt. Alteration of external K⁺ concentration caused changes in the temporal and spatial pattern of [Ca²⁺]_cyt increase, providing evidence for Na⁺-induced Ca²⁺ influx across the plasma membrane. The effects of various pharmacological agents on NaCl-induced increases in [Ca²⁺]_cyt indicate that NaCl may induce influx of Ca²⁺ through both plasma membrane and intracellular Ca²⁺-permeable channels. Analysis of spatiotemporal [Ca²⁺]_cyt dynamics using photon-counting imaging revealed additional levels of complexity in the [Ca²⁺]_cyt signal that may reflect the oscillatory nature of NaCl-induced changes in single cells.     

Differential fading of inhibitory and excitatory B2 bradykinin receptor responses in rat sympathetic neurons: a role for protein kinase C

Through inhibitory and excitatory effects on sympathetic neurons, B₂ bradykinin receptors contribute to protective and noxious cardiovascular mechanisms. Presynaptic inhibition of sympathetic transmitter release involves an inhibition of Ca_V2 channels, neuronal excitation an inhibition of K_V7 channels. To investigate which of these mechanisms prevail over time, the respective currents were determined. The inhibition of Ca²⁺ currents by bradykinin reached a maximum of 50%, started to fade within the first minute, and became attenuated significantly after ≥ 4 min. The inhibition of K⁺ currents reached a maximum of 85%, started to fade after > 3 min, and became attenuated significantly after ≥ 7 min. Blocking Ca²⁺-independent protein kinase C (PKC) enhanced the inhibition of Ca²⁺ currents by bradykinin and delayed its fading, left the inhibition of K⁺ currents and its fading unaltered, and enhanced the reduction of noradrenaline release and slowed its fading. Conversely, direct activation of PKC abolished the inhibition of noradrenaline release and largely attenuated the inhibition of Ca²⁺ currents. These results show that the inhibitory effects of bradykinin in sympathetic neurons are outweighed over time by its excitatory actions because of more rapid, PKC-dependent fading of the inhibitory response.     

Ca2+-Dependent Inactivation of P-Type Calcium Channels in Nerve Terminals

Abstract: Rapid Ca²⁺ signals evoked by K⁺ depolarization of rat cerebral cortical synaptosomes were measured by dual-channel Ca²⁺ spectrofluorometry coupled to a stopped-flow device. Kinetic analysis of the signal rise phase at various extracellular Ca²⁺ concentrations revealed that the responsible voltage-dependent Ca²⁺ channels, previously identified as P-type Ca²⁺ channels, inactivate owing to the rise in intracellular Ca²⁺ levels. At millimolar extracellular Ca²⁺ concentrations the channels were inactivated very rapidly and the rate was dependent on the high influx rate of Ca²⁺, thus limiting the Ca²⁺ signal amplitudes to 500–600 n M. A slower, probably voltage-dependent regulation appears to be effective at lower Ca²⁺ influx rates, leading to submaximal Ca²⁺ signal amplitudes. The functional feedback regulation of calcium channels via a sensor for intracellular Ca²⁺ levels appears to be responsible for the different inhibition characteristics of Cd²⁺ versus ω-agatoxin IVa.     

Fast-activating cation channel in barley mesophyll vacuoles. Inhibition by calcium

In contrast to the vacuolar ion channels which are gated open by an increase of cytosolic Ca²⁺ the vacuolar ion currents at resting cytosolic Ca²⁺are poorly explored. Therefore, this study was performed to investigate the properties of the so-called fast-activating vacuolar (FV) current which dominates the electrical characteristics of the tonoplast at physiological free Ca²⁺ concentrations. Patch—clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. Single ion channels were identified, which, based on their selectivity, activation kinetics, Ca²⁺- and voltage-dependence, carry the whole-vacuole FV current. Reversal potential determinations indicated a K⁺ overs C⁻ permeability ratio of about 30. Both inward and outward whole-vacuole currents as well as the activity of single FV channels were inhibited by an increase of cytosolic Ca²⁺, with a K_d≈ 6 µM. At physiological vacuolar Ca²⁺ activities, the FV channel is an outward-rectifying potassium channel. The FV channel was activated in less than a few milliseconds both by negative and positive potential steps, having a minimal activity that is 40 mV negative of the K⁺ equilibrium potential. It is proposed that transport of K⁺ through this cation channel controls the electrical potential difference across the tonoplast.     

Alcohol Inhibits the Depolarization-Induced Stimulation of Oxidative Phosphorylation in Synaptosomes

Abstract: The effects of alcohol and Ca²⁺ transport inhibitors on depolarization-induced stimulation of oxidative phosphorylation and free-Ca²⁺ concentrations in rat synaptosomes were investigated. Glucose oxidation was stimulated by depolarization with K⁺ or veratridine and by the Ca²⁺ ionophore ionomycin. The stimulation by K⁺, veratridine, and ionomycin was correlated with elevation of synaptosomal free Ca²⁺. Depolarization-stimulated respiration was inhibited by verapamil, Cd²⁺, and ruthenium red but not by diltiazem. Synaptosomal Ca²⁺ elevation was inhibited by verapamil but not by ruthenium red. These results indicate that the stimulation depends on elevation of mitochondrial free Ca²⁺. Ethanol, at pharmacological concentrations (50–200 m M ), inhibited the Ca²⁺-dependent stimulation of oxidative phosphorylation. This inhibition resulted, in part, from the inhibition of voltage-gated Ca²⁺ channels, which inhibited the elevation of synaptosomal free Ca²⁺, and, in part, from the stimulation of the mitochondrial Ca²⁺/Na⁺ antiporter, which inhibited the elevation of the mitochondrial matrix free Ca²⁺. The inhibition by ethanol of the excitation-induced stimulation of oxidative phosphorylation in the synapse may contribute to the depressant and narcotic effects of alcohol and enhance excitotoxicity.     

α-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of α-synuclein (α-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α-synuclein gene ( SNCA ) are associated with familial PD. Since Ca²⁺ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca²⁺ homeostasis in cells stably transfected with either wild-type α-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca²⁺ influx evoked by exposure of cells to 50 mM K⁺ was enhanced in cells expressing all three forms of α-syn, an effect which was due specifically to increased Ca²⁺ entry via L-type Ca²⁺ channels. Mobilization of Ca²⁺ by muscarine was not strikingly modified by any of the α-syn forms, but they all reduced capacitative Ca²⁺ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca²⁺]_i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca²⁺ content was unaffected by any form of α-synuclein. However, only WT α-syn transfected cells displayed significantly impaired viability. Our findings suggest that α-syn regulates Ca²⁺ entry pathways and, consequently, that abnormal α-syn levels may promote neuronal damage through dysregulation of Ca²⁺ homeostasis.     

Amyloid β Protein Potentiates Ca2+ Influx Through L-Type Voltage-Sensitive Ca2+ Channels: A Possible Involvement of Free Radicals

Abstract: Amyloid β protein (Aβ), the central constituent of senile plaques in Alzheimer's disease (AD) brain, is known to exert toxic effects on cultured neurons. The role of the voltage-sensitive Ca²⁺ channel (VSCC) in β(25–35) neurotoxicity was examined using rat cultured cortical and hippocampal neurons. When L-type VSCCs were blocked by application of nimodipine, β(25–35) neurotoxicity was attenuated, whereas application of ω-conotoxin GVIA (ω-CgTX-GVIA) or ω-agatoxin IVA (ω-Aga-IVA), the blocker for N- or P/Q-type VSCCs, had no effects. Whole-cell patch-clamp studies indicated that the Ca²⁺ current density of β(25–35)-treated neurons is about twofold higher than that of control neurons. Also, β(25–35) increased Ca²⁺ uptake, which was sensitive to nimodipine. The 2',7'-dichlorofluorescin diacetate assay showed the ability of β(25–35) to produce reactive oxygen species. Nimodipine had no effect on the level of free radicals. In contrast, vitamin E, a radical scavenger, reduced the level of free radicals, neurotoxicity, and Ca²⁺ uptake. These results suggest that β(25–35) generates free radicals, which in turn, increase Ca²⁺ influx via the L-type VSCC, thereby inducing neurotoxicity.     

The phytotoxin syringomycin elicits Ca2+-dependent callose synthesis in suspension-cultured cells of Catharanthus roseus

Deposition of the 1,3-β-glucan callose onto the cell wall represents one of the defence reactions of plants against pathogens. This process can be induced in suspension cells of Catharanthus roseus by subtoxic concentrations of the bacterial phytotoxin syringomycin and is associated with a slight increase in Ca²⁺ uptake and some K⁺ release. Under these conditions callose formation can be prevented by complexing external Ca²⁺, indicating that some Ca²⁺ uptake is essential as a signal. However, higher syringomycin concentrations elicit increased Ca²⁺ uptake without increasing callose formation, although the potential for callose synthesis is not exhausted – as shown using digitonin as an additional elicitor. These results suggest that, superimposed on Ca²⁺, another, yet unknown signal is also involved in the regulation of callose synthesis.     

Internalization of Voltage-Dependent Sodium Channels in Fetal Rat Brain Neurons: A Study of the Regulation of Endocytosis

Abstract: In fetal rat brain neurons, activation of voltage-dependent Na⁺ channels induced their own internalization, probably triggered by an increase in intracellular Na⁺ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na⁺ channel activation. An increase in intracellular Ca²⁺ concentration induced either by thapsigargin, a Ca²⁺-ATPase blocker, or by A23187, a Ca²⁺ ionophore, was unable to provoke Na⁺ channel internalization. However, Ca²⁺ seems to be necessary because both neurotoxin- and amphotericin B-induced Na⁺ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na⁺ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na⁺ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na⁺ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na⁺ channel internalization in neurons.     

A Toxin Fraction (FTX) from the Funnel-Web Spider Poison Inhibits Dihydropyridine-Insensitive Ca2+ Channels Coupled to Catecholamine Release in Bovine Adrenal Chromaffin Cells

Abstract: In adrenal chromaffin cells, depolarization-evoked Ca²⁺ influx and catecholamine release are partially blocked by blockers of L-type voltage-sensitive Ca²⁺ channels. We have now evaluated the sensitivity of the dihydropyridine-resistant components of Ca²⁺ influx and catecholamine release to a toxin fraction (FTX) from the funnel-web spider poison, which is known to block P-type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K⁺-depolarization-induced Ca²⁺ influx by 50%, as monitored with fura-2, whereas nitrendipine (0.1–1 μ M ) and FTX (3:3), a synthetic FTX analogue (1 m M ), blocked the [Ca²⁺]_i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca²⁺]_i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by ∼80 and 70%, respectively, but both substances together abolished the K⁺-evoked catecholamine release, as measured by HPLC. The ω-conotoxin GVIA (0.5 μ M ) was without effect on K⁺-stimulated ⁴⁵Ca²⁺ uptake. Our results indicate that FTX blocks dihydropyridine- and ω-conotoxin-insensitive Ca²⁺ channels that, together with L-type voltage-sensitive Ca²⁺ channels, are coupled to catecholamine release.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

Endoplasmic reticulum Ca(2+) homeostasis and neuronal death

The endoplasmic reticulum (ER) is a universal signalling organelle, which regulates a wide range of neuronal functional responses. Calcium release from the ER underlies various forms of intracellular Ca²⁺ signalling by either amplifying Ca²⁺ entry through voltage-gated Ca²⁺ channels by Ca²⁺-induced Ca²⁺ release (CICR) or by producing local or global cytosolic calcium fluctuations following stimulation of metabotropic receptors through inositol-1,4,5-trisphosphate-induced Ca²⁺ release (IICR). The ER Ca²⁺ store emerges as a single interconnected pool, thus allowing for a long-range Ca²⁺ signalling via intra-ER tunnels. The fluctuations of intra-ER free Ca²⁺ concentration regulate the activity of numerous ER resident proteins responsible for post-translational protein folding and modification. Disruption of ER Ca²⁺ homeostasis results in the developing of ER stress response, which in turn controls neuronal survival. Altered ER Ca²⁺ handling may be involved in pathogenesis of various, neurodegenerative diseases including brain ischemia and Alzheimer dementia.