Multiple modes of calcium-induced calcium release in sympathetic neurons II: a [Ca2+](i)- and location-dependent transition from endoplasmic reticulum Ca accumulation to net Ca release.

CICR from an intracellular store, here directly characterized as the ER, usually refers to net Ca(2)+ release that amplifies evoked elevations in cytosolic free calcium [Ca2+](i). However, the companion paper (Albrecht, M.A., S.L. Colegrove, J. Hongpaisan, N.B. Pivovarova, S.B. Andrews, and D.D. Friel. 2001. J. Gen. Physiol. 118:83-100) shows that in sympathetic neurons, small [Ca2+](i) elevations evoked by weak depolarization stimulate ER Ca accumulation, but at a rate attenuated by activation of a ryanodine-sensitive CICR pathway. Here, we have measured depolarization-evoked changes in total ER Ca concentration ([Ca](ER)) as a function of [Ca2+](i), and found that progressively larger [Ca2+](i) elevations cause a graded transition from ER Ca accumulation to net release, consistent with the expression of multiple modes of CICR. [Ca](ER) is relatively high at rest (12.8 +/- 0.9 mmol/kg dry weight, mean +/- SEM) and is reduced by thapsigargin or ryanodine (5.5 +/- 0.7 and 4.7 +/- 1.1 mmol/kg, respectively). [Ca](ER) rises during weak depolarization (to 17.0 +/- 1.6 mmol/kg over 120s, [Ca2+](i) less than approximately 350 nM), changes little in response to stronger depolarization (12.1 +/- 1.1 mmol/kg, [Ca2+](i) approximately 700 nM), and declines (to 6.5 +/- 1.0 mmol/kg) with larger [Ca2+](i) elevations (>1 microM) evoked by the same depolarization when mitochondrial Ca2+ uptake is inhibited (FCCP). Thus, net ER Ca2+ transport exhibits a biphasic dependence on [Ca2+](i). With mitochondrial Ca2+ uptake enabled, [Ca](ER) rises after repolarization (to 16.6 +/- 1.8 mmol/kg at 15 min) as [Ca2+](i) falls within the permissive range for ER Ca accumulation over a period lengthened by mitochondrial Ca2+ release. Finally, although spatially averaged [Ca](ER) is unchanged during strong depolarization, net ER Ca2+ release still occurs, but only in the outermost approximately 5-microm cytoplasmic shell where [Ca2+](i) should reach its highest levels. Since mitochondrial Ca accumulation occurs preferentially in peripheral cytoplasm, as demonstrated here by electron energy loss Ca maps, the Ca content of ER and mitochondria exhibit reciprocal dependencies on proximity to sites of Ca2+ entry, possibly reflecting indirect mitochondrial regulation of ER Ca(2)+ transport.     

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca²⁺ inside the ER ([Ca²⁺]_ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca²⁺]_ER (EC₅₀) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca²⁺]^ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 μM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 μM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca²⁺. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 107–112, March–April, 2007.     

Dissection of mitochondrial Ca2+ uptake and release fluxes in situ after depolarization-evoked [Ca2+](i) elevations in sympathetic neurons

We studied how mitochondrial Ca2+ transport influences [Ca2+](i) dynamics in sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca2+ accumulation by SERCA pumps and depolarized to elevate [Ca2+(i); the recovery that followed repolarization was then examined. The total Ca2+ flux responsible for the [Ca2+](i) recovery was separated into mitochondrial and nonmitochondrial components based on sensitivity to the proton ionophore FCCP, a selective inhibitor of mitochondrial Ca2+ transport in these cells. The nonmitochondrial flux, representing net Ca2+ extrusion across the plasma membrane, has a simple dependence on [Ca2+](i), while the net mitochondrial flux (J(mito)) is biphasic, indicative of Ca+) accumulation during the initial phase of recovery when [Ca2+](i) is high, and net Ca2+ release during later phases of recovery. During each phase, mitochondrial Ca2+ transport has distinct effects on recovery kinetics. J(mito) was separated into components representing mitochondrial Ca2+ uptake and release based on sensitivity to the specific mitochondrial Na(+)/Ca2+ exchange inhibitor, CGP 37157 (CGP). The CGP-resistant (uptake) component of J(mito) increases steeply with [Ca2+](i), as expected for transport by the mitochondrial uniporter. The CGP-sensitive (release) component is inhibited by lowering the intracellular Na(+) concentration and depends on both intra- and extramitochondrial Ca2+ concentration, as expected for the Na(+)/Ca2+ exchanger. Above approximately 400 nM [Ca2+](i), net mitochondrial Ca2+ transport is dominated by uptake and is largely insensitive to CGP. When [Ca2+](i) is approximately 200-300 nM, the net mitochondrial flux is small but represents the sum of much larger uptake and release fluxes that largely cancel. Thus, mitochondrial Ca2+ transport occurs in situ at much lower concentrations than previously thought, and may provide a mechanism for quantitative control of ATP production after brief or low frequency stimuli that raise [Ca(2+)](i) to levels below approximately 500 nM.     

Modulation of STIM1 and capacitative Ca2+ entry by the endoplasmic reticulum luminal oxidoreductase ERp57

STIM1 is an endoplasmic reticulum (ER) membrane Ca(2+) sensor responsible for activation of store-operated Ca(2+) influx. We discovered that STIM1 oligomerization and store-operated Ca(2+) entry (SOC) are modulated by the ER oxidoreductase ERp57. ERp57 interacts with the ER luminal domain of STIM1, with this interaction involving two conserved cysteine residues, C(49) and C(56). SOC is accelerated in the absence of ERp57 and inhibited in C(49) and C(56) mutants of STIM1. We show that ERp57, by ER luminal interaction with STIM1, has a modulatory role in capacitative Ca(2+) entry. This is the first demonstration of a protein involved in ER intraluminal regulation of STIM1.     

Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors identify a novel calcium pool in the central nervous system

Ca2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/Mg2+-dependent 45Ca2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca2+ imaging in living cells we distinguish two ER Ca2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45Ca2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca2+ ATPase and [32P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca2+ levels. TG-R Ca2+ accumulation represents a distinct Ca2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.     

Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ

Ca(2+) signaling is the astrocyte form of excitability and the endoplasmic reticulum (ER) plays an important role as an intracellular Ca(2+) store. Since the subcellular distribution of the ER influences Ca(2+) signaling, we compared the arrangement of ER in astrocytes of hippocampus tissue and astrocytes in cell culture by electron microscopy. While the ER was usually located in close apposition to the plasma membrane in astrocytes in situ, the ER in cultured astrocytes was close to the nuclear membrane. Activation of metabotropic receptors linked to release of Ca(2+) from ER stores triggered distinct responses in cultured and in situ astrocytes. In culture, Ca(2+) signals were commonly first recorded close to the nucleus and with a delay at peripheral regions of the cells. Store-operated Ca(2+) entry (SOC) as a route to refill the Ca(2+) stores could be easily identified in cultured astrocytes as the Zn(2+)-sensitive component of the Ca(2+) signal. In contrast, such a Zn(2+)-sensitive component was not recorded in astrocytes from hippocampal slices despite of evidence for SOC. Our data indicate that both, astrocytes in situ and in vitro express SOC necessary to refill stores, but that a SOC-related signal is not recorded in the cytoplasm of astrocytes in situ since the stores are close to the plasma membrane and the refill does not affect cytoplasmic Ca(2+) levels.     

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1)

Ryanodine receptor type 1 (RyR1) releases Ca²⁺ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca²⁺, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca²⁺, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [³H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca²⁺-binding site to sensitize RyR1 to Ca²⁺. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca²⁺. However, enhanced RyR1 activation occurred in the presence of Ca²⁺, AMPPCP, and Caf. In the absence of Ca²⁺, Na⁺ inhibited [³H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca²⁺-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca²⁺, Na⁺ is predicted to inhibit RyR1 by interacting with the Ca²⁺-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca²⁺-binding site, and conversely, Ca²⁺ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca²⁺, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca²⁺-, ATP-, and Caf-binding sites.     

The endoplasmic reticulum Ca2+-pump SERCA2b interacts with G protein-coupled receptors and enhances their expression at the cell surface

Calcium (Ca(2+)) plays a pivotal role in both cellular signaling and protein synthesis. However, it is not well understood how calcium metabolism and synthesis of secreted and membrane-bound proteins are related. Here we demonstrate that the sarco(endo)plasmic reticulum Ca(2+) ATPase 2b (SERCA2b), which maintains high Ca(2+) concentration in the lumen of the endoplasmic reticulum, interacts specifically with the human delta opioid receptor during early steps of receptor biogenesis in human embryonic kidney 293 cells. The interaction involves newly synthesized incompletely folded receptor precursors, because the association between the delta opioid receptor and SERCA2b (i) was short-lived and took place soon after receptor translation, (ii) was not affected by misfolding of the receptor, and (iii) decreased if receptor folding was enhanced by opioid receptor pharmacological chaperone. The physical association with SERCA2b was found to be a universal feature among G protein-coupled receptors within family A and was shown to occur also between the endogenously expressed luteinizing hormone receptor and SERCA2b in rat ovaries. Importantly, active SERCA2b rather than undisturbed Ca(2+) homeostasis was found to be essential for delta opioid receptor biogenesis, as inhibition of its Ca(2+) pumping activity by thapsigargin reduced the interaction and impaired the efficiency of receptor maturation, two phenomena that were not affected by a Ca(2+) ionophore A23187. Nevertheless, inhibition of SERCA2b did not compromise the functionality of receptors that were able to mature. Thus, we propose that the association with SERCA2b is required for efficient folding and/or membrane integration of G protein-coupled receptors.     

A quantitative model for linking Na+/Ca2+ exchanger to SERCA during refilling of the sarcoplasmic reticulum to sustain [Ca2+] oscillations in vascular smooth muscle

We have developed a quantitative model for the creation of cytoplasmic Ca²⁺ gradients near the inner surface of the plasma membrane (PM). In particular we simulated the refilling of the sarcoplasmic reticulum (SR) via PM–SR junctions during asynchronous [Ca²⁺]_i oscillations in smooth muscle cells of the rabbit inferior vena cava. We have combined confocal microscopy data on the [Ca²⁺]_i oscillations, force transduction data from cell contraction studies and electron microscopic images to build a basis for computational simulations that model the transport of calcium ions from Na⁺/Ca²⁺ exchangers (NCX) on the PM to sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pumps on the SR as a three-dimensional random walk through the PM–SR junctional cytoplasmic spaces. Electron microscopic ultrastructural images of the smooth muscle cells were elaborated with software algorithms to produce a very clear and dimensionally accurate picture of the PM–SR junctions. From this study, we conclude that it is plausible and possible for enough Ca²⁺ to pass through the PM–SR junctions to replete the SR during the regenerative Ca²⁺ release, which underlies agonist induced asynchronous Ca²⁺ oscillations in vascular smooth muscle.