BiP-mediated closing of the Sec61 channel limits Ca(2+) leakage from the ER

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein-conducting channel, the Sec61 complex. Previous work has characterized the Sec61 channel as a potential ER Ca(2+) leak channel and identified calmodulin as limiting Ca(2+) leakage in a Ca(2+)-dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca(2+) imaging to monitor the effects of reduced levels of BiP on ER Ca(2+) leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca(2+) leakage via the Sec61 complex. When we replaced wild-type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca(2+) leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca(2+) leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

BiP Modulates the Affinity of Its Co-chaperone ERj1 for Ribosomes

Ribosomes synthesizing secretory and membrane proteins are bound to the endoplasmic reticulum (ER) membrane and attach to ribosome-associated membrane proteins such as the Sec61 complex, which forms the protein-conducting channel in the membrane. The ER membrane-resident Hsp40 protein ERj1 was characterized as being able to recruit BiP to ribosomes in solution and to regulate protein synthesis in a BiP-dependent manner. Here, we show that ERj1 and Sec61 are associated with ribosomes at the ER of human cells and that the binding of ERj1 to ribosomes occurs with a binding constant in the picomolar range and is prevented by pretreatment of ribosomes with RNase. However, the affinity of ERj1 for ribosomes dramatically changes upon binding of BiP. This modulation by BiP may be responsible for the dual role of ERj1 at the ribosome, i.e. acting as a recruiting factor for BiP and regulating translation.     

Calmodulin regulation of the calcium-leak channel Sec61 is unique to vertebrates

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, such as those formed by Sec61 complexes in the ER membrane, would interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism of intracellular signaling. We identified a calmodulin (CaM) binding motif in the cytosolic N-terminus of Sec61α from Canis familiaris that binds CaM, but not Ca²⁺-free apo-CaM, with nanomolar affinity and sequence specificity. In single channel lipid bilayer measurements, CaM potently mediated Sec61-channel closure in a Ca²⁺-dependent manner. No functional CaM binding motif was identified in the corresponding region of Sec61p from Saccharomyces cerevisiae, and no channel closure occurred in the presence of CaM and Ca²⁺. Therefore, CaM binding to the cytosolic N-terminus of Sec61α is involved in limiting Ca²⁺-leakage from the ER in C. familiaris but not S. cerevisiae.     

Plasma Membrane Ca2+-ATPase Overexpression Depletes Both Mitochondrial and Endoplasmic Reticulum Ca2+ Stores and Triggers Apoptosis in Insulin-secreting BRIN-BD11 Cells

Ca²⁺ may trigger apoptosis in β-cells. Hence, the control of intracellular Ca²⁺ may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca²⁺-ATPase (PMCA) overexpression on Ca²⁺-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca²⁺] using a combination of aequorins with different Ca²⁺ affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. Overexpression of PMCA decreased [Ca²⁺] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. PMCA overexpression depletes intracellular [Ca²⁺] stores and, despite a decrease in mitochondrial [Ca²⁺], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca²⁺ homeostasis that could decrease β-cell apoptosis in diabetes.     

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.     

The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae

We studied the molecular nature of the interaction between the integral membrane protein Sec63p and the lumenal Hsp70 BiP to elucidate their role in the process of precursor transit into the ER of Saccharomyces cerevisiae. A lumenal stretch of Sec63p with homology to the Escherichia coli protein DnaJ is the likely region of interface between Sec63p and BiP. This domain, purified as a fusion protein (63Jp) with glutathione S–transferase (GST), mediated a stable ATP-dependent binding interaction between 63Jp and BiP and stimulated the ATPase activity of BiP. The interaction was highly selective because only BiP was retained on immobilized 63Jp when detergent-solubilized microsomes were mixed with ATP and the fusion protein. GST alone was inactive in these assays. Additionally, a GST fusion containing a point mutation in the lumenal domain of Sec63p did not interact with BiP. Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein. We conclude that the lumenal domain of Sec63p is sufficient to mediate enzymatic interaction with BiP and that this interaction positioned at the translocation apparatus or translocon at the lumenal face of the ER is vital for protein translocation into the ER.     

TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum

Ca²⁺ is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca²⁺ influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca²⁺‐dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca²⁺ concentration in the endoplasmic reticulum (ER). ER‐resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca²⁺ ATPase by increasing the ER Ca²⁺ permeability. This results in diminished cytosolic and mitochondrial Ca²⁺ signals upon stimulation of inositol 1,4,5‐trisphosphate receptors and reduces Ca²⁺ release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca²⁺ release and augments sensitivity to apoptosis. Our findings indicate an important function of ER‐resident TRPP2 in the modulation of intracellular Ca²⁺ signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.     

Calcium-activated K+ channels of mouse beta-cells are controlled by both store and cytoplasmic Ca2+: experimental and theoretical studies

A novel calcium-dependent potassium current (K_slow) that slowly activates in response to a simulated islet burst was identified recently in mouse pancreatic β-cells (Göpel, S.O., T. Kanno, S. Barg, L. Eliasson, J. Galvanovskis, E. Renström, and P. Rorsman. 1999. J. Gen. Physiol. 114:759–769). K_slow activation may help terminate the cyclic bursts of Ca²⁺-dependent action potentials that drive Ca²⁺ influx and insulin secretion in β-cells. Here, we report that when [Ca²⁺]_i handling was disrupted by blocking Ca²⁺ uptake into the ER with two separate agents reported to block the sarco/endoplasmic calcium ATPase (SERCA), thapsigargin (1–5 μM) or insulin (200 nM), K_slow was transiently potentiated and then inhibited. K_slow amplitude could also be inhibited by increasing extracellular glucose concentration from 5 to 10 mM. The biphasic modulation of K_slow by SERCA blockers could not be explained by a minimal mathematical model in which [Ca²⁺]_i is divided between two compartments, the cytosol and the ER, and K_slow activation mirrors changes in cytosolic calcium induced by the burst protocol. However, the experimental findings were reproduced by a model in which K_slow activation is mediated by a localized pool of [Ca²⁺] in a subspace located between the ER and the plasma membrane. In this model, the subspace [Ca²⁺] follows changes in cytosolic [Ca²⁺] but with a gradient that reflects Ca²⁺ efflux from the ER. Slow modulation of this gradient as the ER empties and fills may enhance the role of K_slow and [Ca²⁺] handling in influencing β-cell electrical activity and insulin secretion.     

4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer

Calcium (Ca²⁺) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca²⁺ release was associated with the ER Ca²⁺ release channels, inositol 1,4,5?triphosphate receptor (IP₃R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca²⁺ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca²⁺ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca²⁺ concentration ([Ca²⁺]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca²⁺ release mechanism distinct from classical Ca²⁺ release channels.     

Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca²⁺ leak using synthetic Ca²⁺ indicators that report changes in both the cytoplasmic ([Ca²⁺]_i) and the luminal ER ([Ca²⁺]_ER) Ca²⁺ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca²⁺ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca²⁺]_ER but did not decrease the ER Ca²⁺ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP₃R and only Orai3 channel supported the 2-APB-induced ER Ca²⁺ leak, while Orai1 and Orai2 inhibited this type of ER Ca²⁺ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca²⁺ leak but did not modify the ER Ca²⁺ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca²⁺]_i response after TG but only when the ER store had been overloaded with Ca²⁺ by eliminating the acidic internal Ca²⁺ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca²⁺ leak but forms an ER Ca²⁺ leak channel that is limiting the overloading with Ca²⁺ of the ER store.     

Live cell calcium imaging combined with siRNA mediated gene silencing identifies Ca²⁺ leak channels in the ER membrane and their regulatory mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca(2+) leak channels. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca(2+) from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca(2+) leak channel and to characterize its putative regulatory mechanisms. This system combines siRNA mediated gene silencing and live cell Ca(2+) imaging. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca(2+)-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca(2+) concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca(2+) also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca(2+) leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca(2+) efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca(2+) efflux, and iii) study the effects of disease linked mutations in the relevant components.     

Sec61p is part of the endoplasmic reticulum‐associated degradation machinery

Endoplasmic reticulum‐associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro‐translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd‐Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY^* proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins.     

Knockdown of STIM1 inhibits 6-hydroxydopamine-induced oxidative stress through attenuating calcium-dependent ER stress and mitochondrial dysfunction in undifferentiated PC12 cells

Abstract

Stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca²⁺ signaling systems and are considered to be important players in regulating neuronal Ca²⁺ homeostasis under normal ageing and pathological conditions. Here, we investigated the potential role of STIM1 in 6-hydroxydopamine (6-OHDA)-induced toxicity in undifferentiated PC12 cell lines. Cells exposed to 6-OHDA demonstrated alterations in the generation of reactive oxygen species (ROS) in a Ca²⁺-dependent manner. Downregulation of STIM1 expression by specific small interfering RNA (siRNA) attenuated apoptotic cell death, reduced intracellular ROS production, and partially prevented the impaired endogenous antioxidant enzyme activities after 6-OHDA treatment. Furthermore, STIM1 knockdown significantly attenuated 6-OHDA-induced intracellular Ca²⁺ overload by inhibiting endogenous store-operated calcium entry (SOCE). The effect of STIM1 siNRA on SOCE was related to orai1 and L-type Ca²⁺ channels, but not to transient receptor potential canonical type 1 (TRPC1) channel. In addition, silencing of STIM1 increased the Ca²⁺ buffering capacity of the endoplasmic reticulum (ER) in 6-OHDA-injured cells. ER vacuoles formed from the destruction of ER structural integrity and activation of ER-related apoptotic factors (CHOP and Caspase-12) were partially prevented by STIM1 knockdown. Moreover, STIM1 knockdown attenuated 6-OHDA-induced mitochondrial Ca²⁺ uptake and mitochondrial dysfunction, including the collapse of mitochondrial membrane potential (MMP) and the decrease of ATP generation. Taken together, our data provide the first evidence that inhibition of STIM1-meditated intracellular Ca²⁺ dyshomeostasis protects undifferentiated PC12 cells against 6-OHDA toxicity and indicate that STIM1 may be responsible for neuronal oxidative stress induced by ER stress and mitochondrial dysfunction in PD.     

Di-arginine signals and the K-rich domain retain the Ca²⁺ sensor STIM1 in the endoplasmic reticulum

STIM1 is a core component of the store‐operated Ca²⁺‐entry channel involved in Ca²⁺‐signaling with an important role in the activation of immune cells and many other cell types. In response to cell activation, STIM1 protein senses low Ca²⁺ concentration in the lumen of the endoplasmic reticulum (ER) and activates the channel protein Orai1 in the plasma membrane by direct physical contact. The related protein STIM2 functions similar but its physiological role is less well defined. We found that STIM2, but not STIM1, contains a di‐lysine ER‐retention signal. This restricts the function of STIM2 as Ca²⁺ sensor to the ER while STIM1 can reach the plasma membrane. The intracellular distribution of STIM1 is regulated in a cell‐cycle‐dependent manner with cell surface expression of STIM1 during mitosis. Efficient retention of STIM1 in the ER during interphase depends on its lysine‐rich domain and a di‐arginine ER retention signal. Store‐operated Ca²⁺‐entry enhanced ER retention, suggesting that trafficking of STIM1 is regulated and this regulation contributes to STIM1s role as multifunctional component in Ca²⁺‐signaling.