Sarcoplasmic Reticulum/endoplasmic Reticulum Research Articles

Establishment and evaluation of targeted molecular screening model for the ryanodine receptor or sarco/endoplasmic reticulum calcium ATPase.

Endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is crucial for maintaining intracellular calcium homeostasis due to the calcium-signaling-related proteins on its membrane. While ryanodine receptors (RyR) on insect ER/SR membranes are well-known as targets for diamide insecticides, little is known about other calcium channels. Given the resistance of diamide insecticides, the establishment of molecular screening models targeting RyR or sarco/endoplasmic reticulum calcium ATPase (SERCA) is conducive to the discovery of new insecticidal molecules. The morphological features of Mythimna separata SR have closed vesicles with integrity and high density. The 282 proteins in the SR component contained RyR and SERCA. A measurement model for the release and uptake of calcium was successfully established by detecting calcium ions outside the SR membrane using a fluorescence spectrophotometer. In vitro testing systems using SR vesicles found that diamide insecticides could activate dose-dependently RyR, with EC50 values of 0.14 μM (Chlorantraniliprole), 0.21 μM (Flubendiamide), and 0.57 μM (Cyantraniliprole), respectively. However, dantrolene inhibited RyR-mediated calcium release with an IC50 value of 353.9 μM, suggesting that dantrolene can weakly antagonize RyR. Moreover, cyclopiazonic acid significantly reduced the enzyme activity and calcium uptake capacity of SERCA. On the contrary, CDN1163 markedly activated the enzyme activity and improved the calcium transport capacity of SERCA. SR vesicles can be used to study the function of unknown proteins on the SR membranes, as well as for high-throughput screening of highly active compounds targeting RyR or SERCA. © 2024 Society of Chemical Industry.

Disruption of ER ion homeostasis maintained by an ER anion channel CLCC1 contributes to ALS-like pathologies

Although anion channel activities have been demonstrated in sarcoplasmic reticulum/endoplasmic reticulum (SR/ER), their molecular identities and functions remain unclear. Here, we link rare variants of Chloride Channel CLIC Like 1 (CLCC1) to amyotrophic lateral sclerosis (ALS)-like pathologies. We demonstrate that CLCC1 is a pore-forming component of an ER anion channel and that ALS-associated mutations impair channel conductance. CLCC1 forms homomultimers and its channel activity is inhibited by luminal Ca2+ but facilitated by phosphatidylinositol 4,5-bisphosphate (PIP2). We identified conserved residues D25 and D181 in CLCC1 N-terminus responsible for Ca2+ binding and luminal Ca2+-mediated inhibition on channel open probability and K298 in CLCC1 intraluminal loop as the critical PIP2-sensing residue. CLCC1 maintains steady-state [Cl–]ER and [K+]ER and ER morphology and regulates ER Ca2+ homeostasis, including internal Ca2+ release and steady-state [Ca2+]ER. ALS-associated mutant forms of CLCC1 increase steady-state [Cl–]ER and impair ER Ca2+ homeostasis, and animals with the ALS-associated mutations are sensitized to stress challenge-induced protein misfolding. Phenotypic comparisons of multiple Clcc1 loss-of-function alleles, including ALS-associated mutations, reveal a CLCC1 dosage dependence in the severity of disease phenotypes in vivo. Similar to CLCC1 rare variations dominant in ALS, 10% of K298A heterozygous mice developed ALS-like symptoms, pointing to a mechanism of channelopathy dominant-negatively induced by a loss-of-function mutation. Conditional knockout of Clcc1 cell-autonomously causes motor neuron loss and ER stress, misfolded protein accumulation, and characteristic ALS pathologies in the spinal cord. Thus, our findings support that disruption of ER ion homeostasis maintained by CLCC1 contributes to ALS-like pathologies.

Ryanodine receptor 2 (RYR2) dysfunction activates the unfolded protein response and perturbs cardiomyocyte maturation.

Calcium-handling capacity is a major gauge of cardiomyocyte maturity. Ryanodine receptor 2 (RYR2) is the pre-dominant calcium channel that releases calcium from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) to activate cardiomyocyte contraction. Although RYR2 was previously implied as a key regulator of cardiomyocyte maturation, the mechanisms remain unclear. The aim of this study is to solve this problem. We performed Cas9/AAV9-mediated somatic mutagenesis to knockout RYR2 specifically in cardiomyocytes in mice. We conducted a genetic mosaic analysis to dissect the cell-autonomous function of RYR2 during cardiomyocyte maturation. We found that RYR2 depletion triggered ultrastructural and transcriptomic defects relevant to cardiomyocyte maturation. These phenotypes were associated with the drastic activation of ER stress pathways. The ER stress alleviator tauroursodeoxycholic acid partially rescued the defects in RYR2-depleted cardiomyocytes. Overexpression of ATF4, a key ER stress transcription factor, recapitulated defects in RYR2-depleted cells. Integrative analysis of RNA-Seq and bioChIP-Seq data revealed that protein biosynthesis-related genes are the major direct downstream targets of ATF4. RYR2-regulated ER homeostasis is essential for cardiomyocyte maturation. Severe ER stress perturbs cardiomyocyte maturation primarily through ATF4 activation. The major downstream effector genes of ATF4 are related to protein biosynthesis.

Microdomain elements of airway smooth muscle in calcium regulation and cell proliferation.

Airway remodeling manifested by hyperplasia of airway smooth muscle cells (ASMCs) and other structural and functional changes is a pathological condition in asthma not addressed by current treatment. Ca2+ signaling is crucial for ASMC proliferation. Inositol-1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) mediate Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum (ER/SR). Upon sensing the depletion of Ca2+ in ER/SR, stromal interaction molecule 1 (STIM1) aggregates and redistributes at the microdomain of ER/SR-plasma membrane (PM) and activates Orai1, a component of the store-operated Ca2+ (SOC) channels, to initiate Ca2+ influx. The STIM1/Orai1-mediated SOC entry is the main cause of a sustained intracellular calcium ([Ca2+]i) elevation, which is different from a transient rise of [Ca2+]i mediated by IP3R and RyR. Extended-synaptotagmin 1 (E-Syt1) is recruited to the ER/SR-PM junction and anchors to the PM lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in a SOC-dependent manner. The subsequent strengthening of the ER/SR-PM connection by E-Syt1 facilitates the phosphatidylinositol (PI) transfer protein, Nir2, to supplement PI, a PI(4,5)P2 substrate, for the generation of IP3 and the propagation of Ca2+ signaling. Calcineurin and nuclear factor of activated T cells are the downstream signaling factors of elevated [Ca2+]i contributing to ASMC proliferation. Mitochondrial Ca2+ uptake/efflux, mitochondrial fission/fusion and mitochondrial-ER/SR coupling also play important roles in modulating [Ca2+]i and ASMC proliferation. Together, these pathways and mechanisms represent new therapeutic targets for airway remodeling. The present review provides an overview of our current understanding of the mechanisms of ASMC proliferation involving Ca2+ and highlights potential directions to control airway remodeling in asthma.

Calsequestrin, a new modulator of unfolded protein response in skeletal and cardiac muscle

Cellular responses to stress are an integral part of muscle physiology and pathology. To deal with stress, cardiac and skeletal muscle cells utilize various mechanisms, engaging intercellular organelles including the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) [1]. The disruptions of ER functions are often observed in ischemic, hypertrophic and failing hearts [1]. However, the molecular mechanisms governing muscle ER stress regulation in the muscle are still unclear. Disruption of ER/SR hemostasis with physiological and pathological stimuli triggers a coping response, so called unfolded protein response (UPR). One of the UPR signaling pathways involves stress sensor inositol‐requiring enzyme 1α (IRE1α), an integral ER/SR membrane protein kinase and mRNA splicing enzyme. Here, we discovered that calsequestrin, a muscle Ca2+ binding protein localized to junctional SR, interacts with UPR stress sensor IRE1α, and contribute to the modulation of stress responses in the muscle. Both skeletal and cardiac isoforms of calsequestrin bind directly to IRE1α as measured by Microscale Thermophoresis (MST), Surface Plasmon Resonance (SPR), and His‐tag Ni‐NTA‐agarose pull down techniques. Although calsequestrin contains three thioredoxin‐like domains its interaction with IRE1α did not involve cysteine residues in IRE1α. Iodixanol gradient fractionation and immunostaining of skeletal muscle sections or isolated cardiomyocytes showed enrichment of IRE1α protein in junctional SR where it is colocalized with calsequestrin. MST analysis revealed that calsequestrin binding to IRE1α has inhibitory effect on IRE1α by preventing dimerization/oligomerization IRE1α required for X‐box binding protein 1 (XBP1) splicing activity. Consequently, cardiac calsequestrin‐deficient cardiomyocytes have increased activity of IRE1α. We concluded that calsequestrin is a new IRE1α binding partner and the protein plays a role in modulation of stress responses in skeletal and cardiac muscle.Support or Funding InformationCanada Institutes of Health Research (CIHR)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of galanin receptor 2 stimulates large conductance Ca2+-dependent K+ (BK) channels through the IP3 pathway in human embryonic kidney (HEK293) cells

The large conductance Ca2+-activated K+ (BK) channels are widely distributed in the brain, and act as intracellular calcium sensors in neurons. They play an important feedback role in controlling Ca2+ flux and Ca2+-dependent processes, including neurotransmitter release and cellular excitability. In this study, the effects of the neuropeptide galanin on BK channels were examined by determining the whole-cell currents and single-channel activities in human embryonic kidney (HEK293) cells co-expressing GalR2 and the BK alpha subunit. Galanin enhanced the currents of BK channels, in a concentration-dependent and PTX-independent manner, with an ED50 value of 71.8±16.9nM. This activation was mediated by GalR2, since its agonist AR-M1896 mimicked the effect of galanin, and since galanin did not facilitate BK currents in cells co-expressing cDNAs of BK and GalR1 or GalR3. The galanin-induced BK current persisted after replacement with Ca2+-free solution, suggesting that extracellular Ca2+ is not essential. Chelating intracellular Ca2+ by either the slow Ca2+ buffer EGTA or the fast Ca2+ buffer BAPTA abolished galanin-mediated activation of BK channels, indicating the important role of intracellular Ca2+. The role of Ca2+ efflux from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) was confirmed by application of thapsigargin, an irreversible inhibitor that depletes Ca2+ from SR/ER. Moreover, the inositol-1,4,5-triphosphate receptor (IP3R) was identified as the mediator responsible for increased intracellular Ca2+ activating BK channels. Taken together, activation of GalR2 leads to elevation of intracellular Ca2+ is due to Ca2+ efflux from ER through IP3R sequentially opening BK channels.

Is the lack of adiponectin associated with increased ER/SR stress and inflammation in the heart?

Objective To study whether there is an association between adiponectin and endoplasmic reticulum/sarcoplasmic reticulum (ERSR) stress. Research design Eleven-month-old male wild-type (WT) and adiponectin knockout (ADKO) mice were placed on chow or high fat diet for 12 weeks. The changes in ER stress and inflammatory genes were determined in the epididymal adipose, as well as heart tissue of adult WT and ADKO mice. To understand the role of ER/SR stress in the regulation of adiponectin, we studied the effect of tunicamycin or palmitate on H9C2 cardiomyoblasts in culture. To demonstrate the protective role of adiponectin, we studied the effect of purified adiponectin on the regulation of ERSR stress genes and inflammation in H9C2 cardiomyoblasts. Results (1) High fat diet increased TNFα in adipose tissue of ADKO mice. (2) ERSR stress genes, HSPa5, ERN1, and GADD34, and inflammation response genes, TNFα and CD68, were increased in heart of ADKO mice. High fat diet did not further increase the effect. (3) Induction of ERSR stress by tunicamycin in H9C2 resulted in the upregulation of ERSR stress response genes along with downregulation of adiponectin, adiponectin receptors 1 and 2, and Serca2A. ER stress was accompanied by down regulation of Iкβα and an increase in HSPa5 proteins. (4) Adiponectin decreased ERSR stress and inflammation response genes and increased Serca2A in to H9C2 cardiomyoblasts. Conclusion The lack of adiponectin is associated with increased ER/SR stress and inflammation in the heart. Adiponectin provides a protective effect by lowering inflammation and ER/SR stress along with increasing Serca2A in H9C2 cells.

Tail-anchored membrane protein SLMAP is a novel regulator of cardiac function at the sarcoplasmic reticulum

Sarcolemmal membrane-associated proteins (SLMAPs) are components of cardiac membranes involved in excitation-contraction (E-C) coupling. Here, we assessed the role of SLMAP in cardiac structure and function. We generated transgenic (Tg) mice with cardiac-restricted overexpression of SLMAP1 bearing the transmembrane domain 2 (TM2) to potentially interfere with endogenous SLMAP through homodimerization and subcellular targeting. Histological examination revealed vacuolated myocardium; the severity of which correlated with the expression level of SLMAP1-TM2. High resolution microscopy showed dilation of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) and confocal imaging combined with biochemical analysis indicated targeting of SLMAP1-TM2 to the SR/ER membranes and inappropriate homodimerization. Older (28 wk of age) Tg mice exhibited reduced contractility with impaired relaxation as assessed by left ventricle pressure monitoring. The ventricular dysfunction was associated with electrophysiological abnormalities (elongated QT interval). Younger (5 wk of age) Tg mice also exhibited an elongated QT interval with minimal functional disturbances associated with the activation of the fetal gene program. They were less responsive to isoproterenol challenge (ΔdP/dt(max)) and developed electrical and left ventricular pressure alternans. The altered electrophysiological and functional disturbances in Tg mice were associated with diminished expression level of calcium cycling proteins of the sarcoplasmic reticulum such as the ryanodine receptor, Ca(2+)-ATPase, calsequestrin, and triadin (but not phospholamban), as well as significantly reduced calcium uptake in microsomal fractions. These data demonstrate that SLMAP is a regulator of E-C coupling at the level of the SR and its perturbation results in progressive deterioration of cardiac electrophysiology and function.

GRP94 hyperglycosylation and phosphorylation in Sf21 cells

GRP94 is an inducible resident endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) glycoprotein that functions as a protein chaperone and Ca 2+ regulator. GRP94 has been reported to be a substrate for protein kinase CK2 in vitro, although its phosphorylation in intact cells remains unreported. In Sf21 insect cells, overexpression of canine GRP94 led to the appearance of a multiplet of three or more molecular-mass isoforms which was reduced to a single mobility form following treatment of cells with tunicamycin, suggesting stable accumulations of consecutively modified protein. Metabolic labeling of Sf21 cells with 32P i led to a constitutive phosphorylation of GRP94 which, based upon phosphopeptide mapping, occurred specifically on CK2-sensitive sites. Among the GRP94 multiplet, however, only the lowest mobility form of GRP94 was phosphorylated, even though in vitro phosphorylation of GRP94 by CK2 led to phosphorylation of all glycosylated forms. The 32P i incorporation into GRP94 indicated a slow turnover of phosphate incorporation that was unaffected by inhibition of biosynthesis, resulting in a steady-state level of phospho-GRP94 on CK2 sites. These data support a role for protein kinase CK2 in the cell biology for GRP94 and other resident ER/SR proteins that may occur in ER compartments.

Regulation of vascular tone: cross-talk between sarcoplasmic reticulum and plasmalemma

Selected topics on the roles of sarcoplasmic reticulum (SR) in the control of vascular smooth muscle (VSM) tone are briefly reviewed with particular reference to the regulation of cytosolic concentration of free calcium ions, [Ca2+]i. Although morphological evidence and subcellular membrane studies indicate a relatively meager quantity of SR in VSM and of endoplasmic reticulum (ER) in endothelial cells (ECs) compared with skeletal muscle and cardiac muscle, contractility studies suggest that vascular tone is, to a large extent, regulated by the intracellular Ca2+ stores in smooth muscle and endothelial cells. Cytosolic Ca2+ levels control myosin light chain phosphorylation and contraction in VSM and activation of NO synthase and phospholipase A2 in ECs to regulate nitric oxide (NO) and prostaglandin I2 formation. Understanding of the importance of SR or ER in modulating the [Ca2+]i in VSM and ECs has been further advanced as a result of the new development and refinement of biophysical techniques in the measurement of cellular Ca2+ concentrations and ion currents, such as fluorescent Ca2+ indicators and patch-clamp techniques. Experimental evidence has accumulated in support of the existence of cross-talk between SR-ER and the plasma membrane (PM). Novel pharmacological tool drugs selective for the SR-ER Ca2+ pump, such as thapsigargin and cyclopiazonic acid, as well as for SR-ER Ca2+ channels, such as ryanodine (for the Ca(2+)-induced Ca2+ release channel) and inositol polyphosphates and heparin (for the inositol-1,4,5-trisphosphate activated Ca2+ channel), together with the use of blockers for selective PM Ca2+ channels have enabled better formulation and elucidation of the mechanisms of cross-talk between SR-ER and PM.(ABSTRACT TRUNCATED AT 250 WORDS)

SCE in human ‘metabolizing system’. The assessment of DNA damage caused by cytostatic therapy and its modification by a dihydroquinoline-type antioxidant

The supposition that all eukaryotic cell types contain a plasma membrane (PM)-type Ca pump (i.e. a Ca pump which is directly-stimulated by calmodulin and located exclusively at the PM) has been questioned by recent data from higher plant cells. These studies suggest the presence of Ca pumps directly stimulated by calmodulin associated with an intracellular membrane (probably the endoplasmic reticulum, ER) in a variety of monocotelydonous and dicotelydonous species. Thus plants have a ‘PM-type’ Ca pump at an intracellular membrane. The evidence for this includes studies on isolated membranes, purification and functional reconstitution and phosphorylated intermediate formation. Plant cells also contain a homologue of the sarcoplasmic reticulum/endoplasmic reticulum (SRER) Ca pump, probably located at the ER. The implications of these new data for our appreciation of the structure, function and location of eukaryotic Ca pumps are discussed, together with recent data from the use of inhibitors specific to mammalian ERSR Ca pumps.