Cell calcium最新文献

There have been in the last three decades repeated publications indicating that the inositol 1,4,5-trisphosphate receptor (IP₃R) is regulated not only by cytosolic Ca²⁺ but also by intraluminal Ca²⁺. Although most studies indicated that a decreasing intraluminal Ca²⁺ level led to an inhibition of the IP₃R, a number of publications reported exactly the opposite effect, i.e. an inhibition of the IP₃R by high intraluminal Ca²⁺ levels. Although intraluminal Ca²⁺-binding sites on the IP₃Rs were reported, a regulatory role for them was not demonstrated. It is also well known that the IP₃R is regulated by a vast array of associated proteins, but only relatively recently proteins were identified that can be linked to the regulation of the IP₃R by intraluminal Ca²⁺. The first to be reported was annexin A1 that is proposed to associate with the second intraluminal loop of the IP₃R at high intraluminal Ca²⁺ levels and to inhibit the IP₃R. More recently, ERdj5/PDIA19 reductase was described to reduce an intraluminal disulfide bridge of IP₃R1 only at low intraluminal Ca²⁺ levels and thereby to inhibit the IP₃R. Annexin A1 and ERdj5/PDIA19 can therefore explain most of the experimental results on the regulation of the IP₃R by intraluminal Ca²⁺. Further studies are needed to provide a fuller understanding of the regulation of the IP₃R from the intraluminal side. These findings underscore the importance of the state of the endoplasmic reticulum in the control of IP₃R activity.

Calcium is a universal intracellular messenger and proper Ca²⁺concentrations ([Ca²⁺]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca²⁺ homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca²⁺ levels result in malfunction and disease. Selective intraorganellar Ca²⁺measurements are best achieved by using targeted genetically encoded Ca²⁺ indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca²⁺] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions. We report here a procedure to calibrate the ratiometric signal of GAP (GFP-Aequorin Protein) targeted to the endo-sarcoplasmic reticulum (ER/SR) into [Ca²⁺]_ER/SR based on imaging of fluorescence after heating the tissue at 50–52 °C, since this value coincides with that obtained in the absence of Ca²⁺ (R_min). Knowledge of the dynamic range (R_max/R_min) and the Ca²⁺-affinity (K_D) of the indicator permits calculation of [Ca²⁺] by applying a simple algorithm. We have validated this procedure in vitro using several cell types (HeLa, HEK 293T and mouse astrocytes), as well as in vivo in Drosophila. Moreover, this methodology is applicable to other low Ca²⁺ affinity green and red GECIs.

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is frequently caused by mutations in the β-cardiac myosin heavy chain gene (MYH7). Abnormal calcium handling and diastolic dysfunction are archetypical features of HCM caused by MYH7 gene mutations. However, the mechanism of how MYH7 mutations leads to these features remains unclear, which inhibits the development of effective therapies. Initially, cardiomyocytes were generated from induced pluripotent stem cells from an eight-year-old girl diagnosed with HCM carrying a MYH7(C.1063 G>A) heterozygous mutation(mutant-iPSC-CMs) and mutation-corrected isogenic iPSCs(control-iPSC-CMs) in the present study. Next, we compared phenotype of mutant-iPSC-CMs to that of control-iPSC-CMs, by assessing their morphology, hypertrophy-related genes expression, calcium handling, diastolic function and myofilament calcium sensitivity at days 15 and 40 respectively. Finally, to better understand increased myofilament Ca²⁺ sensitivity as a central mechanism of central pathogenicity in HCM, inhibition of calcium sensitivity with mavacamten can improveed cardiomyocyte hypertrophy. Mutant-iPSC-CMs exhibited enlarged areas, increased sarcomere disarray, enhanced expression of hypertrophy-related genes proteins, abnormal calcium handling, diastolic dysfunction and increased myofilament calcium sensitivity at day 40, but only significant increase in calcium sensitivity and mild diastolic dysfunction at day 15. Increased calcium sensitivity by levosimendan aggravates cardiomyocyte hypertrophy phenotypes such as expression of hypertrophy-related genes, abnormal calcium handling and diastolic dysfunction, while inhibition of calcium sensitivity significantly improves cardiomyocyte hypertrophy phenotypes in mutant-iPSC-CMs, suggesting increased myofilament calcium sensitivity is the primary mechanisms for MYH7 mutations pathogenesis. Our studies have uncovered a pathogenic mechanism of HCM caused by MYH7 gene mutations through which enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction. Correction of the myofilament calcium sensitivity was found to be an effective method for treating the development of HCM phenotype in vitro.

Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream protein kinases, including CaMKI, CaMKIV, PKB/Akt, and AMPK; thus, regulates various Ca²⁺-dependent physiological and pathophysiological pathways. Further, CaMKKβ/2 in mammalian species comprises multiple alternatively spliced variants; however, their functional differences or redundancy remain unclear. In this study, we aimed to characterize mouse CaMKKβ/2 splice variants (CaMKKβ-3 and β-3x). RT-PCR analyses revealed that mouse CaMKKβ-1, consisting of 17 exons, was predominantly expressed in the brain; whereas, mouse CaMKKβ-3 and β-3x, lacking exon 16 and exons 14/16, respectively, were primarily expressed in peripheral tissues. At the protein level, the CaMKKβ-3 or β-3x variants showed high expression levels in mouse cerebrum and testes. This was consistent with the localization of CaMKKβ-3/-3x in spermatids in seminiferous tubules, but not the localization of CaMKKβ-1. We also observed the co-localization of CaMKKβ-3/-3x with a target kinase, CaMKIV, in elongating spermatids. Biochemical characterization further revealed that CaMKKβ-3 exhibited Ca²⁺/CaM-induced kinase activity similar to CaMKKβ-1. Conversely, we noted that CaMKKβ-3x impaired Ca²⁺/CaM-binding ability, but exhibited significantly weak autonomous activity (approximately 500-fold lower than CaMKKβ-1 or β-3) due to the absence of C-terminal of the catalytic domain and a putative residue (Ile478) responsible for the kinase autoinhibition. Nevertheless, CaMKKβ-3x showed the ability to phosphorylate downstream kinases, including CaMKIα, CaMKIV, and AMPKα in transfected cells comparable to CaMKKβ-1 and β-3. Collectively, CaMKKβ-3/-3x were identified as functionally active and could be bona fide CaMKIV-kinases in testes involved in the activation of the CaMKIV cascade in spermatids, resulting in the regulation of spermiogenesis.

Ryanodine receptors (RyR) are intracellular Ca²⁺ channels localized in the endoplasmic reticulum, where they act as critical mediators of Ca²⁺-induced Ca²⁺ calcium release (CICR). In the brain, mammals express in both neurons, and non-neuronal cells, a combination of the three RyR-isoforms (RyR1–3). Pharmacological approaches, which do not distinguish between isoforms, have indicated that RyR-isoforms contribute to brain function. However, isoform-specific manipulations have revealed that RyR-isoforms display different subcellular localizations and are differentially associated with neuronal function. These findings raise the need to understand RyR-isoform specific transcriptional regulation, as this knowledge will help to elucidate the causes of neuronal dysfunction for a growing list of brain disorders that show altered RyR channel expression and function.

Neuronal activity and neurochemical stimulation trigger spatio-temporal changes in the cytoplasmic concentration of Na⁺ ions in astrocytes. These changes constitute the substrate for Na⁺ signalling and are fundamental for astrocytic excitability. Astrocytic Na⁺ signals are generated by Na⁺ influx through neurotransmitter transporters, with primary contribution of glutamate transporters, and through cationic channels; whereas recovery from Na⁺ transients is mediated mainly by the plasmalemmal Na⁺/K⁺ ATPase. Astrocytic Na⁺ signals regulate the activity of plasmalemmal transporters critical for homeostatic function of astrocytes, thus providing real-time coordination between neuronal activity and astrocytic support.

NCX1, NCX2, and NCX3 gene isoforms and their splice variants are characteristically expressed in different regions of the brain. The tissue-specific splice variants of NCX1–3 isoforms show specific expression profiles in neurons and astrocytes, whereas the relevant NCX isoform/splice variants exhibit diverse allosteric modes of Na⁺- and Ca²⁺-dependent regulation. In general, overexpression of NCX1–3 genes leads to neuroprotective effects, whereas their ablation gains the opposite results. At this end, the partial contributions of NCX isoform/splice variants to neuroprotective effects remain unresolved. The glutamate-dependent Na⁺ entry generates Na⁺ transients (in response to neuronal cell activities), whereas the Na⁺-driven Ca²⁺ entry (through the reverse NCX mode) raises Ca²⁺ transients. This special mode of signal coupling translates Na⁺ transients into the Ca²⁺ signals while being a part of synaptic neurotransmission. This mechanism is of general interest since disease-related conditions (ischemia, metabolic stress, and stroke among many others) trigger Na⁺ and Ca²⁺ overload with deadly outcomes of downstream apoptosis and excitotoxicity. The recently discovered mechanisms of NCX allosteric regulation indicate that some NCX variants might play a critical role in the dynamic coupling of Na⁺-driven Ca²⁺ entry. In contrast, the others are less important or even could be dangerous under altered conditions (e.g., metabolic stress). This working hypothesis can be tested by applying advanced experimental approaches and highly focused computational simulations. This may allow the development of structure-based blockers/activators that can selectively modulate predefined NCX variants to lessen the life-threatening outcomes of excitotoxicity, ischemia, apoptosis, metabolic deprivation, brain injury, and stroke.

Canonical TRP (TRPC) channels are a still enigmatic family of signaling molecules with multimodal sensing features. These channels enable Ca²⁺ influx through the plasma membrane to control a diverse range of cellular functions. Based on both regulatory- and recently uncovered structural features, TRPC channels are considered to coordinate Ca²⁺ and other divalent cations not only within the permeation path but also at additional sensory sites. Analysis of TRPC structures by cryo-EM identified multiple regulatory ion binding pockets. With this review, we aim at an overview and a critical discussion of the current concepts of divalent sensing by TRPC channels.