Roles of I(f) and intracellular Ca2+ release in spontaneous activity of ventricular cardiomyocytes during murine embryonic development

In recent years, the contribution of I(f), an important pacemaker current, and intracellular Ca²⁺ release (ICR) from sarcoplasmic reticulum to pacemaking and arrhythmia has been intensively studied. However, their functional roles in embryonic heart remain uncertain. Using patch clamp, Ca²⁺ imaging, and RT‐PCR, we found that I(f) regulated the firing rate in early and late stage embryonic ventricular cells, as ivabradine (30 µM), a specific blocker of I(f), slowed down action potential (AP) frequency. This inhibitory effect was even stronger in late stage cells, though I(f) was down‐regulated. In contrast to I(f), ICR was found to be indispensable for the occurrence of APs in ventricular cells of different stages, because abolishment of ICR with ryanodine and 2‐aminoethoxydiphenyl borate (2‐APB), specific blockers of ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), completely abolished APs. In addition, we noticed that RyR‐ and IP3R‐mediated ICR coexisted in early‐stage ventricular cells and RyRs functionally dominated. While at late stage RyRs, but not IP3Rs, mediated ICR. In both early and late stage ventricular cells, Na‐Ca exchanger current (I_Na/Ca) mediated ICR‐triggered depolarization of membrane potential and resulted in the initiation of APs. We also observed that different from I(f), which presented as the substantial component of the earlier diastolic depolarization current, application of ryanodine, and/or 2‐APB slowed the late phase of diastolic depolarization. Thus, we conclude that in murine embryonic ventricular cells I(f) regulates firing rate, while RyRs and IP3Rs (early stage) or RyRs (late stage)‐mediated ICR determines the occurrence of APs. J. Cell. Biochem. 114: 1852–1862, 2013. © 2013 Wiley Periodicals, Inc.     

Extracellular ATP-induced nuclear Ca transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic β-cells

Extracellular ATP (eATP) induces an intracellular Ca²⁺ transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca²⁺ release from intracellular stores in mouse pancreatic β-cells. Using laser scanning confocal microscopy, Ca²⁺ indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca²⁺ release in pancreatic β-cell nuclei. eATP induced a higher nuclear Ca²⁺ transient in pancreatic β-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca²⁺ transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca²⁺-ATPase (PMCA) or the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) by LaCl₃ or by replacement of Na⁺ with N-Methyl-Glucosamine. eATP-induced nuclear Ca²⁺ transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic β-cells. These results indicate that eATP triggers nuclear Ca²⁺ transients by mobilizing a nuclear Ca²⁺ store via nuclear IP3Rs.     

Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons

Progesterone (P4) is a steroid hormone that plays multiple roles in the central nervous system (CNS) including promoting neuroprotection. However, the precise mechanisms involved in its neuroprotective effects are still unknown. Given that the regulation of the intracellular calcium (Ca²⁺) concentration is critical for cell survival, we determined if inositol 1, 4, 5-trisphosphate receptors (IP₃Rs) are relevant targets of P4. Using primary hippocampal neurons, we tested the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca²⁺ signaling in neurons and characterized the subcellular distribution and phosphorylation of potential signaling intermediates involved in P4s actions. Our results reveal that P4 treatment altered the intensity and distribution of IP3R immunoreactivity and induced the nuclear translocation of phosphorylated Akt. Further, P4 potentiated IP₃R-mediated intracellular Ca²⁺ responses. These results suggest a potential involvement of P4 in particular and of steroid hormone signaling pathways in general in the control of intracellular Ca²⁺ signaling and its related functions.     

Bradykinin‐mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c‐Kit+ progenitor cells

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c‐Kit⁺ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin‐mediated Ca²⁺ signalling participates in regulating cellular functions in cultured human cardiac c‐Kit⁺ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca²⁺ () by triggering a transient Ca²⁺ release from ER IP3Rs followed by sustained Ca²⁺ influx through store‐operated Ca²⁺ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca²⁺ release and Ca²⁺ influx. It is interesting to note that the bradykinin‐induced cell cycle progression and migration were not observed in cells with siRNA‐silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin‐induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin‐mediated increase in free via ER‐IP3R3 Ca²⁺ release followed by Ca²⁺ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c‐Kit⁺ progenitor cells.     

Keeping zombies alive: The ER-mitochondria Ca2+ transfer in cellular senescence

Cellular senescence generates a permanent cell cycle arrest, characterized by apoptosis resistance and a pro-inflammatory senescence-associated secretory phenotype (SASP). Physiologically, senescent cells promote tissue remodeling during development and after injury. However, when accumulated over a certain threshold as happens during aging or after cellular stress, senescent cells contribute to the functional decline of tissues, participating in the generation of several diseases. Cellular senescence is accompanied by increased mitochondrial metabolism. How mitochondrial function is regulated and what role it plays in senescent cell homeostasis is poorly understood. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca²⁺) storage organelle in mammalian cells, through special domains known as mitochondria-ER contacts (MERCs). In this domain, the release of Ca²⁺ from the ER is mainly regulated by inositol 1,4,5-trisphosphate receptors (IP3Rs), a family of three Ca²⁺ release channels activated by a ligand (IP3). IP3R-mediated Ca²⁺ release is transferred to mitochondria through the mitochondrial Ca²⁺ uniporter (MCU), where it modulates the activity of several enzymes and transporters impacting its bioenergetic and biosynthetic function. Here, we review the possible connection between ER to mitochondria Ca²⁺ transfer and senescence.Understanding the pathways that contribute to senescence is essential to reveal new therapeutic targets that allow either delaying senescent cell accumulation or reduce senescent cell burden to alleviate multiple diseases.     

An efficient reduced-lattice model of IP3R for probing Ca2+ dynamics

Numerous cellular processes are regulated by Ca²⁺ signals, and the endoplasmic reticulum (ER) membrane's inositol triphosphate receptor (IP₃R) is critical for modulating intracellular Ca²⁺ dynamics. The IP₃Rs are seen to be clustered in a variety of cell types. The combination of IP₃Rs clustering and IP₃Rs-mediated Ca²⁺-induced Ca²⁺ release results in the hierarchical organization of the Ca²⁺ signals, which challenges the numerical simulation given the multiple spatial and temporal scales that must be covered. The previous methods rather ignore the spatial feature of IP₃Rs or fail to coordinate the conflicts between the real biological relevance and the computational cost. In this work, a general and efficient reduced-lattice model is presented for the simulation of IP₃Rs-mediated multiscale Ca²⁺ dynamics. The model highlights biological details that make up the majority of the calcium events, including IP₃Rs clustering and calcium domains, and it reduces the complexity by approximating the minor details. The model's extensibility provides fresh insights into the function of IP₃Rs in producing global Ca²⁺ events and supports the research under more physiological circumstances. Our work contributes to a novel toolkit for modeling multiscale Ca²⁺ dynamics and advances knowledge of Ca²⁺ signals.     

Inositol (1,4,5)-Trisphosphate Receptor Microarchitecture Shapes Ca Puff Kinetics

Inositol (1,4,5)-trisphosphate receptors (IP₃Rs) release intracellular Ca²⁺ as localized Ca²⁺ signals (Ca²⁺ puffs) that represent the activity of small numbers of clustered IP₃Rs spaced throughout the endoplasmic reticulum. Although much emphasis has been placed on estimating the number of active Ca²⁺ release channels supporting Ca²⁺ puffs, less attention has been placed on understanding the role of cluster microarchitecture. This is important as recent data underscores the dynamic nature of IP₃R transitions between heterogeneous cellular architectures and the differential behavior of IP₃Rs socialized into clusters. Here, we applied a high-resolution model incorporating stochastically gating IP₃Rs within a three-dimensional cytoplasmic space to demonstrate: 1), Ca²⁺ puffs are supported by a broad range of clustered IP₃R microarchitectures; 2), cluster ultrastructure shapes Ca²⁺ puff characteristics; and 3), loosely corralled IP₃R clusters (>200 nm interchannel separation) fail to coordinate Ca²⁺ puffs, owing to inefficient triggering and impaired coupling due to reduced Ca²⁺-induced Ca²⁺ release microwave velocity (<10 nm/s) throughout the channel array. Dynamic microarchitectural considerations may therefore influence Ca²⁺ puff occurrence/properties in intact cells, contrasting with a more minimal role for channel number over the same simulated conditions in shaping local Ca²⁺ dynamics.     

Coupling switch of P2Y-IP<Subscript>3</Subscript> receptors mediates differential Ca<Superscript>2+</Superscript> signaling in human embryonic stem cells and derived cardiovascular progenitor cells

Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However, how it mediates Ca²⁺ signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here, we aimed to determine the role of P2Rs in mediating Ca²⁺ mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP₃Rs) was analyzed by quantitative/RT-PCR. IP₃R3 knockdown (KD) or IP₃R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations, respectively. Confocal imaging revealed that Ca²⁺ responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently, the gene expression levels of most P2YRs except P2Y₁ were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca²⁺ transients in hESCs but only partially inhibited those in CVPCs. Moreover, the P2Y₁ receptor-specific antagonist MRS2279 abolished most ATP-induced Ca²⁺ signals in hESCs but not in CVPCs. P2Y₁ receptor-specific agonist MRS2365 induced Ca²⁺ transients only in hESCs but not in CVPCs. Furthermore, IP₃R2KO but not IP₃R3KD decreased the proportion of hESCs responding to MRS2365. In contrast, both IP₃R2 and IP₃R3 contributed to UTP-induced Ca²⁺ responses while ATP-induced Ca²⁺ responses were more dependent on IP₃R2 in the CVPCs. In conclusion, a predominant role of P2Y₁ receptors in hESCs and a transition of P2Y-IP₃R coupling in derived CVPCs are responsible for the differential Ca²⁺ mobilization between these cells.     

The formation of Ca<Superscript>2+</Superscript> gradients at the cleavage furrows during cytokinesis of Zebrafish embryos

In dividing embryos, a localized elevation in intracellular Ca²⁺ ([Ca²⁺]_i) at the cleavage furrow has been shown to be essential for cytokinesis. However, the underlying mechanisms for generating and maintaining these [Ca²⁺]_i gradients throughout cytokinesis are not fully understood. In the present study, we analyzed the role of inositol 1,4,5-trisphosphate receptors (IP₃Rs) and endoplasmic reticulum (ER) distribution in determining the intracellular Ca²⁺ gradients in early zebrafish blastomeres. Application of the injected Ca²⁺ indicator, Indo-1, showed that during the first cell division a standing Ca²⁺ gradient was formed ∼35 min after fertilization, with the [Ca²⁺]_i spatially decaying from 500–600 nmol/L at the cleavage furrow to 100–200 nmol/L around the nucleus. While the IP3R immunohistochemical fluorescence was relatively concentrated in the peri-furrow region, ER labeling was relatively enriched in both peri-furrow and peri-nuclear regions. Numeric simulation suggested that a divergence in the spatial distribution of IP3R and the locations of Ca²⁺ uptake within the ER was essential for the formation of a standing Ca²⁺ gradient, and the Ca²⁺ gradient could only be well-established under an optimal stoichiometry of Ca²⁺ uptake and release. Indeed, while inhibition of IP3R Ca²⁺ release blocked the generation of the Ca²⁺ gradient at a lower [Ca²⁺]_i level, both Ca²⁺ release stimulation by inositol 1,4,5-trisphosphate (IP3) injection and ER Ca²⁺ pump inhibition by cyclopiazonic acid also eliminated the Ca²⁺ gradients at higher [Ca²⁺]_i levels. Our results suggest a dynamic relationship between ER-mediated Ca²⁺ release and uptake that underlies the maintenance of the perifurrow Ca²⁺ gradient and is essential for cytokinesis of zebrafish embryos.     

Interaction of 2-APB,dantrolene, and TDMT with IP3R and RyR modulates ER stress-induced programmed cell death I and II in neuron-like PC12 cells: an experimental and computational investigation

Ca²⁺ is an essential second messenger, playing a fundamental role in maintaining cell viability and neuronal activity. Two specific endoplasmic reticulum calcium channels, ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) play an important role in Ca²⁺ regulation. In the present study, we provided a 3D structure of RyR and IP3R by homology modeling, and we predicted their interactions with a known neuroprotective compound, 3-thiomethyl-5,6-(dimethoxyphenyl)-1,2,4-triazine (TDMT), as well as two inhibitors, dantrolene and 2-aminoethoxydiphenyl borate (2-APB). Interestingly, we found that dantrolene and 2-APB can bind to the IP3-binding domain of IP3R and RyR, while TDMT may directly block both channels by interacting with the putative resident domains in the pore. Cell culture experiments showed that these compounds could protect PC12 cells against H₂O₂-induced apoptosis and activate autophagic pathways. Collectively, our computational (in silico) and cell culture studies suggest that RyR and IP3R are novel and promising targets to be used against neurodegenerative diseases.     

IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) serve to discharge Ca²⁺ from ER stores in response to agonist stimulation. The present review summarizes the role of these receptors in models of Ca²⁺-dependent apoptosis. In particular we focus on the regulation of IP₃Rs by caspase-3 cleavage, cytochrome c, anti-apoptotic proteins and Akt kinase. We also address the evidence that some of the effects of IP₃Rs in apoptosis may be independent of their ion-channel function. The role of IP₃Rs in delivering Ca²⁺ to the mitochondria is discussed from the perspective of the factors determining inter-organellar dynamics and the spatial proximity of mitochondria and ER membranes.     

Bcl-xL acts as an inhibitor of IP3R channels,thereby antagonizing Ca2+-driven apoptosis

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca²⁺ dynamics by controlling IP₃ receptor (IP₃R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP₃Rs and preventing pro-apoptotic Ca²⁺ release and Bcl-xL sensitizing IP₃Rs to low [IP₃] and promoting pro-survival Ca²⁺ oscillations. We here demonstrate that Bcl-xL too inhibits IP₃R-mediated Ca²⁺ release by interacting with the same IP₃R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2’s IP₃R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP₃R and abrogated Bcl-xL’s inhibitory effect on IP₃Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL^K87D, suppressed IP₃R single-channel openings stimulated by sub-maximal and threshold [IP₃]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP₃Rs contributes to its anti-apoptotic properties against Ca²⁺-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca²⁺ elevations in wild-type but not in IP₃R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca²⁺ signals and cell death, while Bcl-xL^K87D was much less effective in doing so. In the absence of IP₃Rs, Bcl-xL and Bcl-xL^K87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP₃R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP₃R-mediated Ca²⁺ release and increased the sensitivity towards STS, without altering the ER Ca²⁺ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca²⁺-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP₃R-mediated Ca²⁺ release and IP₃R-driven cell death. Our work further underpins that IP₃R inhibition is an integral part of Bcl-xL’s anti-apoptotic function.Subject terms: Cancer, Cell biology, Molecular biology     

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP₃R) are the most widely expressed intracellular Ca²⁺ release channels. Their activation by IP₃ and Ca²⁺ allows Ca²⁺ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca²⁺] may directly regulate cytosolic effectors or fuel Ca²⁺ uptake by other organelles, while the decrease in ER luminal [Ca²⁺] stimulates store-operated Ca²⁺ entry (SOCE). We are close to understanding the structural basis of both IP₃R activation, and the interactions between the ER Ca²⁺-sensor, STIM, and the plasma membrane Ca²⁺ channel, Orai, that lead to SOCE. IP₃Rs are the usual means through which extracellular stimuli, through ER Ca²⁺ release, stimulate SOCE. Here, we review evidence that the IP₃Rs most likely to respond to IP₃ are optimally placed to allow regulation of SOCE. We also consider evidence that IP₃Rs may regulate SOCE downstream of their ability to deplete ER Ca²⁺ stores. Finally, we review evidence that IP₃Rs in the plasma membrane can also directly mediate Ca²⁺ entry in some cells.     

Stimulus-Dependent Regulation of Nuclear Ca2+ Signaling in Cardiomyocytes: A Role of Neuronal Calcium Sensor-1

In cardiomyocytes, intracellular calcium (Ca²⁺) transients are elicited by electrical and receptor stimulations, leading to muscle contraction and gene expression, respectively. Although such elevations of Ca²⁺levels ([Ca²⁺]) also occur in the nucleus, the precise mechanism of nuclear [Ca²⁺] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca²⁺] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca²⁺ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca²⁺ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca²⁺ transients are slower than cytoplasmic Ca²⁺ transients, and chelating cytoplasmic Ca²⁺ abolished nuclear Ca²⁺ transients, suggesting that nuclear Ca²⁺ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca²⁺] compared to cytoplasmic [Ca²⁺]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca²⁺ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca²⁺ transient amplitude was significantly lower in myocytes lacking neuronal Ca²⁺ sensor-1 (NCS-1), a Ca²⁺ binding protein implicated in IP₃R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP₃R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca²⁺] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca²⁺ signal regulation.     

CaMKIIα interacts with M4 muscarinic receptors to control receptor and psychomotor function

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the mammalian brain and are essential for neuronal functions. These receptors are believed to be actively regulated by intracellular signals, although the underlying mechanisms are largely unknown. In this study, we show that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) binds directly and selectively to one of five mAChR subtypes, M4 receptors (M4Rs), at their C‐terminal regions of second intracellular loops. This binding relies on Ca²⁺ activation of the kinase and leads to the phosphorylation of M4Rs at a specific threonine site (Thr145). Complementary in vivo studies in rat striatal neurons enriched with M4Rs confirm that rising Ca²⁺ recruits CaMKIIα to M4Rs to potentiate receptor signalling, which controls behavioural sensitivity to dopamine stimulation in an activity‐dependent manner. Our data identify a new model of protein–protein interactions. In a Ca²⁺‐sensitive manner, CaMKIIα regulates M4R efficacy and controls the acetylcholine–dopamine balance in the basal ganglia and also the dynamics of movement.     

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca²⁺-activated-K⁺ (BK_Ca) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BK_Ca channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca²⁺ concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca²⁺ abolished STOCs activity. Ca²⁺ ionophore A23187 (10 μmol/L) increased STOCs activity significantly. Verapamil (20 μmol/L) and CdCl₂ (200 μmol/L), two kinds of organic L-type voltage-dependent Ca²⁺ channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 μmol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) by 2APB (40 μmol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BK_Ca channels. Extracellular Ca²⁺ is essential for STOCs activity, while Ca²⁺ entry through L-VDCCs has little effect on STOCs. Intracellular Ca²⁺ release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.     

Termination of calcium puffs and coupled closings of inositol trisphosphate receptor channels

Calcium puffs are localized Ca²⁺ signals mediated by Ca²⁺ release from the endoplasmic reticulum (ER) through clusters of inositol trisphosphate receptor (IP₃R) channels. The recruitment of IP₃R channels during puffs depends on Ca²⁺-induced Ca²⁺ release, a regenerative process that must be terminated to maintain control of cell signaling and prevent Ca²⁺ cytotoxicity. Here, we studied puff termination using total internal reflection microscopy to resolve the gating of individual IP₃R channels during puffs in intact SH-SY5Y neuroblastoma cells. We find that the kinetics of IP₃R channel closing differ from that expected for independent, stochastic gating, in that multiple channels tend to remain open together longer than predicted from their individual open lifetimes and then close in near-synchrony. This behavior cannot readily be explained by previously proposed termination mechanisms, including Ca²⁺-inhibition of IP₃Rs and local depletion of Ca²⁺ in the ER lumen. Instead, we postulate that the gating of closely adjacent IP₃Rs is coupled, possibly via allosteric interactions, suggesting an important mechanism to ensure robust puff termination in addition to Ca²⁺-inactivation.     

Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival

Bcl-2 family proteins, known for their apoptosis functioning at the mitochondria, have been shown to localize to other cellular compartments to mediate calcium (Ca²⁺) signals. Since the proper supply of Ca²⁺ in cells serves as an important mechanism for cellular survival and bioenergetics, we propose an integrating role for Bcl-2 family proteins in modulating Ca²⁺ signaling. The endoplasmic reticulum (ER) is the main Ca²⁺ storage for the cell and Bcl-2 family proteins competitively regulate its Ca²⁺ concentration. Bcl-2 family proteins also regulate the flux of Ca²⁺ from the ER by physically interacting with inositol 1,4,5-trisphosphate receptors (IP₃Rs) to mediate their opening. Type 1 IP₃Rs reside at the bulk ER to coordinate cytosolic Ca²⁺ signals, while type 3 IP₃Rs reside at mitochondria-associated ER membrane (MAM) to facilitate mitochondrial Ca²⁺ uptake. In healthy cells, mitochondrial Ca²⁺ drives pyruvate into the citric acid (TCA) cycle to facilitate ATP production, while a continuous accumulation of Ca²⁺ can trigger the release of cytochrome c, thus initiating apoptosis. Since multiple organelles and Bcl-2 family proteins are involved in Ca²⁺ signaling, we aim to clarify the role that Bcl-2 family proteins play in facilitating Ca²⁺ signaling and how mitochondrial Ca²⁺ is relevant in both bioenergetics and apoptosis. We also explore how these insights could be useful in controlling bioenergetics in apoptosis-resistant cell lines.     

Sigma receptors [σRs]: biology in normal and diseased states

This review compares the biological and physiological function of Sigma receptors [σRs] and their potential therapeutic roles. Sigma receptors are widespread in the central nervous system and across multiple peripheral tissues. σRs consist of sigma receptor one (σ₁R) and sigma receptor two (σ₂R) and are expressed in numerous regions of the brain. The sigma receptor was originally proposed as a subtype of opioid receptors and was suggested to contribute to the delusions and psychoses induced by benzomorphans such as SKF-10047 and pentazocine. Later studies confirmed that σRs are non-opioid receptors (not an µ opioid receptor) and play a more diverse role in intracellular signaling, apoptosis and metabolic regulation. σ₁Rs are intracellular receptors acting as chaperone proteins that modulate Ca²⁺ signaling through the IP₃ receptor. They dynamically translocate inside cells, hence are transmembrane proteins. The σ₁R receptor, at the mitochondrial-associated endoplasmic reticulum membrane, is responsible for mitochondrial metabolic regulation and promotes mitochondrial energy depletion and apoptosis. Studies have demonstrated that they play a role as a modulator of ion channels (K⁺ channels; N-methyl-d-aspartate receptors [NMDAR]; inositol 1,3,5 triphosphate receptors) and regulate lipid transport and metabolism, neuritogenesis, cellular differentiation and myelination in the brain. σ₁R modulation of Ca²⁺ release, modulation of cardiac myocyte contractility and may have links to G-proteins. It has been proposed that σ₁Rs are intracellular signal transduction amplifiers. This review of the literature examines the mechanism of action of the σRs, their interaction with neurotransmitters, pharmacology, location and adverse effects mediated through them.     

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca2 -activated-K (BKCa) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BKCa channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca2 concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca2 abolished STOCs activity. Ca2 ionophore A23187 (10 μmol/L) increased STOCs activity significantly. Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two kinds of organic L-type voltage-dependent Ca2 channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 μmol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP3Rs) by 2APB (40 μmol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BKCa channels. Extracellular Ca2 is essential for STOCs activity, while Ca2 entry through L-VDCCs has little effect on STOCs. Intracellular Ca2 release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.