Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor impairs ER Ca(2+) buffering and causes neurodegeneration in primary cortical neurons

Disruption of neuronal Ca(2+) homeostasis plays a well-established role in cell death in a number of neurodegenerative disorders. Recent evidence suggests that proteolysis of the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3) R1), a Ca(2+) release channel on the endoplasmic reticulum, generates a dysregulated channel, which may contribute to aberrant Ca(2+) signaling and neurodegeneration in disease states. However, the specific effects of InsP(3) R1 proteolysis on neuronal Ca(2+) homeostasis are unknown, as are the functional contributions of this pathway to neuronal death. This study evaluates the consequences of calpain-mediated InsP(3) R1 proteolysis on neuronal Ca(2+) signaling and survival using adeno-associated viruses to express a recombinant cleaved form of the channel (capn-InsP(3) R1) in rat primary cortical neurons. Here, we demonstrate that expression of capn-InsP(3) R1 in cortical cultures reduced cellular viability. This effect was associated with increased resting cytoplasmic Ca(2+) concentration ([Ca(2+) ](i) ), increased [Ca(2+) ](i) response to glutamate, and enhanced sensitivity to excitotoxic stimuli. Together, our results demonstrate that InsP(3) R1 proteolysis disrupts neuronal Ca(2+) homeostasis, and potentially acts as a feed-forward pathway to initiate or execute neuronal death.     

A new type of Ca(2+) channel blocker that targets Ca(2+) sensors and prevents Ca(2+)-mediated apoptosis.

Calmodulin (CaM), as well as other Ca(2+) binding motifs (i.e., EF hands), have been demonstrated to be Ca(2+) sensors for several ion channel types, usually resulting in an inactivation in a negative feedback manner. This provides a novel target for the regulation of such channels. We have designed peptides that interact with EF hands of CaM in a specific and productive manner. Here we have examined whether these peptides block certain Ca(2+)-permeant channels and inhibit biological activity that is dependent on the influx of Ca(2+). We found that these peptides are able to enter the cell and directly, as well as indirectly (through CaM), block the activity of glutamate receptor channels in cultured neocortical neurons and a nonselective cation channel in Jurkat T cells that is activated by HIV-1 gp120. As a consequence, apoptosis mediated by an influx of Ca(2+) through these channels was also dose-dependently inhibited by these novel peptides. Thus, this new type of Ca(2+) channel blocker may have utility in controlling apoptosis due to HIV infection or neuronal loss due to ischemia.     

Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4.

Modulation of Ca(2+) channels by neurotransmitters provides critical control of neuronal excitability and synaptic strength. Little is known about regulation of the Ca(2+) efflux pathways that counterbalance Ca(2+) influx in neurons. We demonstrate that bradykinin and ATP significantly facilitate removal of action potential-induced Ca(2+) loads by stimulating plasma membrane Ca(2+)-ATPases (PMCAs) in rat sensory neurons. This effect was mimicked in the soma and axonal varicosities by phorbol esters and was blocked by antagonists of protein kinase C (PKC). Reduced expression of PMCA isoform 4 abolished, and overexpression of isoform 4b enhanced, PKC-dependent facilitation of Ca(2+) efflux. This acceleration of PMCA4 underlies the shortening of the action potential afterhyperpolarization produced by activation of bradykinin and purinergic receptors. Thus, isoform-specific modulation of PMCA-mediated Ca(2+) efflux represents a novel mechanism to control excitability in sensory neurons.     

Glucocorticoids prolong Ca(2+) transients in hippocampal-derived H19-7 neurons by repressing the plasma membrane Ca(2+)-ATPase-1

Calcium ions (Ca(2+)) play an important role in mediating an array of structural and functional responses in cells. In hippocampal neurons, elevated glucocorticoid (GC) levels, as seen during stress, perturb calcium homeostasis and result in altered neuronal excitability and viability. Ligand- and voltage-gated calcium channels have been the presumed targets of hormonal regulation; however, circumstantial evidence has suggested the possibility that calcium extrusion might be an important target of GC regulation. Here we demonstrate that GC-induced repression of the plasma membrane Ca(2+)-ATPase-1 (PMCA1) is an essential determinant of intracellular Ca(2+) levels ([Ca(2+)](i)) in cultured hippocampal H19-7 cells. In particular, GC treatment caused a prolongation of agonist-evoked elevation of [Ca(2+)](i) that was prevented by the expression of exogenous PMCA1. Furthermore, selective inhibition of PMCA1 using the RNA interference technique caused prolongation of Ca(2+) transients in the absence of GC treatment. Taken together, these observations suggest that GC-mediated repression of PMCA1 is both necessary and sufficient to increase agonist-evoked Ca(2+) transients by down-regulating Ca(2+) extrusion mechanisms in the absence of effects on calcium channels. Prolonged exposure to GCs, resulting in concomitant accumulation of [Ca(2+)](i), is likely to compromise neuronal function and viability.     

M6a acts as a nerve growth factor-gated Ca(2+) channel in neuronal differentiation

To elucidate the function of M6a, which is a neuron-specific membrane glycoprotein of the brain and possesses putative phosphorylation sites for protein kinase C (PKC), we established rat M6a cDNA expression vector-transfected PC12 cells. These transfectants exhibited high susceptibilities to nerve growth factor (NGF) for neuronal differentiation. Interestingly, we found that Ca(2+) influx in these transfectants was significantly augmented by the treatment of NGF, but not epidermal growth factor (EGF), which stimulates PC12 cell growth. NGF-dependent augmentation of Ca(2+) influx was detected within 3h and severely inhibited by EGTA- and PKC-specific inhibitors. Anti-M6 antibody suppressed both NGF-triggered Ca(2+) influx and neuronal differentiation. These results support the idea that M6a implicates in neuronal differentiation as a novel Ca(2+) channel gated selectively by phosphorylation with PKC in the downstream of NGF signaling pathway.     

Properties of voltage-gated Ca(2+) channels in rabbit ventricular myocytes expressing Ca(2+) channel alpha(1E) cDNA

Using the whole-cell patch-clamp technique, we have studied the properties of alpha(1E) Ca(2+) channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current (I(Ca,L)). Expression of green fluorescent protein significantly decreased the I(Ca,L). By contrast, expression of alpha(1E) with beta(2b) and alpha(2)/delta significantly increased the total Ca(2+) current, and in these cells a Ca(2+) antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni(2+) and was little affected by changing the charge carrier from Ca(2+) to Ba(2+) or by beta-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of alpha(1E) did not generate T-like current in cardiac myocytes. On the other hand, expression of alpha(1E) decreased I(Ca,L) and slowed the I(Ca,L) inactivation. This inactivation slowing was attenuated by the beta(2b) coexpression, suggesting that the alpha(1E) may slow the inactivation of I(Ca,L) by scrambling with alpha(1C) for intrinsic auxiliary beta.     

Regulation of (1-3)-beta-glucan-stimulated Ca(2+) influx by protein kinase C in NR8383 alveolar macrophages

Stimulation of (1-3)-beta-glucan receptors results in Ca(2+) influx through receptor-operated channels in alveolar macrophages (AMs), but the mechanism(s) regulating Ca(2+) influx is still undefined. In this study we investigated the role of protein kinase C (PKC) regulation of Ca(2+) influx in the NR8383 AM cell line using the particulate (1-3)-beta-glucan receptor agonist zymosan. PKC inhibition with calphostin C (CC) or bisindolymaleimide I (BSM) significantly reduced zymosan-induced Ca(2+) influx, whereas activation of PKC with phorbol-12-myristate 13-acetate (PMA) or 1, 2-dioctanoyl-sn-glycerol (DOG) mimicked zymosan, inducing a concentration-dependent Ca(2+) influx. This influx was dependent on extracellular Ca(2+) and inhibited by the receptor-operated Ca(2+) channel blocker SK&F96365, indicating that zymosan and PKC activate Ca(2+) influx through a similar pathway. NR8383 AMs expressed one new PKC isoform (delta) and two atypical PKC isoforms (iota and lambda), but conventional PKC isoforms were not present. Stimulation with zymosan resulted in a translocation of PKC-delta from the cytosol to the membrane fraction. Furthermore, inhibition of protein tyrosine kinases (PTKs) with genistein prevented zymosan-stimulated Ca(2+) influx and PKC-delta translocation. These results suggest that PKC-delta plays a critical role in regulating (1-3)-beta-glucan receptor activated Ca(2+) influx in NR8383 AMs and PKC-delta translocation is possibly dependent on PTK activity.     

The role of Ca2+ channel modulation in the neuroprotective actions of estrogen in beta-amyloid protein and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) cytotoxic models

Physiologically relevant concentrations of 17beta-estradiol (E2) are neuroprotective in both beta-amyloid protein 25-35 (Abeta) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced cytotoxicity in SK-N-SH cells. MPTP, but not Abeta, induces apoptosis in this cell line. The L-type calcium channel blocker nifedipine or decreased extracellular Ca(2+) concentration blocked Abeta-induced cell death, but not MPTP-induced cell death. Other blockers selective for different Ca(2+) channel subtypes had no effects on either Abeta or MPTP induced death. Western blot analysis for L-type Ca(2+) channel alpha(1)-subunits demonstrated that Abeta increases the expression of the neuronal alpha(1C) and alpha(1D) subunits of L-type channels. Both E2 and nifedipine inhibit the increase in expression of these by Abeta. MPTP also increases expression of alpha(1C) and alpha(1D), but the increases were not influenced by E2 or nifedipine. These observations suggested that Abeta cytotoxicity in SK-N-SH cells may involve increased availability of calcium to cells, whereas MPTP induced cytotoxicity does not require extracellular Ca(2+). Both cytotoxic models were associated with increased expression of Ca(2+) channel alpha(1) subunits, and neuroprotection associated with inhibition of that increase. These studies reveal that nifedipine, in addition to its direct action of nifedipine on Ca(2+) channels, may also protect neurons from Abeta toxicity through the suppression of the channel protein overexpression. A new action of dihydropyridines (DHPs) may be considered in the regulation of calcium homeostasis.     

Activity- and Ca(2+)-dependent modulation of surface expression of brain-derived neurotrophic factor receptors in hippocampal neurons

Brain-derived neurotrophic factor (BDNF) has been shown to regulate neuronal survival and synaptic plasticity in the central nervous system (CNS) in an activity-dependent manner, but the underlying mechanisms remain unclear. Here we report that the number of BDNF receptor TrkB on the surface of hippocampal neurons can be enhanced by high frequency neuronal activity and synaptic transmission, and this effect is mediated by Ca(2+) influx. Using membrane protein biotinylation as well as receptor binding assays, we show that field electric stimulation increased the number of TrkB on the surface of cultured hippocampal neurons. Immunofluorescence staining suggests that the electric stimulation facilitated the movement of TrkB from intracellular pool to the cell surface, particularly on neuronal processes. The number of surface TrkB was regulated only by high frequency tetanic stimulation, but not by low frequency stimulation. The activity dependent modulation appears to require Ca(2+) influx, since treatment of the neurons with blockers of voltage-gated Ca(2+) channels or NMDA receptors, or removal of extracellular Ca(2+), severely attenuated the effect of electric stimulation. Moreover, inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) significantly reduced the effectiveness of the tetanic stimulation. These findings may help us to understand the role of neuronal activity in neurotrophin function and the mechanism for receptor tyrosine kinase signaling.     

Human group IIA secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels in cultured rat cortical neurons

Mammalian group IIA secretory phospholipase A2 (sPLA2-IIA) generates prostaglandin D2 (PGD2) and triggers apoptosis in cortical neurons. However, mechanisms of PGD2 generation and apoptosis have not yet been established. Therefore, we examined how second messengers are involved in the sPLA2-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons. sPLA2-IIA potentiated a marked influx of Ca2+ into neurons before apoptosis. A calcium chelator and a blocker of the L-type voltage-sensitive Ca2+ channel (L-VSCC) prevented neurons from sPLA2-IIA-induced neuronal cell death in a concentration-dependent manner. Furthermore, the L-VSCC blocker ameliorated sPLA2-IIA-induced morphologic alterations and apoptotic features such as condensed chromatin and fragmented DNA. Other blockers of VSCCs such as N type and P/Q types did not affect the neurotoxicity of sPLA2-IIA. Blockers of L-VSCC significantly suppressed sPLA2-IIA-enhanced Ca2+ influx into neurons. Moreover, reactive oxygen species (ROS) were generated prior to apoptosis. Radical scavengers reduced not only ROS generation, but also the sPLA2-IIA-induced Ca2+ influx and apoptosis. In conclusion, we demonstrated that sPLA2-IIA potentiates the influx of Ca2+ into neurons via L-VSCC. Furthermore, the present study suggested that eicosanoids and ROS generated during arachidonic acid oxidative metabolism are involved in sPLA2-IIA-induced apoptosis in cooperation with Ca2+.     

Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.     

Unconventional neuroprotection against Ca2+ -dependent insults by metalloporphyrin catalytic antioxidants

We evaluated whether both inert and catalytically active metalloporphyrin antioxidants, meso-substituted with either phenyl-based or N-alkylpyridinium-based groups, suppress Ca(2+)-dependent neurotoxicity in cell culture models of relevance to cerebral ischemia. Representatives from both metalloporphyrin classes, regardless of antioxidant strength, protected cultured cortical neurons or PC-12 cultures against the Ca(2+) ionophores ionomycin or A23187, by suppressing neurotoxic Ca(2+) influx. Some metalloporphyrins suppressed excitotoxic Ca(2+) influx indirectly induced by the Ca(2+) ionophores in cortical neurons. Metalloporphyrins did not quench intracellular fluorescence, suggesting localization to the plasma membrane interface and/or interference with Ca(2+) ionophores. Metalloporphyrins suppressed ionomycin-induced Mn(2+) influx, but did not protect cortical neurons against pyrithione, a Zn(2+) ionophore. In other Ca(2+)-dependent paradigms, Ca(2+) influx via plasma membrane depolarization, but not through reversal of plasmalemmal Na(+)/Ca(2+) exchangers, was modestly suppressed by Mn(III)meso-tetrakis(4-benzoic acid)porphyrin (Mn(III)TBAP) or by an inert analog, Zn(II)TBAP. Mn(III)TBAP and Zn(II)TBAP potently protected cortical neurons against long-duration oxygen-glucose deprivation (OGD), performed in the presence of antagonists of NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and L-type voltage-gated Ca(2+) channels, raising the possibility of an unconventional mode of blockade of transient receptor protein melastatin 7 channels by a metalloTBAP family of metalloporphyrins. The present study extends the range of Ca(2+)-dependent insults for which metalloporphyrins demonstrate unconventional neuroprotection. MetalloTBAPs appear capable of targeting an OGD temporal continuum.     

Topiramate reduces AMPA-induced Ca(2+) transients and inhibits GluR1 subunit phosphorylation in astrocytes from primary cultures

Topiramate (TPM) is a structurally novel broad spectrum anticonvulsant known to have a negative modulatory effect on the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate subtypes of glutamate receptors and some types of voltage-gated Na(+) and Ca(2+) channels, and a positive modulatory effect on some types of gamma-aminobutyric acid(A) (GABA(A)) receptors and at least one type of K(+) channels in neurons. In an earlier work, we showed that the negative modulatory effect of TPM (100 mum) on AMPA/kainate receptors in neurons is dependent on TPM modulation of the phosphorylation state of these receptors. In this work, we investigated the effect of TPM on AMPA-induced intracellular calcium ([Ca(2+)](i)) responses in cultured rat cortical astrocytes, with special interest in intracellular mechanisms. Here, we report that the ability of TPM (1-100 mum) to inhibit AMPA-induced accumulation of Ca(2+) in astrocytes is inversely related to the level of protein kinase A (PKA) -mediated phosphorylation of channels activated by AMPA. The level of receptor phosphorylation was further determined with western blot using phosphorylation specific antibodies that recognize the glutamate receptor 1 (GluR1) subunit phosphorylated on Ser845. These results demonstrated that, even in cultured cortical astrocytes, TPM significantly reduced the phophorylation level of GluR1 subunits. Furthermore, it was shown that TPM binds to AMPA receptors in the dephosphorylated state and thereby exerts an allosteric modulatory effect on the ion channel.     

Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.     

Alpha-latrotoxin Triggers Extracellular Ca^2＋-dependent Exocytosis and Sensitizes Fusion Machinery in Endocrine Cells

α-Latrotoxin from the venom of black widow spider induces and augments neurotransmitterand hormone release by way of extracellular Ca~(2 ) influx and cellular signal transduction pathways.By usingwhole cell current and capacitance recording,the photolysis of card Ca~(2 ),and Ca~(2 ) microfluorometry andamperometry,we investigated the stimulating effect and mechá(?)ism of α-latrotoxin on exocytosis in ratpancreatic β cells,LβT2 cells and latrophilin plasmid-transfected INS-1 cells.Our data indicated that:(1)α-latrotoxin increased cytosolic Ca~(2 ) concentration through the formation of cation-permitting pores and sub-sequent Ca~(2 ) influx with the presence of extracellular Ca~(2 );(2)α-latrotoxin stimulated exocytosis in normalbath solution and its stimulating effect on secretion was eradicated in Ca~(2 )-free bath solution; and (3)α-latrotoxin sensitized the molecular machinery of fusion through activation of protein kinase C and increasedthe response of cells to Ca~(2 ) photolysed by a flash of ultraviolet light.In summary,α-latrotoxin inducedexocytosis by way of Ca~(2 ) influx and accelerated vesicle fusion by the sensitization of fusion machinery.     

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) has InsP(3)-independent gating and disrupts intracellular Ca(2+) homeostasis

The type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) is a ubiquitous intracellular Ca(2+) release channel that is vital to intracellular Ca(2+) signaling. InsP(3)R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca(2+) homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP(3)R1 and utilized a recombinant truncated form of the channel (capn-InsP(3)R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP(3)R1 revealed InsP(3)-independent gating and high open probability (P(o)) under optimal cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) conditions. However, some [Ca(2+)](i) regulation of the cleaved channel remained, with a lower P(o) in suboptimal and inhibitory [Ca(2+)](i). Expression of capn-InsP(3)R1 in N2a cells reduced the Ca(2+) content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca(2+) loading compared with control cells expressing full-length InsP(3)R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP(3)R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP(3)R1 generates a dysregulated channel that disrupts cellular Ca(2+) homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP(3)R1 in a clinically relevant injury model, suggesting that Ca(2+) leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.     

Ca(2+) homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hFis1

Mitochondria modulate Ca(2+) signals by taking up, buffering, and releasing Ca(2+) at key locations near Ca(2+) release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca(2+) homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca(2+) released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca(2+) entering across plasma membrane channels, because the Ca(2+) ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca(2+) entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/rotenone reduced CCE by approximately 35%. These observations indicate that close contact to Ca(2+) influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca(2+) propagation within interconnected mitochondria.     

Voltage- and calcium-dependent inactivation in high voltage-gated Ca(2+) channels

Calcium influx into cardiac myocytes via voltage-gated Ca channels is a key step in initiating the contractile response. During prolonged depolarizations, toxic Ca(2+) overload is prevented by channel inactivation occurring through two different processes identified by their primary trigger: voltage or intracellular Ca(2+). In physiological situations, cardiac L-type (Ca(V)1.2) Ca(2+) channels inactivate primarily via Ca(2+)-dependent inactivation (CDI), while neuronal P/Q (Ca(V)2.1) Ca(2+) channels use preferentially voltage-dependent inactivation (VDI). In certain situations however, these two types of channels have been shown to be able to inactivate by both processes. From a structural view point, the rearrangement occurring during CDI and VDI is not precisely known, but functional studies have underlined the role played by at least 2 channel sequences: a C-terminal binding site for the Ca(2+) sensor calmodulin, essential for CDI, and the loop connecting domains I and II, essential for VDI. The conserved regulation of VDI and CDI by the auxiliary channel beta subunit strongly suggests that these two mechanisms may use a set of common protein-protein interactions that are influenced by the auxiliary subunit. We will review our current knowledge of these interactions. New data are presented on L-P/Q (Ca(V)1.2/Ca(V)2.1) channel chimera that confirm the role of the I-II loop in VDI and CDI, and reveal some of the essential steps in Ca(2+) channel inactivation.