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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.37 n.4 Santiago  2004 


Biol Res 37: 693-699, 2004


Endoplasmic reticulum calcium signaling in nerve cells


School of Biological Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK

Dirección para Correspondencia


The endoplasmic reticulum (ER) is an important organelle involved in various types of signaling in nerve cells. The ER serves as a dynamic Ca2+ pool being thus involved in rapid signaling events associated with cell stimulation by either electrical (action potential) or chemical (neurotransmitters) signals. This function is supported by Ca2+ release channels (InsP3 and ryanodine receptors) and SERCA Ca2+ pumps residing in the endomembrane. In addition the ER provides a specific environment for the posttranslational protein processing and transport of various molecules towards their final destination. In parallel, the ER acts as a "calcium tunnel," which facilitates Ca2+ movements within the cell by avoiding cytoplasmic routes. Finally the ER appears as a source of numerous signals aimed at the nucleus and involved in long-lasting adaptive cellular responses. All these important functions are controlled by intra-ER free Ca2+ which integrates various signaling events and establishes a link between fast signaling, associated with ER Ca2+ release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. Disruption of ER Ca2+ homeostasis triggers several forms of cellular stress response and is intimately involved in neurodegeneration and neuronal cell death.

Key words: calcium signaling; endoplasmic reticulum; ryanodine receptors; InsP3 receptors.

ABBREVIATIONS: ER - endoplasmic reticulum; SERCA - Sarco(Endo)Plasmic reticulum Calcium ATPase; InsP3 - inositol-(1,4,5)-trisphosphate; RyR - ryanodine receptors; InsP3Rs - InsP3 receptors; GFP - green fluorescent protein; [Ca2+]L - intraluminal free Ca2+ concentration; [Ca2+]i - cytosolic free Ca2+ concentration; CICR - Ca2+-indiced Ca2+ release.


The endomembrane, organized into an interconnected mesh of microtubulae and cisternae, forms the endoplasmic reticulum (ER), which is found in all types of eukaryotic cells (7). In neural cells ER is present in both glial cells (16; 74) and in neurons where it forms a continuous network extending from soma towards cellular processes (40; 73; 75). In neurons the ER serves several vital functions, being a common transport route for numerous proteins seeking their final destinations, providing a specialized environment for post-translational protein processing and finally acting as a dynamic calcium store (7; 9; 19; 23; 41; 73). In the latter guise, the ER appears as an intracellular reservoir of free Ca2+ ions, ready to be released in consequence of cell stimulation by either electrical (action potential) or chemical (neurotransmitter) signals. This rapid signaling function is supported by several families of proteins residing in the endomembrane or within the ER lumen. These proteins are responsible for: (i) active Ca2+ accumulation into the ER lumen; (ii) Ca2+ storage in the ER; and (iii) Ca2+ release in response to appropriate stimulation. The first task is accomplished by Ca2+ pumps of the SERCA family (79), which constantly pump Ca2+ against its concentration gradient. Interestingly, the activity of SERCA pumps is controlled by intra-ER Ca2+ content, so that any lowering of [Ca2+]L immediately increases incoming Ca2+ flux through ER Ca2+ pumps (43; 64). The SERCA mediated Ca2+ influx into the ER together with intra-ER Ca2+ buffers, which have a rather low affinity to Ca2+ (KD ~ 0.5-1.0 mM) determines high levels of resting [Ca2+]L, which according to various estimating methods, vary between several hundreds mM to > 1 mM (5; 14; 43; 64).

Such a high concentration of intra-ER free Ca2+ creates a large concentration gradient aimed from the ER to the cytosol, which drives Ca2+ release from the ER lumen. The latter occurs through specific intracellular Ca2+ channels, expressed in the ER membrane. The main subtypes of these channels are represented by ryanodine receptors (RyRs), or Ca2+-gated Ca2+ channels (20; 53), and InsP3 receptors, or InsP3-gated Ca2+ channels (18; 66), which are co-expressed in almost all types of neurons. Several other types of Ca2+ release channels have been discovered in recent years (22; 36; 80), although their functional expression in nerve cells awaits final clarification.

Internally, the ER most likely is constituted as a single, continuous space, so that Ca2+ ions may diffuse relatively freely within the lumen of the reticulum thus creating "calcium tunnels," which facilitate Ca2+ movements within the cell by avoiding cytoplasmic routes (42; 47; 52; 73; 76). Although the idea of a single, continuous ER calcium store is not generally embraced (see e.g., 8), most of the currently available morphological and functional data argue in its favor. That is, injection of cerebellar Purkinje neurons in brain slices and cultured hippocampal neurons with the ER-specific fluorescent lipophylic compound 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbo cyanine perchlorate (DiIC16) clearly revealed a continuity of the endomembrane. The DiIC16 stained the ER structures in living neurons as well as in fixed cells, therefore demonstrating that the dye spread through the ER membrane is due to diffusion and not via intra-ER trafficking (67). Similarly various large molecules, such as fluorescent Ca2+ indicators (47) or ER-targeted green fluorescent protein (15; 65), can freely diffuse through the ER lumen. Furthermore, free Ca2+ rapidly equilibrates within the endoplasmic reticulum lumen following local uncaging of caged calcium (47).

The majority of functional experiments also point out to the existence of a single calcium pool shared by both types of Ca2+release channels. In principle, these experiments have demonstrated that in neuronal preparations the Ca2+ store can be completely depleted by activating either InsP3-dependent or caffeine/ryanodine-dependent Ca2+ release routes. For example, depletion of ryanodine-sensitive Ca2+ stores in Purkinje neurons studied in brain slices completely abolished [Ca2+]i elevations in response to photo-released InsP3, indicating that both receptors share the same interconnected Ca2+ pool (33). Similarly, InsP3Rs and RyRs share a single pool in rat adrenal chromaffine cells (30), cerebellar granule neurons (31; 60), as well as in cultured myenteric (34) and in dissociated hippocampal (29) neurons. Furthermore, when directly monitoring [Ca2+]L in sensory neurons, it was found that activation of RyRs by caffeine or low concentration of ryanodine, deplete the very same Ca2+ pool (63). This pool was also depleted by inhibition of SERCA pumps by either thapsigargin or cyclopiazonic acid. These phenomena of single continuous ER calcium store seem to be widespread in eukaryotic cells, as a very same single Ca2+ pool was clearly identified not only in neurons but also in various non-excitable cells (see e.g., 9; 42; 47; 52).


Currently, three main groups of calcium indicators are used for dynamic [Ca2+]L recordings. Historically, photoproteins were the first probes for free calcium measurements in living cells, and a number of [Ca2+]L recordings were performed using ER-targeted derivatives of the chemiluminescent protein (photoprotein) aequorin, which emits visible light in a calcium-dependent manner. Aequorins with relatively low KD were used for ER Ca2+ measurements in many cell types, including chromaffin cells, which possess many neuron-like properties (3-5). To target aequorin to the ER, several chimeric cDNAs encoding a recombinant polypeptide composed of a photoprotein and an ER-targeting sequence have been constructed and successfully deployed (e.g., 10; 11; 44). The use of ER-targeted aequorin provides an excellent signal-to-noise ratio (as no luminescent proteins are naturally present in mammalian cells) and clear association of the probe with the ER lumen. At the same time, low concentrations of the probe in the transfected cells results in an exceedingly small light emission, which prevents real time imaging and combination of [Ca2+]L recordings with electrophysiology (see 62 for review).

The second group of ER-specific Ca2+ probes is represented by fluorescent indicators based on ER-targeted variants of green fluorescent protein. These GFP-derived [Ca2+]L indicators comprise the camgaroo and pericam probes, based on a circularly permutated GFP, and the cameleon probes, which rely on the fluorescence resonance energy transfer (FRET) between two GFP mutants of different colors. The advantages and problems associated with ER Ca2+ measurements using GFP-based probes have been recently reviewed in an excellent paper by N. Demaurex and M. Frieden (17).

Finally, the third approach followed for [Ca2+]L recordings is the use of conventional Ca2+ fluorescent probes, such as Mag-Fura-2/5, Mag-Fluo-4, Oregon Green BAPTA 5N, etc. These Ca2+ probes have a sufficiently low KD (~20-100 mM), thus allowing them to accurately report high intra-ER Ca2+ levels. This technique was initially proposed by A. Hofer and her coworkers (24-26) and has been used since to monitor [Ca2+]L in non-excitable (e.g., 43; 47) and excitable cells (e.g., 59; 61; 64; 71; 78). The main problem in using this technique is associated with the necessity of removing the cytosolic portion of the dye, which is usually achieved by either intracellular dialysis or by permeabilization of the plasma membrane with various detergents, such as digitonin or saponin. In certain cell types, such a permeabilization is not necessary as the dye is either highly compartmentalized within the endoplasmic reticulum or the cytosolic Ca2+ signals are small enough not to be detected by a low-affinity probe (see 48; 62, for review).


Caffeine-induced Ca2+ release

The initial discovery of RyR-mediated intracellular Ca2+ release in nerve cells was achieved by using caffeine, the ability of which to activate ryanodine receptors became known in the mid-1970s. The very first indication of intracellular Ca2+ release in neurons derived from voltage-clamp experiments, which have demonstrated that application of caffeine in millimolar concentrations results in cell hyperpolarization due to an activation of Ca2+-dependent K+ channels (see e.g., 13; 37; 45). Several years later, this caffeine-induced intracellular Ca2+ release was directly demonstrated in sensory neurons injected with the cytosolic Ca2+ probe, aequorin (46). Within the next decade, this caffeine-induced Ca2+ release was found in many types of neurons, and its pharmacology (i.e., sensitivity to ryanodine, thapsigargin, dantrolene, etc.) was shown to be identical to the pharmacology of CICR in muscle cells (32; 35; 38; 54-58; 72; 77).

Physiologically-activated CICR

The role of intracellular Ca2+ release in shaping cytosolic Ca2+ signals results from the balance between two opposed Ca2+ fluxes, that is by Ca2+ accumulation onto the ER mediated by SERCA pumps and by Ca2+ efflux from the ER through Ca2+ release channels, as well as through Ca2+ leakage pathways. Ca2+ uptake into the ER lumen reduces [Ca2+]i, whereas Ca2+ efflux amplifies [Ca2+]i elevations, these two opposite effects being the consequence of the dual role of the ER Ca2+ store, which acts simultaneously as a Ca2+ sink and a Ca2+ source (21). These two functions are directly controlled by actual Ca2+ levels in the cytosol and within the ER lumen, as [Ca2+]i regulates the activation of Ca2+ release channels and provides Ca2+ for SERCA pumps, whereas [Ca2+]L. governs the rate of SERCA-mediated pumping and controls the sensitivity of Ca2+ release channels to their appropriate agonists.

As far as neuronal CICR is concerned, the relation between Ca2+ entry and hence the amount of "trigger" (i.e., CICR-activating) [Ca2+]i elevation from one side and [Ca2+]L and SERCA-mediated Ca2+ pumping directly determines the net effect on the cytosolic Ca2+ signal. This net effect, which allows the ER to work in two different modes either providing for Ca2+ uptake at weak stimulation or for Ca2+ release at strong stimulation, was indeed recently demonstrated in sympathetic neurons (1; 2; 27).

Yet, precise characterization of CICR mechanisms in neurons and further understanding of the relations between Ca2+ entry, [Ca2+]i elevation, and Ca2+ release required simultaneous and direct measurements of all parameters concerned, i.e. of [Ca2+]i, [Ca2+]L and transmembrane Ca2+ current. The initial attempt to demonstrate decrease in [Ca2+]L triggered by Ca2+ entry in neuronal preparation employed low-affinity ER-targeted aequorin transfected into cultured bovine chromaffin cells (4). These experiments clearly demonstrated that high-K+ depolarization of chromaffin cells triggered transient decreases in [Ca2+]L, thus directly demonstrating CICR process. Yet, this technique did not allow for simultaneous monitoring of [Ca2+]i and voltage-clamp recordings of Ca2+ currents, thus limiting the quantification of Ca2+ entry/CICR relations. These relations were investigated in much more detail in experiments on primary cultured sensory neurons. These neurons were loaded with Mag-Fura-2/AM and subsequently dialyzed under whole-cell voltage clamp conditions; furthermore, the intrapipette solution was supplemented with the high-affinity Ca2+ indicator Mag-Fura-2, so that [Ca2+]L, [Ca2+]i and Ca2+ currents could be recorded simultaneously from the same cell (62-64). This experimental approach has shown that Ca2+ currents activated by cell depolarization resulted in a transient decrease in [Ca2+]L, the latter being indicative of physiological CICR. These [Ca2+]L transients in response to depolarization-induced Ca2+ entry were blocked by high (50 mM) concentrations of ryanodine and potentiated by low (1 mM) concentrations of caffeine. Most importantly however, the linear relation between Ca2+ entry and the amplitude of [Ca2+]L transient decrease was revealed, indicating the gradual activation of CICR in neurons under physiological conditions (64).


Calcium ions accumulated within the ER lumen not only control fast signaling events associated with the activation of Ca2+ release, but they also regulate numerous ER-residing enzymes, responsible for postranslational protein processing. These enzymes, known as chaperones, are intimately involved in folding of proteins, during which they acquire their tertiary and quaternary structures. Many chaperons (e.g. calreticulin, calnexin and calmegin) are endowed with numerous Ca2+-binding sites and their functional activity is tightly regulated by intraluminal free Ca2+ concentration (12; 41). Thus, [Ca2+]L integrates various signaling events and establishes a link between fast signaling, associated with ER Ca2+release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. Most importantly, disturbances in ER Ca2+ homeostasis affect post-translational protein processing resulting in the appearance of specific conditions known as ER stress response (49-51; 76). The latter appears as a fundamental adaptive cell reaction which has two features: one is cytoprotective when signaling systems try to overcome the endoplasmic reticulum stress by over-expression of relevant chaperones and tuning the overall protein synthesis. Another feature is cytotoxic: when the cell can not cope with the stress, the endoplasmic reticulum releases pro-apoptotic factors, thereby eliminating the cell in the manner least harmful for its neighbors. Yet in many conditions disrupted ER Ca2+ homeostasis may initiate cell death, which is particularly important for neurodegenerative processes (23; 49; 50). In particular, impairment of ER Ca2+ homeostasis may be the triggering step in initiation of many forms of neuropathology, such as brain ischemia and glutamate excitotoxicity (6; 68; 69), Alzheimer disease (39; 70) and diabetic neuropathies (28).


In conclusion, the dynamic changes in free Ca2+ concentration within the lumen of ER are involved in both rapid and long-lasting signaling events, which control various aspects of neuronal physiology. Impairment of ER Ca2+ homeostasis may result in neuropathies, neurodegeneration and neuronal death being thus important as an ethiopathological factor in many forms of brain disease.


The author acknowledges support from The Wellcome Trust and BBSRC, UK.


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Corresponding author: Prof. A. Verkhratsky, The University of Manchester, School of Biological Sciences, 1124 Stopford Building, Oxford Road, Manchester M13 9PT, UK. Tel: (44-161) 275-5414, Fax: (44-161) 275-5948, E-mail:

Received: January 5, 2004. Accepted: March 3, 2004.


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