The endoplasmic reticulum and neuronal calcium signalling

The endoplasmic reticulum (ER) is a multifunctional signalling organelle regulating a wide range of neuronal functional responses. The ER is intimately involved in intracellular Ca(2+) signalling, producing local or global cytosolic calcium fluctuations via Ca(2+)-induced Ca(2+) release (CICR) or inositol-1,4,5-trisphosphate-induced Ca(2+) release (IICR). The CICR and IICR are controlled by two subsets of Ca(2+) release channels residing in the ER membrane, the Ca(2+)-gated Ca(2+) release channels, generally known as ryanodine receptors (RyRs) and InsP(3)-gated Ca(2+) release channels, referred to as InsP(3)-receptors (InsP(3)Rs). Both types of Ca(2+) release channels are expressed abundantly in nerve cells and their activation triggers cytoplasmic Ca(2+) signals important for synaptic transmission and plasticity. The RyRs and InsP(3)Rs show heterogeneous localisation in distinct cellular sub-compartments, conferring thus specificity in local Ca(2+) signals. At the same time, the ER Ca(2+) store emerges as a single interconnected pool fenced by the endomembrane. The continuity of the ER Ca(2+) store could play an important role in various aspects of neuronal signalling. For example, Ca(2+) ions may diffuse within the ER lumen with comparative ease, endowing this organelle with the capacity for "Ca(2+) tunnelling". Thus, continuous intra-ER Ca(2+) highways may be very important for the rapid replenishment of parts of the pool subjected to excessive stimulation (e.g. in small compartments within dendritic spines), the facilitated removal of localised Ca(2+) loads, and finally in conveying Ca(2+) signals from the site of entry towards the cell interior and nucleus.     

Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress and cell death in liver cells

Chronic exposure to elevated free fatty acids, in particular long chain saturated fatty acids, provokes endoplasmic reticulum (ER) stress and cell death in a number of cell types. The perturbations to the ER that instigate ER stress and activation of the unfolded protein in response to fatty acids in hepatocytes have not been identified. The present study employed H4IIE liver cells and primary rat hepatocytes to examine the hypothesis that saturated fatty acids induce ER stress via effects on ER luminal calcium stores. Exposure of H4IIE liver cells and primary hepatocytes to palmitate and stearate reduced thapsigargin-sensitive calcium stores and increased biochemical markers of ER stress over similar time courses (6 h). These changes preceded cell death, which was only observed at later time points (16 h). Co-incubation with oleate prevented the reduction in calcium stores, induction of ER stress markers and cell death observed in response to palmitate. Inclusion of calcium chelators, BAPTA-AM or EGTA, reduced palmitate- and stearate-mediated enrichment of cytochrome c in post-mitochondrial supernatant fractions and cell death. These data suggest that redistribution of ER luminal calcium contributes to long chain saturated fatty acid-mediated ER stress and cell death.     

Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria.

Transmission of cytosolic [Ca2+] ([Ca2+]c) oscillations into the mitochondrial matrix is thought to be supported by local calcium control between IP3 receptor Ca2+ channels (IP3R) and mitochondria, but study of the coupling mechanisms has been difficult. We established a permeabilized cell model in which the Ca2+ coupling between endoplasmic reticulum (ER) and mitochondria is retained, and mitochondrial [Ca2+] ([Ca2+]m) can be monitored by fluorescence imaging. We demonstrate that maximal activation of mitochondrial Ca2+ uptake is evoked by IP3-induced perimitochondrial [Ca2+] elevations, which appear to reach values >20-fold higher than the global increases of [Ca2+]c. Incremental doses of IP3 elicited [Ca2+]m elevations that followed the quantal pattern of Ca2+ mobilization, even at the level of individual mitochondria. In contrast, gradual increases of IP3 evoked relatively small [Ca2+]m responses despite eliciting similar [Ca2+]c increases. We conclude that each mitochondrial Ca2+ uptake site faces multiple IP3R, a concurrent activation of which is required for optimal activation of mitochondrial Ca2+ uptake. This architecture explains why calcium oscillations evoked by synchronized periodic activation of IP3R are particularly effective in establishing dynamic control over mitochondrial metabolism. Furthermore, our data reveal fundamental functional similarities between ER-mitochondrial Ca2+ coupling and synaptic transmission.     

The endoplasmic reticulum network in the ascidian egg: localization and calcium content

In contrast with most other eggs, where the endoplasmic reticulum is mixed with many other organelles, in ascidians, continuous sheets and tubes of endoplasmic reticulum constitute the only prominent organelle in the immediate layer (0.5-1μm) beneath the plasma membrane, and occupies 16–20% of the cortical volume. We took advantage of this unusual stratification of the organelles in the ascidian egg, to carry X-ray microanalysis. Our measurements provide the first estimate of the calcium content of the endoplasmic reticulum network in an egg, and show it is the main calcium store.     

Energy-dependent calcium transport in endoplasmic reticulum of adipocytes.

The endoplasmic reticulum from isolated rat adipocytes has the ability to actively accumulate calcium. The calcium uptake was characterized using the 20,000 X g supernatant (S1 fraction) of total cellular homogenate. Endoplasmic reticulum vesicles isolated from the S1 fraction as a 160,000 X g microsomal pellet prior to testing demonstrated little ability to accumulate calcium. The calcium uptake in the S1 fraction was localized to the endoplasmic reticulum vesicles by morphologic appearance, by the use of selective inhibitors of calcium uptake, and by high speed sedimentation of the accumulated calcium. The uptake was MgATP- and temperature-dependent and was sustained by the oxalate used as the intravesicular trapping agent. Uptake was linear with time for at least 30 min at all calcium concentrations tested (3 to 100 muM) and exhibited a pH optimum of approximately 7.0. The sulfhydryl inhibitor p-chloromercuribenzene sulfonate produced a dose-dependent inhibition of calcium uptake with total inhibition at 0.07 mumol/mg protein. Ruthenium red and sodium azide inhibited less than 5% of the uptake at concentrations (5 muM and 10 mM, respectively) which completely blocked calcium uptake by mitochondria isolated from the same cells. The Km for calcium uptake was 10 muM total calcium which corresponded to approximately 3.6 muM ionized calcium in the assay system. The maximum velocity of the uptake was 5.0 nmol (mg of microsomal protein)-1 (min)-1 at 24 degrees under the assay conditions used and exhibited a Q10 of 1.8. The uptake activity of the endoplasmic reticulum vesicles in the S1 fraction exhibited a marked time- and temperature-dependent lability which might account in part for the lack of uptake in the isolated microsomal fraction. This energy-dependent calcium uptake system would appear to be of physiologic importance to the regulation of intracellular calcium.     

Remodelling of the endoplasmic reticulum during store-operated calcium entry

SOCE (store-operated calcium entry) is a ubiquitous cellular mechanism linking the calcium depletion of the ER (endoplasmic reticulum) to the activation of PM (plasma membrane) Ca2+-permeable channels. The activation of SOCE channels favours the entry of extracellular Ca2+ into the cytosol, thereby promoting the refilling of the depleted ER Ca2+ stores as well as the generation of long-lasting calcium signals. The molecules that govern SOCE activation comprise ER Ca2+ sensors [STIM1 (stromal interaction molecule 1) and STIM2], PM Ca2+-permeable channels {Orai and TRPC [TRP (transient receptor potential) canonical]} and regulatory Ca2+-sensitive cytosolic proteins {CRACR2 [CRAC (Ca2+ release-activated Ca2+ current) regulator 2]}. Upon Ca2+ depletion of the ER, STIM molecules move towards the PM to bind and activate Orai or TRPC channels, initiating calcium entry and store refilling. This molecular rearrangement is accompanied by the formation of specialized compartments derived from the ER, the pre-cER (cortical ER) and cER. The pre-cER appears on the electron microscope as thin ER tubules enriched in STIM1 that extend along microtubules and that are devoid of contacts with the PM. The cER is located in immediate proximity to the PM and comprises thinner sections enriched in STIM1 and devoid of chaperones that might be dedicated to calcium signalling. Here, we review the molecular interactions and the morphological changes in ER structure that occur during the SOCE process.     

Autophagy regulates endoplasmic reticulum homeostasis and calcium mobilization in T lymphocytes

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular bulk degradation pathway that plays critical roles in eliminating intracellular pathogens, presenting endogenous Ags, and regulating T lymphocyte survival and proliferation. In this study, we have investigated the role of autophagy in regulating the endoplasmic reticulum (ER) compartment in T lymphocytes. We found that ER content is expanded in mature autophagy-related protein (Atg) 7-deficient T lymphocytes. Atg7-deficient T cells stimulated through the TCR display impaired influx, but not efflux, of calcium, and ER calcium stores are increased in Atg7-deficient T cells. Treatment with the ER sarco/ER Ca(2+)-ATPase pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T lymphocytes, suggesting that this impairment is caused by an intrinsic defect in ER. Furthermore, we found that the stimulation-induced redistribution of stromal interaction molecule-1, a critical event for the store-operated Ca(2+) release-activated Ca(2+) channel opening, is impaired in Atg7-deficient T cells. Together, these findings indicate that the expanded ER compartment in Atg7-deficient T cells contains increased calcium stores, and the inability of these stores to be depleted causes defective calcium influx in these cells. Our results demonstrate that autophagy plays an important role in maintaining ER and calcium homeostasis in T lymphocytes.     

Mitochondria, calcium, and endoplasmic reticulum stress in Parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta (SNPC) and the presence of intracytoplasmatic inclusions known as Lewy bodies, largely composed of alpha-synuclein (α-syn). PD is a multifactorial disease and its etiology remains largely elusive. Although more than 90% of the cases are sporadic, mutations in several nuclear encoded genes have been linked to the development of autosomal recessive and dominant familial parkinsonian syndromes (Bogaerts et al. (2008) Genes Brain Behav 7, 129-151), enhancing our understanding of biochemical and cellular mechanisms contributing to the disease. Many cellular mechanisms are thought to be involved in the dopaminergic neuronal death in PD, including oxidative stress, intracellular Ca(2+) homeostasis impairment, and mitochondrial dysfunctions. Furthermore, endoplasmic reticulum (ER) stress together with abnormal protein degradation by the ubiquitin proteasome system is considered to contribute to the PD pathogenesis. This review covers all the aspects related to the molecular mechanisms underlying the interplay between mitochondria, ER, and proteasome system in PD-associated neurodegeneration.     

An endoplasmic reticulum storage disease causing congenital goiter with hypothyroidism

In humans, deficient thyroglobulin (Tg, the thyroid prohormone) is an important cause of congenital hypothyroid goiter; further, homozygous mice expressing two cog/cog alleles (linked to the Tg locus) exhibit the same phenotype. Tg mutations might affect multiple different steps in thyroid hormone synthesis; however, the microscopic and biochemical phenotype tends to involve enlargement of the thyroid ER and accumulation of protein bands of M(r) < 100. To explore further the cell biology of this autosomal recessive illness, we have examined the folding and intracellular transport of newly synthesized Tg in cog/cog thyroid tissue. We find that mutant mice synthesize a full-length Tg, which appears to undergo normal N-linked glycosylation and glucose trimming. Nevertheless, in the mutant, Tg is deficient in the folding that leads to homodimerization, and there is a deficiency in the quantity of intracellular Tg transported to the distal portion of the secretory pathway. Indeed, we find that the underlying disorder in cog/cog mice is a thyroid ER storage disease, in which a temperature- sensitive Tg folding defect, in conjunction with normal ER quality control mechanisms, leads to defective Tg export. In relation to quality control, we find that the physiological response in this illness includes the specific induction of five molecular chaperones in the thyroid ER. Based on the pattern of chaperone binding, different potential roles for individual chaperones are suggested in glycoprotein folding, retention, and degradation in this ER storage disease.     

The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes

Ca²⁺ oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca²⁺ homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Mater^tm/tm (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Mater^tm/tm oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca²⁺ oscillations was altered in Mater^tm/tm oocytes after fertilization in vitro. Intriguingly, Ca²⁺ oscillations in Mater^tm/tm oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca²⁺ oscillation defect in Mater^tm/tm oocytes was likely caused by a reduced amount of Ca²⁺ in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca²⁺ homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.     

Properties of the smooth muscle cell endoplasmic reticulum calcium pump

In experiments with 45Ca2+ conducted on digitonin-treated (0.1 mg/ml) myometrium cells suspension, the properties of ruthenium red-insensitive, oxalate- or phosphate-stimulated and thapsigargin- or cyclopiasonic acid-suppressed Mg2+, ATP-dependent calcium pump of myometrium sarcoplasmic reticulum was studied. The Ca2+ accumulation increased linearly in time up to 10 min, the average initial rate was 80-130 pmol Ca2+/10(6) cells per min. In the presence of 10 mM oxalate the values of the activation constant KMg for Mg2+ and K(m) for ATP were 0.6 and 1.0 mM, respectively. The relative efficiency of the different cations in insuring of the ATP-dependent Ca2+ accumulation was Mg2+ > Mn2+ = Co2+ > Ni2+; the Ca2+ accumulation was not observed in the presence of 3 mM Zn2+ or Cu2+. We observed the suppression of calcium pump activity by different inhibitors such as thapsigargin, cyclopiazonic acid, p-chloromercuribenzoic acid, eosin Y ad Na3 VO4: the values of K0.5 were 2.0 nM, 0.3 microM, 0.6 microM, 0.8 microM and 45 microM respectively. The conclusion was made that suspension of myometrial cells treated with digitonin represent a suitable experimental model for studying the properties of myometrium sarcoplasmic reticulum calcium pump.     

Intraluminal calcium as a primary regulator of endoplasmic reticulum function

The concentration of Ca2+ inside the lumen of endoplasmic reticulum (ER) regulates a vast array of spatiotemporally distinct cellular processes, from intracellular Ca2+ signals to intra-ER protein processing and cell death. This review summarises recent data on the mechanisms of luminal Ca2+-dependent regulation of Ca2+ release and uptake as well as ER regulation of cellular adaptive processes. In addition we discuss general biophysical properties of the ER membrane, as trans-endomembrane Ca2+ fluxes are subject to basic electrical forces, determined by factors such as the membrane potential of the ER and the ease with which Ca2+ fluxes are able to change this potential (i.e. the resistance of the ER membrane). Although these electrical forces undoubtedly play a fundamental role in shaping [Ca2+](ER) dynamics, at present there is very little direct experimental information about the biophysical properties of the ER membrane. Further studies of how intraluminal [Ca2+] is regulated, best carried out with direct measurements, are vital for understanding how Ca2+ orchestrates cell function. Direct monitoring of [Ca2+](ER) under conditions where the cytosolic [Ca2+] is known may also help to capture elusive biophysical information about the ER, such as the potential difference across the ER membrane.     

Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brain

Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/-) mice by utilizing ATP/Mg(2+)-dependent (45)Ca(2+) uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/-) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive (45)Ca(2+) uptake as that seen in wild-type mouse brain, the Anx7 (+/-) mouse brain Ca(2+) pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca(2+)-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca(2+) from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca(2+) release.     

The effects of lipolytic hormones on calcium uptake in endoplasmic reticulum of adipocytes

To explore interrelationship between the roles of cAMP and calcium ion in hormone-stimulated lipolysis, cAMP accumulation in rat adipocytes and calcium binding in the endoplasmic reticulum were investigated with special reference to the effects of lipolytic hormones under various conditions. ACTH, isoproterenol, DBcAMP and aminophylline significantly increased ATP-dependent calcium uptake in adipocyte endoplasmic reticulum, but only after they were incubated with intact cells and not when they were added after homogenization. In vivo dexamethasone treatment and A-23187 accelerated, while 2.4-dinitrophenol blunted ACTH-stimulated lipolysis, cAMP accumulation and microsomal calcium uptake in parallel. Adrenalectomy, Mn2+ and adenosine enhanced ACTH-stimulated cAMP accumulation in adipocytes but lowered the calcium uptake and lipolysis. Thus, there was consistent parallelism between hormone-stimulated lipolysis and microsomal calcium uptake throughout the study. These data suggest that changes in the microsomal calcium uptake plays a crucial role in the regulation of hormone-induced lipolysis, irrespective of whether or not the intracellular cAMP concentration is involved in the lipolytic mechanism.