Sec61 complex/translocon: The role of an atypical ER Ca2+-leak channel in health and disease

International audienceThe heterotrimeric Sec61 protein complex forms the functional core of the so-called translocon that forms an aqueous channel in the endoplasmic reticulum (ER). The primary role of the Sec61 complex is to allow protein import in the ER during translation. Surprisingly, a completely different function in intracellular Ca 2+ homeostasis has emerged for the Sec61 complex, and the latter is now accepted as one of the major Ca 2+ -leak pathways of the ER. In this review, we first discuss the structure of the Sec61 complex and focus on the pharmacology and regulation of the Sec61 complex as a Ca 2+ -leak channel. Subsequently, we will pay particular attention to pathologies that are linked to Sec61 mutations, such as plasma cell deficiency and congenital neutropenia. Finally, we will explore the relevance of the Sec61 complex as a Ca 2+ -leak channel in various pathophysiological (ER stress, apoptosis, ischemia-reperfusion) and pathological (type 2 diabetes, cancer) settings

Pathophysiological Role of Trpv1 In Malignant Hyperthermia: Identification of New Variants

International audienceMalignant hyperthermia is a pharmacogenetic disorder caused by volatile anesthetics that trigger severe muscle contraction and a hypermetabolic state. Outside triggered crisis, patients are asymptomatic. The molecular mechanisms of MH involve an uncontrolled increase of cytosolic Ca2+ concentration in skeletal muscles, which contributes to excessive muscle contraction and rigidity, increased body temperature, severe rhabdomyolysis and a generalized acidosis and hypermetabolic state. Mutations altering the function of the 2 main calcium channels involved in muscle contraction were so far linked to Malignant Hyperthermia. Recently, we showed that mutations in the Transient Receptor Potential Vanilloid 1 (TRPV1) cation channel could also be involved in MH. We propose that TRPV1, acting as a Ca2+ leak channel, is a target of volatile anesthetics such as isoflurane as well as a mechanism that could explain its implication in MH

Ribosome-translocon complex mediates calcium leakage from endoplasmic reticulum stores.

Under resting conditions, the endoplasmic reticulum (ER) intraluminal free calcium concentration ([Ca(2+)](ER)) reflects a balance between active uptake by Ca(2+)-ATPases and passive efflux via 'leak channels'. Despite their physiological importance and ubiquitous leak pathway mechanism, very little is known about the molecular nature of these channels. As it has been suggested that the open translocon pore complex of the ER is permeable to ions and neutral molecules, we hypothesized that the ribosome-bound translocon would be permeable to calcium after treatment with puromycin, a translation inhibitor that specifically releases polypeptide chains. At this time, the translocon channel is left open. We measured the fluctuations in cytoplasmic and luminal calcium concentrations using fluorescent dyes (fura-2 and magfura-2, respectively). The calcium release induced by thapsigargin (a Ca(2+)-ATPase inhibitor) was lower after puromycin treatment. Puromycin also reduced the [Ca(2+)](ER) level when perfused into the medium, but was ineffective after anisomycin pre-treatment (an inhibitor of the peptidyl transferase). Puromycin had a similar effect in the presence of heparin and ryanodine. This puromycin-evoked [Ca(2+)](ER) decrease was specific to the translocon. We conclude that the translocon complex is a major calcium leak channel. This work reveals a new role for the translocon which is involved in the control of the [Ca(2+)](ER) and could therefore supervise many physiological processes, including gene expression and apoptosis

Passive calcium leak via translocon is a first step for iPLA2-pathway regulated store operated channels activation.

Calcium concentration within the endoplasmic reticulum (ER) plays an essential role in cell physiopathology. One of the most enigmatic mechanisms responsible for Ca2+ concentration in the ER is passive calcium leak. Previous studies have shown that the translocon complex is permeable to calcium. However, the involvement of the translocon in the passive calcium leak has not been directly demonstrated. Furthermore, the question whether the passive store depletion via the translocon could activate SOC (store operated channels) replenishing the ER, remains still unresolved. In this study, for the first time, we show that thapsigargin and calcium chelators deplete ER via translocon. Indeed, using confocal imaging, we demonstrate that when the number of opened translocons was lowered neither thapsigargin nor calcium chelators could induce ER store depletion. We also demonstrate that calcium leakage occurring via the translocon activates store-operated current, which is, by its kinetic and pharmacology, similar to that evoked by thapsigargin and EGTA (but not IP3), thus highlighting our hypothesis that calcium leakage via the translocon is a first step for activation of the specific iPLA2-regulated SOC. As the translocon is present in yeast and mammalian cells, our findings suggest that translocon-related calcium signaling is a common phenomenon

Transformation of local Ca(2+) spikes to global Ca(2+) transients: the combinatorial roles of multiple Ca(2+) releasing messengers

In pancreatic acinar cells, low, threshold concentrations of acetylcholine (ACh) or cholecystokinin (CCK) induce repetitive local cytosolic Ca(2+) spikes in the apical pole, while higher concentrations elicit global signals. We have investigated the process that transforms local Ca(2+) spikes to global Ca(2+) transients, focusing on the interactions of multiple intracellular messengers. ACh-elicited local Ca(2+) spikes were transformed into a global sustained Ca(2+) response by cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), whereas inositol 1,4,5-trisphosphate (IP(3)) had a much weaker effect. In contrast, the response elicited by a low CCK concentration was strongly potentiated by IP(3), whereas cADPR and NAADP had little effect. Experiments with messenger mixtures revealed a local interaction between IP(3) and NAADP and a stronger global potentiating interaction between cADPR and NAADP. NAADP strongly amplified the local Ca(2+) release evoked by a cADPR/IP(3) mixture eliciting a vigorous global Ca(2+) response. Different combinations of Ca(2+) releasing messengers can shape the spatio-temporal patterns of cytosolic Ca(2+) signals. NAADP and cADPR are emerging as key messengers in the globalization of Ca(2+) signals

Basal and physiological Ca2+ leak from the endoplasmic reticulum of pancreatic acinar cells: second messenger-activated channels and translocons

We have studied the Ca2+ leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca2+ in the endoplasmic reticulum ([Ca2+]ER). Cytosolic Ca2+ ([Ca2+]C) was clamped to the resting level by a BAPTA-Ca2+ mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of [Ca2+]ER, and the rate of Ca2+ release generated by 10 pm cholecystokinin is at least 3× as fast as the basal Ca2+ leak revealed by inhibition of the endoplasmic reticulum Ca2+-ATPase. Acetylcholine also evokes a dose-dependent decrease of [Ca2+]ER, with an EC50 of 0.98 ± 0.06 μm. Inhibition of receptors for inositol 1,4,5-trisphosphate (IP3) by heparin or flunarizine blocks the effect of acetylcholine but only partly blocks the effect of cholecystokinin. 8-NH2 cyclic ADP-ribose (20 μm) inhibits the action of cholecystokinin, but not of acetylcholine. The basal Ca2+ leak from the endoplasmic reticulum is not blocked by antagonists of the IP3 receptor, the ryanodine receptor, or the receptor for nicotinic acid adenine dinucleotide phosphate. However, treatment with puromycin (0.1–1 mm) to remove nascent polypeptides from ribosomes increases Ca2+leak from the endoplasmic reticulum by a mechanism independent of the endoplasmic reticulum Ca2+ pumps and of the receptors for IP3 or ryanodine

Bcl-2-dependent modulation of Ca2+ homeostasis and store-operated channels in prostate cancer cells

AbstractAntiapoptotic oncoprotein Bcl-2 has extramitochondrial actions due to its localization on the endoplasmic reticulum (ER); however, the specific mechanisms of such actions remain unclear. Here we show that Bcl-2 overexpression in LNCaP prostate cancer epithelial cells results in downregulation of store-operated Ca2+ current by decreasing the number of functional channels and inhibiting ER Ca2+ uptake through a reduction in the expression of calreticulin and SERCA2b, two key proteins controlling ER Ca2+ content. Furthermore, we demonstrate that Ca2+ store depletion by itself is not sufficient to induce apoptosis in Bcl-2 overexpressing cells, and that sustained Ca2+ entry via activated store-operated channels (SOCs) is required as well. Our data therefore suggest the pivotal role of SOCs in apoptosis and cancer progression

Characterization of Functional TRPV1 Channels in the Sarcoplasmic Reticulum of Mouse Skeletal Muscle

<div><p>TRPV1 represents a non-selective cation channel activated by capsaicin, acidosis and high temperature. In the central nervous system where TRPV1 is highly expressed, its physiological role in nociception is clearly identified. In skeletal muscle, TRPV1 appears implicated in energy metabolism and exercise endurance. However, how as a Ca²⁺ channel, it contributes to intracellular calcium concentration ([Ca²⁺]_i) maintenance and muscle contraction remains unknown. Here, as in rats, we report that TRPV1 is functionally expressed in mouse skeletal muscle. In contrast to earlier reports, our analysis show TRPV1 presence only at the sarcoplasmic reticulum (SR) membrane (preferably at the longitudinal part) in the proximity of SERCA1 pumps. Using intracellular Ca²⁺ imaging, we directly accessed to the channel functionality in intact FDB mouse fibers. Capsaicin and resiniferatoxin, both agonists as well as high temperature (45°C) elicited an increase in [Ca²⁺]_i. TRPV1-inhibition by capsazepine resulted in a strong inhibition of TRPV1-mediated functional responses and abolished channel activation. Blocking the SR release (with ryanodine or dantrolene) led to a reduced capsaicin-induced Ca²⁺ elevation suggesting that TRPV1 may participate to a secondary SR Ca²⁺ liberation of greater amplitude. In conclusion, our experiments point out that TRPV1 is a functional SR Ca²⁺ leak channel and may crosstalk with RyR1 in adult mouse muscle fibers.</p> </div