The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca(2+) levels to α-synuclein toxicity in Parkinson's disease models.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons, which arises from a yet elusive concurrence between genetic and environmental factors. The protein α-synuclein (αSyn), the principle toxic effector in PD, has been shown to interfere with neuronal Ca(2+) fluxes, arguing for an involvement of deregulated Ca(2+) homeostasis in this neuronal demise. Here, we identify the Golgi-resident Ca(2+)/Mn(2+) ATPase PMR1 (plasma membrane-related Ca(2+)-ATPase 1) as a phylogenetically conserved mediator of αSyn-driven changes in Ca(2+) homeostasis and cytotoxicity. Expression of αSyn in yeast resulted in elevated cytosolic Ca(2+) levels and increased cell death, both of which could be inhibited by deletion of PMR1. Accordingly, absence of PMR1 prevented αSyn-induced loss of dopaminergic neurons in nematodes and flies. In addition, αSyn failed to compromise locomotion and survival of flies when PMR1 was absent. In conclusion, the αSyn-driven rise of cytosolic Ca(2+) levels is pivotal for its cytotoxicity and requires PMR1.

Glutamate sensing with enzyme-modified floating-gate field effect transistors.

Neurotransmitter release is the key factor of chemical messaging in the brain. Fast, sensitive and in situ detection of single cell neurotransmitter release is essential for the investigation of synaptic transmission under physiological or pathophysiological conditions. Although various techniques have been developed for detecting neurotransmitter release both in vitro and in vivo, the sensing of such events still remains challenging. First of all, the amount of neurotransmitter released during synaptic transmission is unknown because of the limited number of molecules released and the fast diffusion and reuptake of these molecules after release. On the other hand, advances in microelectronic biosensor devices have made possible the fast detection of various analytes with high sensitivity and selectivity. Specifically, enzyme-modified field-effect (ENFET) devices are attractive for such applications due to their fast response, small dimensions and the possibility to integrate a large number of sensors on the same chip. In this paper, we present a floating-gate FET device coated with glutamate oxidase (GLOD) layer. The surface chemistry was optimized for maximal enzyme loading and long-term stability, and characterized by quartz crystal microbalance and colorimetric assays. Enzyme loading was largest on poly-L-lysin-based surfaces combined with glutaraldehyde. The surface chemistry showed excellent stability for at least one month in Tris buffers stored at 4 degrees C. A glutamate detection limit of 10(-7) M has been obtained with the GLOD-coated FET and our sensor proved to be selective to glutamate only. We show that this biosensor is a promising tool for the in vitro detection of glutamate and can be extended to other neurotransmitters.

Differential contribution of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons.

Plasma membrane chloride (Cl(-)) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active Cl(-) uptake mechanism) in Cl(-) handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin-perforated patch-clamp recordings revealed that DRGNs accumulate intracellular Cl(-) through a bumetanide- and Na(+)-sensitive mechanism, indicative of the functional expression of NKCC1. Western blotting confirmed the expression of NKCC1 in both DRGNs and MNs, but immunocytochemistry experiments showed a restricted expression in dendrites of MNs, which contrasts with a homogeneous expression in DRGNs. Both MNs and DRGNs could be readily loaded with or depleted of Cl(-) during GABA(A) receptor activation at depolarizing or hyperpolarizing membrane potentials. After loading, the rate of recovery to the resting Cl(-) concentration (i.e., [Cl(-)](i) decrease) was similar in both cell types and was unaffected by lowering the extracellular Na(+) concentration. In contrast, the recovery on depletion (i.e., [Cl(-)](i) increase) was significantly faster in DRGNs in control conditions but not in low extracellular Na(+). The experimental observations could be reproduced by a mathematical model for intracellular Cl(-) kinetics, in which DRGNs show higher NKCC1 activity and smaller Cl(-)-handling volume than MNs. On the basis of these results, we conclude that embryonic DRGNs show a higher somatic functional expression of NKCC1 than embryonic MNs. The high NKCC1 activity in DRGNs is important for maintaining high [Cl(-)](i), whereas lower NKCC1 activity in MNs allows large [Cl(-)](i) variations during neuronal activity.

Deficiency in apoptotic effectors Bax and Bak reveals an autophagic cell death pathway initiated by photodamage to the endoplasmic reticulum.

Efficient exploitation of cell death mechanisms for therapeutic purpose requires the identification of the molecular events committing cancer cells to death and the intracellular elements of the pro-death and pro-survival machinery activated in response to the anticancer therapy. Photodynamic therapy (PDT) is a paradigm of anticancer therapy utilizing the generation of reactive oxygen species to kill the cancer cells. In this study we have identified the photodamage to the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) pump and consequent loss in the ER-Ca(2+) homeostasis as the most apical molecular events leading to cell death in hypericin-photosensitized cells. Downstream of the ER-Ca(2+) emptying, both caspase-dependent and -independent pathways are activated to ensure cell demise. The induction of apoptosis as a cell death modality is dependent on the availability of proapopototic Bax and Bak proteins, which are essential effectors of the mitochondrial outer membrane permeabilization (MOMP) and subsequent caspase activation. In Bax(-/-)/Bak(-/-) cells a nonapoptotic pathway dependent on sustained autophagy commits the oxidatively damaged cells to death. These results argue that the decision to die in this paradigm of oxidative stress is taken upstream of Bax-dependent MOMP and that the irreversible photodamage to the ER induced by hypericin-PDT acts as a trigger for an autophagic cell death pathway in apoptosis-deficient cells.

Cytosolic Ca2+ signals depending on the functional state of the Golgi in HeLa cells.

The Golgi apparatus is, like the endoplasmic reticulum, an inositol-1,4,5-trisphosphate-sensitive Ca2+ store, but its role in setting up Ca2+ signals is not well understood. We have now measured histamine-induced Ca2+ signals in HeLa cells pretreated with brefeldin A, a fungal metabolite that leads to the fragmentation and subsequent disappearance of the Golgi apparatus by its reabsorption within the endoplasmic reticulum. Ca2+ responses in which the free cytoplasmic Ca2+ concentration returned to resting levels during the histamine stimulation (mainly baseline Ca2+ oscillations or a single Ca2+ peak) occurred more often in brefeldin A pretreated cells, resulting in a lower Ca2+ plateau in population measurements. The latencies before the onset of the Ca2+ signals were longer after brefeldin A pretreatment. These results suggest that the integrity of the Golgi apparatus contributes to the shaping of intracellular Ca2+ signals.

[Intracellular calcium-releasing channels as cellular targets for immunophilins: a molecular, functional and structural analysis]. / Intracellulaire calcium-vrijzettingskanalen als cellulaire doelwitten voor immunofilines: een moleculaire, functionele en structurele analyse.

In this study, the FKBP12-binding properties of IP3Rs and RyRs were compared. Although the primary sequence of IP3Rs en RyRs contained a putative FKBP12-binding site, the functional, molecular and structural properties of these sites appeared to be completely different. For RyRs, FKBPs appear to function as associated proteins that are important for the functional regulation of the channel, thereby stabilizing the RyR complex. For IP3Rs, FKBPs might be involved in the de novo protein synthesis of the IP3Rs and the folding of the peptide chain to a functional IP3R protein, thereby functioning as helper enzymes. Hence, it is very unlikely that they function as associated regulatory proteins of the IP3R. In addition, we provided evidence that FKBP 12 is also an important regulating protein of the Ca(2+)-flux properties of the RyR3. FKBP12 clearly modulated both RyR3-mediated global and local Ca(2+)-responses.

Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments.

Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.

Inositol trisphosphate producing agonists do not mobilize the thapsigargin-insensitive part of the endoplasmic-reticulum and Golgi Ca2+ store.

Non-mitochondrial intracellular Ca2+ stores contain both thapsigargin-sensitive sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and thapsigargin-insensitive secretory-pathway Ca2+-ATPases (SPCA1). We now have studied the Ca2+-release properties of the compartments associated with these pumps in intact, i.e. non-permeabilized, cells of different origin (HeLa, keratinocytes, 16HBE14o-, COS-1, A7r5) and with different approaches (45Ca2+ fluxes, Ca2+ imaging and measurements of the free luminal [Ca2+] in the endoplasmic-reticulum and the Golgi apparatus using targeted aequorin). Application of an extracellular agonist in the absence of thapsigargin induced in all cells a Ca2+ release from both the endoplasmic-reticulum and the Golgi apparatus. The agonists were not able to release Ca2+ in the presence of 10 microM thapsigargin, except in COS-1 cells overexpressing SPCA1, where this pump not only appeared in the Golgi compartment but also overflowed into the agonist-sensitive part of the endoplasmic-reticulum. We conclude that the subcompartments of the endoplasmic-reticulum and of the Golgi complex that endogenously express SPCA1 are insensitive to agonist stimulation.

Calcineurin and intracellular Ca2+-release channels: regulation or association?

The Ca(2+)- and calmodulin-dependent phosphatase calcineurin was reported to interact with the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor (RyR) and to modulate their phosphorylation status and activity. However, controversial data on the molecular mechanisms involved and on the functional relevance of calcineurin for these channel-complexes have been described. Hence, we will focus on the functional importance of calcineurin for IP(3)R and RyR function and on the different mechanisms by which Ca(2+)-dependent dephosphorylation can affect the gating of those intracellular Ca(2+)-release channels. Since many studies made use of immunosuppressive drugs that are inhibiting calcineurin activity, we will also have to take the different side effects of these drugs into account for the proper interpretation of the effects of calcineurin on intracellular Ca(2+)-release channels. In addition, it became recently known that various other phosphatases and kinases can associate with these channels, thereby forming macromolecular complexes. The relevance of these enzymes for IP(3)R and RyR functioning will be reviewed since in some cases they could interfere with the effects ascribed to calcineurin. Finally, we will discuss the downstream effects of calcineurin on the regulation of the expression levels of intracellular Ca(2+)-release channels as well as the relation between IP(3)R- and RyR-mediated Ca(2+) release and calcineurin-dependent gene expression.

Chloride influx aggravates Ca2+-dependent AMPA receptor-mediated motoneuron death.

AMPA receptor-mediated excitotoxicity has been implicated in the pathogenesis of stroke, neurotrauma, epilepsy, and many neurodegenerative diseases such as motoneuron disease. We studied the role of Cl- in AMPA receptor-mediated Ca2+-dependent excitotoxicity in cultured rat spinal motoneurons. Using the gramicidin perforated patch-clamp technique, the intracellular Cl- concentration could be calculated from the reversal potential of the GABA-induced current. The membrane depolarization caused by AMPA receptor stimulation resulted in Cl- influx through 5-nitro-2(3-phenylpropyl-amino) benzoic acid- and niflumic acid-sensitive Cl- channels. Cl- influx during AMPA receptor stimulation aggravated excitotoxic motoneuron death by two mechanisms: an increase of AMPA receptor conductance and an elevation of the Ca2+ driving force through a partial repolarization. The Cl- influx during AMPA receptor stimulation was enhanced by coadministration of GABA. This resulted in an increased Ca2+ influx and an enhanced cell death, suggesting that concomitant GABAergic stimulation may aggravate excitotoxic motoneuron death.

Similar Ca(2+)-signaling properties in keratinocytes and in COS-1 cells overexpressing the secretory-pathway Ca(2+)-ATPase SPCA1.

Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.

Pharmacology of inositol trisphosphate receptors.

In almost all cells, cytosolic Ca(2+) is a crucial intracellular messenger, regulating many cellular processes. In non-excitable as well as in some excitable cells, Ca(2+) release from the intracellular stores into the cytoplasm is primarily initiated by the second messenger inositol 1,4,5-trisphosphate (IP(3)), which interacts with the IP(3) receptor (IP(3)R), a tetrameric intracellular Ca(2+)-release channel. This review focuses on the pharmacological modulation of the various functionally important sub-domains of the IP(3)R, including the IP(3)-binding domain, calmodulin-binding sites, adenine nucleotide-binding sites and the sites for interaction for FK506-binding proteins and other regulators. We will particularly focus on the pharmacological tools that interfere with these domains and discuss their relative specificity for the IP(3)R, thereby indicating their potential usefulness for unraveling the complex functional regulation of the IP(3)R.

GluR2-dependent properties of AMPA receptors determine the selective vulnerability of motor neurons to excitotoxicity.

AMPA receptor-mediated excitotoxicity has been implicated in the selective motor neuron loss in amyotrophic lateral sclerosis. In some culture models, motor neurons have been shown to be selectively vulnerable to AMPA receptor agonists due to Ca(2+) influx through Ca(2+)-permeable AMPA receptors. Because the absence of GluR2 in AMPA receptors renders them highly permeable to Ca(2+) ions, it has been hypothesized that the selective vulnerability of motor neurons is due to their relative deficiency in GluR2. However, conflicting evidence exists about the in vitro and in vivo expression of GluR2 in motor neurons, both at the mRNA and at the protein level. In this study, we quantified electrophysiological properties of AMPA receptors, known to be dependent on the relative abundance of GluR2: sensitivity to external polyamines, rectification index, and relative Ca(2+) permeability. Cultured rat spinal cord motor neurons were compared with dorsal horn neurons (which are resistant to excitotoxicity) and with motor neurons that survived an excitotoxic insult. Motor neurons had a higher sensitivity to external polyamines, a lower rectification index, and a higher relative Ca(2+) permeability ratio than dorsal horn neurons. These findings confirm that motor neurons are relatively deficient in GluR2. The AMPA receptor properties correlated well with each other and with the selective vulnerability of motor neurons because motor neurons surviving an excitotoxic event had similar characteristics as dorsal horn neurons. These data indicate that the relative abundance of GluR2 in functional AMPA receptors may be a major determinant of the selective vulnerability of motor neurons to excitotoxicity in vitro.

Na(+) entry through AMPA receptors results in voltage-gated k(+) channel blockade in cultured rat spinal cord motoneurons.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents, evoked with the agonist kainate, were studied with the gramicidin perforated-patch-clamp technique in cultured rat spinal cord motoneurons. Kainate-induced currents could be blocked by the AMPA receptor antagonist LY 300164 and displayed an apparent strong inward rectification. This inward rectification was not a genuine property of AMPA receptor currents but was a result of a concomitant decrease in outward current at potentials positive to -40.5 +/- 1.3 mV. The AMPA receptor current itself was nearly linear (rectification index 0.91). The kainate-inhibited outward current had a reversal potential close to the estimated K(+) equilibrium potential and was blocked by 30 mM tetraethylammonium. When voltage steps were applied, it was found that kainate inhibited both the delayed rectifier K(+) current K(V) and the transient outward K(+) current, K(A). The kainate-induced inhibition of K(+) currents was dependent on ion flux through the AMPA receptor, because no change in the membrane conductance was noticed in the presence of LY 300164. Removing extracellular Ca(2+) had no effect, whereas replacing extracellular Na(+) or clamping the membrane close to the estimated Na(+) equilibrium potential during kainate application attenuated the inhibition of the K(+) current. Sustained Na(+) influx induced by application of the Na(+) ionophore monensin could mimic the effect of kainate on K(+) conductance. These findings demonstrate that Na(+) influx through AMPA receptors results in blockade of voltage-gated K(+) channels.

IP(3)-mediated Ca(2+) signals in human neuroblastoma SH-SY5Y cells with exogenous overexpression of type 3 IP(3) receptor.

Human neuroblastoma SH-SY5Y cells, predominantly expressing type 1 inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), were stably transfected with IP(3)R type 3 (IP(3)R3) cDNA. Immunocytochemistry experiments showed a homogeneous cytoplasmic distribution of type 3 IP(3)Rs in transfected and selected high expression cloned cells. Using confocal Ca(2+) imaging, carbachol (CCh)-induced Ca(2+) release signals were studied. Low CCh concentrations (< or = 750 nM) evoked baseline Ca(2+) oscillations. Transfected cells displayed a higher CCh responsiveness than control or cloned cells. Ca(2+) responses varied between fast, large Ca(2+) spikes and slow, small Ca(2+) humps, while in the clone only Ca(2+) humps were observed. Ca(2+) humps in the transfected cells were associated with a high expression level of IP(3)R3. At high CCh concentrations (10 microM) Ca(2+) transients in transfected and cloned cells were similar to those in control cells. In the clone exogenous IP(3)R3 lacked the C-terminal channel domain but IP(3)-binding capacity was preserved. Transfected cells mainly expressed intact type 3 IP(3)Rs but some protein degradation was also observed. We conclude that in transfected cells expression of functional type 3 IP(3)Rs causes an apparent higher affinity for IP(3). In the clone, the presence of degraded receptors leads to an efficient cellular IP(3) buffer and attenuated IP(3)-evoked Ca(2+) release.

Washing out of lipophilic compounds induces a transient increase in the passive Ca(2+) leak in permeabilized A7r5 cells.

We have investigated how the immunosuppressant drug FK506 affected the basal Ca(2+) leak in permeabilized A7r5 cells. Non-mitochondrial Ca(2+) stores loaded to steady state with Ca(2+) slowly lost their accumulated Ca(2+) during incubation in a Ca(2+)-free efflux medium. FK506 up to 100 microM had no effect on the basal Ca(2+) leak. In contrast, the rate of Ca(2+) release proceeded much faster immediately after washing out FK506. The increase in rate of Ca(2+) release after washing out of this compound depended on both its initial concentration and on the time of pre-incubation. A similar effect was also observed after removing another immunosuppressant drug (rapamycin) and after removing the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin C. Since all these substances have a high octanol/H(2)O partition coefficient and accumulate in the endoplasmic reticulum membrane, we suggest that the transient increase in the basal Ca(2+) leak is due to the sudden removal of these lipophilic substances from the membrane.

An alpha-mercaptoacrylic acid derivative (PD150606) inhibits selective motor neuron death via inhibition of kainate-induced Ca2+ influx and not via calpain inhibition.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron death. The exact mechanism responsible for this selectivity is not clear, although it is known that motor neurons are very sensitive to excitotoxicity. This high sensitivity is due to a high density of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors on their surface and to a limited Ca(2+) buffering capacity. Ca(2+) can enter the cell upon stimulation through voltage-operated Ca(2+) channels and through the Ca(2+)-permeable portion of AMPA receptors. How this Ca(2+) kills motor neurons is incompletely understood. In the present study, we report that kainate (KA)-induced motor neuron death is purely mediated through Ca(2+) entering motor neurons through Ca(2+)-permeable AMPA receptors and that voltage-operated Ca(2+) channels play no significant role. In contrast to what has been observed in other neuronal models or after N-methyl-D-aspartate stimulation, NO synthase inhibition and a number of antioxidants did not protect motor neurons from KA-induced death. Only PD150606, derived from alpha-mercaptoacrylic acid and considered as a selective calpain antagonist, inhibited dose-dependently the KA-induced motor neuron death. However, other calmodulin and calpain inhibitors were not effective. At least part of the inhibitory effect of PD150606 is due to an irreversible inhibition of the Ca(2+) influx through the Ca(2+)-permeable AMPA receptor. These results demonstrate the interesting property of PD150606 to interfere with excitotoxicity-dependent motor neuron death and show that PD150606 is not an exclusive calpain/calmodulin antagonist.

The conserved sites for the FK506-binding proteins in ryanodine receptors and inositol 1,4,5-trisphosphate receptors are structurally and functionally different.

We compared the interaction of the FK506-binding protein (FKBP) with the type 3 ryanodine receptor (RyR3) and with the type 1 and type 3 inositol 1,4,5-trisphosphate receptor (IP(3)R1 and IP(3)R3), using a quantitative GST-FKBP12 and GST-FKBP12.6 affinity assay. We first characterized and mapped the interaction of the FKBPs with the RyR3. GST-FKBP12 as well as GST-FKBP12.6 were able to bind approximately 30% of the solubilized RyR3. The interaction was completely abolished by FK506, strengthened by the addition of Mg(2+), and weakened in the absence of Ca(2+) but was not affected by the addition of cyclic ADP-ribose. By using proteolytic mapping and site-directed mutagenesis, we pinpointed Val(2322), located in the central modulatory domain of the RyR3, as a critical residue for the interaction of RyR3 with FKBPs. Substitution of Val(2322) for leucine (as in IP(3)R1) or isoleucine (as in RyR2) decreased the binding efficiency and shifted the selectivity to FKBP12.6; substitution of Val(2322) for aspartate completely abolished the FKBP interaction. Importantly, the occurrence of the valylprolyl residue as alpha-helix breaker was an important determinant of FKBP binding. This secondary structure is conserved among the different RyR isoforms but not in the IP(3)R isoforms. A chimeric RyR3/IP(3)R1, containing the core of the FKBP12-binding site of IP(3)R1 in the RyR3 context, retained this secondary structure and was able to interact with FKBPs. In contrast, IP(3)Rs did not interact with the FKBP isoforms. This indicates that the primary sequence in combination with the local structural environment plays an important role in targeting the FKBPs to the intracellular Ca(2+)-release channels. Structural differences in the FKBP-binding site of RyRs and IP(3)Rs may contribute to the occurrence of a stable interaction between RyR isoforms and FKBPs and to the absence of such interaction with IP(3)Rs.

Baseline cytosolic Ca2+ oscillations derived from a non-endoplasmic reticulum Ca2+ store.

Cytosolic Ca(2+) oscillations can be due to cycles of release and re-uptake of internally stored Ca(2+). To investigate the nature of these Ca(2+) stores, we expressed the Pmr1 Ca(2+) pump of Caenorhabditis elegans in COS-1 cells and pretreated the cells with thapsigargin to prevent Ca(2+) uptake by the sarco(endo)plasmic reticulum Ca(2+)-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca(2+) store that was less leaky for Ca(2+) than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca(2+) with a regenerative Ca(2+) signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca(2+) spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca(2+)-ATPase. Abortive Ca(2+) spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca(2+) store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca(2+) spiking.