Calcium ions (Ca2+) are an archetypical and most versatile second messenger in virtually all cell types. Inspired by the discovery of Ca2+ sparks in the 1990s, vibrant research over the last three decades has unveiled a constellation of Ca2+ microdomains as elementary events of Ca2+ signaling and, more importantly, a digital-analog dualism as the system design principle of Ca2+ signaling. In this brief review, we present a sketchy summary on advances in the field of sparkology, and discuss how the digital subsystem can fulfill physiological roles otherwise impossible for any analog system. In addition, we attempt to address how the digital-analog dualism endows the simple cation messenger with signaling speediness, specificity, efficiency, stability, and unparalleled versatility.
The Golgi apparatus serves as a distinct part of intracellular Ca2+ stores. Here, the early discovery by Professor Shao Bai Xue is reviewed, and the recent progress in the field is outlined. Golgi Ca2+ stores-related functional proteins, such as secretory pathway Ca2+ ATPases (SPCA1/2) and the Golgi-specific Ca2+ releasing channel Golgi anti-apoptotic protein (GAAP), as well as the recently defined Golgi-specific Ca2+ release agent emetine, collectively corroborate the concept of the Golgi apparatus as unique internal Ca2+ stores.
β-adrenergic receptors (βARs) play significant roles in regulating Ca2+ signaling in cardiac myocytes, thus holding a key function in modulating heart performance. βARs regulate the influx of extracellular Ca2+ and the release and uptake of Ca2+ from the sarcoplasmic reticulum (SR) by activating key components such as L-type calcium channels (LTCCs), ryanodine receptors (RyRs) and phospholamban (PLN), mediated by the phosphorylation actions by protein kinase A (PKA). In cardiac myocytes, the presence of β2AR provides a protective mechanism against potential overstimulation of β1AR, which may aid in the restoration of cardiac dysfunctions. Understanding the Ca2+ regulatory signaling pathways of βARs in cardiac myocytes and the differences among various βAR subtypes are crucial in cardiology and hold great potential for developing treatments for heart diseases.
Calcium ions (Ca2+) play a crucial role as secondary messengers in both excitable and non-excitable cells. A complex system of proteins and molecules involved in calcium handling allows Ca2+ signals to be transduced. In cancer cells, mutations, aberrant expression, and dysregulation of these calcium handling toolkit proteins disrupt the normal Ca2+ flux between extracellular space, cytosol, endoplasmic reticulum and mitochondria, as well as the spatio-temporal patterns of Ca2+ signalling. This leads to the dysregulation of calcium-dependent effectors that control key signaling pathways involved in cancer cell proliferation, survival and invasion. Although there has been progressing in understanding the remodelling of calcium homeostasis in cancer cells and identifying key calcium transport molecules that promote malignant phenotypes, much work remains to be done to translate these fundamental findings into new tools for diagnosing and treating cancer by targeting Ca2+ homeostasis.
Channels are typically gated by several factors, including voltage, ligand and mechanical force. Most members of the calcium homeostasis modulator (CALHM) protein family, large-pore ATP release channels, exist in different oligomeric states. Dynamic conversions between CALHM1 heptamers and octamers to gate the channel were proposed. Meanwhile, the latest study observed that the transient receptor potential vanilloid 3 (TRPV3) channel adopts a dynamic transition between pentamers and canonical tetramers in response to small molecule treatment. These results suggest that oligomeric rearrangement may add a new layer to regulate the channel activities.
More and more studies have suggested an essential role of sarcoplasmic reticulum (SR) Ca2+ leak of atrial myocytes in atrial diseases such as atrial fibrillation (AF). The increasing interest in atrial Ca2+ signaling makes it necessary to develop a more accurate approach for Ca2+ measurement in atrial myocytes due to obvious differences between atrial and ventricular Ca2+ handling. In the present study, we proposed a new approach for quantifying total SR Ca2+ leak in atrial myocytes with confocal line-scan Ca2+ images. With a very precious approximation of the histogram of normalized line-scan Ca2+ images by using a modified Gaussian distribution, we separated the signal pixel components from noisy pixels and extracted two new dimensionless parameters, Fsignals and Rsignals, to reflect the summation of signal pixels and their release components, respectively. In the presence of tetracaine blocking SR Ca2+ leak, the two parameters were very close to 0, and in atrial myocytes under normal conditions, the two parameters are well positive correlative with Ca2+ spark frequency and total signal mass, the two classic readouts for SR Ca2+ leak. Consistent with Ca2+ Spark readouts, the two parameters quantified a significant increase of SR Ca2+ leak in atrial myocytes from mice harboring a leaky type 2 ryanodine receptor mutation (RyR2-R2474S+/−) compared to the WT group. Collectively, this study proposed a simple and effective approach to quantify SR Ca2+ leak in atrial myocytes, which may benefit research on calcium signaling in atrial physiology and diseases.
Calcium (Ca2+) is a universal second messenger in eukaryotic cells, and extracellular regulated protein kinases (ERK) are the core component of the mitogen-activated protein kinase (MAPK) signaling cascade. Both are involved in numerous physiological and pathological processes, such as organogenesis, tumorigenesis, proliferation, migration and apoptosis. Over the past decade, it has been found that calcium signaling can regulate the ERK activity through multiple mechanisms, and conversely, ERK signaling transduction can also affect the triggering and intensity of calcium signaling. However, there are few reports on how to perform real-time synchronous detection of these two signals. Here we described a method for dynamically and synchronously recording calcium signals and ERK activity in living cells, utilizing stable expression of multiple genetically-encoded probes and multi-channel synchronous detection technology using confocal microscopy. The protocol can be useful to address the spatiotemporal encoding dynamic mechanism of calcium signaling and ERK activity in single or multiple cells, and to reveal the interaction and causal characteristics of these two signals.
Genetically Encoded Calcium (Ca2+) indicators (GECIs) are indispensable tools for dissecting intracellular Ca2+ signaling and monitoring cellular activities. Mitochondrion acts as a Ca2+ sink and a central player for maintaining Ca2+ homeostasis. Accurately monitoring Ca2+ transients within the mitochondrial matrix that undergo constant pH fluctuations is challenging, as signals of most currently available GECIs suffer from artifacts induced by physiological pH variations. Multiplexed monitoring of optophysiology is also hindered by the limited availability of GECIs with cyan fluorescence. Based on the bright variant of cyan fluorescence protein (CFP), mTurquoise2, we developed a GECI designated as TurCaMP. Results from molecular dynamics simulations and ab initio calculations revealed that the deprotonation of the chromophore may be responsible for the Ca2+-dependent changes in TurCaMP signals. TurCaMP sensors showed inverse response to Ca2+ transients, and their responses were not affected by pH changes within the range of pH 6–9. The high basal fluorescence and insensitivity to physiological pH fluctuations enabled TurCaMP to faithfully monitor mitochondrial Ca2+ responses with a high signal-to-noise ratio. TurCaMP sensors allow simultaneous multi-colored imaging of intracellular Ca2+ signals, expanding the possibility of multiplexed monitoring of Ca2+-dependent physiological events.
The sarcoplasmic reticulum (SR) primarily serves as the intracellular Ca2+ store in cardiac myocytes, mediating cellular function under cardiac physiology and diseases. However, the properties of cardiac SR Ca2+ have not yet been fully determined, particularly in rats and mice, which are the most commonly used experimental species in studies on cardiac physiology and diseases. Here, we developed a spatially detailed numerical model to deduce Ca2+ movements inside the junctional SR (jSR) cisternae of rat cardiomyocytes. Our model accurately reproduced the jSR Ca2+ kinetics of local and global SR Ca2+ releases reported in a recent experimental study. With this model, we revealed that jSR Ca2+ kinetics was mostly determined by the total release flux via type 2 ryanodine receptor (RyR2) channels but not by RyR2 positioning. Although Ca2+ diffusion in global SR was previously reported to be slow, our simulation demonstrated that Ca2+ diffused very quickly inside local jSR cisternae and the decrease in the diffusion coefficient resulted in a significant reduction of jSR Ca2+ depletion amplitude. Intracellular Ca2+ was typically experimentally detected with fluorescence dye. Our simulation revealed that when the dynamical characteristics of fluorescence dye exerted a minimal effect on actual Ca2+ mobility inside jSR, the reaction rate of the dye with Ca2+ could significantly affect apparent jSR Ca2+ kinetics. Therefore, loading a chemical fluorescence dye with fast kinetics, such as Fluo-5N, into SR is important for Ca2+ measurement inside SR. Overall, our model provides new insights into deciphering Ca2+ handling inside nanoscopic jSR cisternae in rat cardiomyocytes.
Gap junction (GJ) intercellular communication is crucial in many physiological and pathological processes. A GJ channel is formed by head-to-head docking of two hexameric hemichannels from two neighboring cells. Heterotypic GJ channels formed by two different homomeric connexin hemichannels often display rectification properties in the current–voltage relationship while the underlying mechanisms are not fully clear. Here we studied heterotypic Cx46/Cx50 GJs at a single GJ channel level. Our data showed unitary Cx46/Cx50 GJ channel conductance (γj) rectification when 5 mmol/L Mg2+ was included in the patch pipette solution, while no γj rectification was observed when no Mg2+ was added. Including 5 mmol/L Mg2+ in pipette solution significantly decreased the γj of homotypic Cx46 GJ with little change in homotypic Cx50 γj. A missense point variant in Cx46 (E43F) reduced the Mg2+-dependent reduction in γj of Cx46 E43F GJ, indicating that E43 might be partially responsible for Mg2+-dependent decrease in γj of Cx46. A comprehensive understanding of Mg2+ modulation of GJ at the individual channel level is useful in understanding factors in modulating GJ-mediated intercellular communication in health and diseases.
