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Calcium microdomains: Organization and function
Abstract
Microdomains of Ca(2+), which are formed at sites where Ca(2+) enters the cytoplasm either at the cell surface or at the internal stores, are a key element of Ca(2+) signalling. The term microdomain includes the elementary events that are the basic building blocks of Ca(2+) signals. As Ca(2+) enters the cytoplasm, it produces a local plume of Ca(2+) that has been given different names (sparks, puffs, sparklets and syntillas). These elementary events can combine to produce larger microdomains. The significance of these localized domains of Ca(2+) is that they can regulate specific cellular processes in different regions of the cell. Such microdomains are particularly evident in neurons where both pre- and postsynaptic events are controlled by highly localized pulses of Ca(2+). The ability of single neurons to process enormous amounts of information depends upon such miniaturization of the Ca(2+) signalling system. Control of cardiac cell contraction and gene transcription provides another example of how the parallel processing of Ca(2+) signalling can occur through microdomains of intracellular Ca(2+).
... Ca 2+ release from neuronal ER stores can be evoked by stimulation of RyRs or IP 3 Rs, and both receptor types can couple to neurotransmitter-gated receptors and voltage-gated Ca 2+ channels on the plasma membrane. This organization enables the ER to function not only as a buffer and source of Ca 2+ in axonal and somatodendritic compartments but also to discriminate between different types of neuronal activity and integrate Ca 2+ signaling between the plasma membrane, cytosol and nucleus (Bardo et al., 2006;Berridge, 2006). RyR1s anchored to the ER in very close proximity to plasmalemmal L-type voltage-dependent Ca 2+ channels engage in a form of voltage-induced Ca 2+ release that is similar to EC coupling in myocytes (De Crescenzo et al., 2006). ...
... RyR channel activity regulates diverse physiological and pathophysiological processes in the nervous system (Berridge, 2006;Lanner et al., 2010;Pessah et al., 2010). RyRs contribute to fundamentally important aspects of neuronal excitability and to both neurochemical and structural aspects of use-dependent synaptic plasticity (Berridge, 1998;Kennedy, 2000;Korkotian and Segal, 1999;Matus, 2000;Segal, 2001). ...
... Consistent with the demonstrated role of RyRs in neurotransmission and synaptic plasticity at the cellular level, ligands that directly modulate RyR activity, such as ryanodine, FK506 and rapamycin, alter functional aspects of neuroplasticity in the hippocampus, including long-term potentiation (LTP) (Wang et al., 1996) and long-term depression (LTD) (Li et al., 1998;Wang et al., 1997). Dynamic changes in [Ca 2+ ] i also play a crucial role in cell proliferation and differentiation, cell movement and cell death in the developing nervous system (Cline, 2001;Moody and Bosma, 2005;Spitzer et al., 2004;Zheng and Poo, 2007) via regulation of Ca 2+ signaling pathways that regulate these neurodevelopmental processes (Berridge, 1998;Berridge, 2006;Pessah et al., 2010). ...
... These ionic transporters are also present on the membranes of intracellular organelles, such as the endoplasmic/sarcoplasmic reticulum, the mitochondria, and the nucleus [16,17]. In addition, calcium micro-and nanodomains are formed at the inner side of the mouth of a transporter that contributes to intracellular calcium release, uptake, and signaling [18][19][20][21][22]. These micro/nanodomains enable the cell to use calcium in a specific part of the cell [18]. ...
... In addition, calcium micro-and nanodomains are formed at the inner side of the mouth of a transporter that contributes to intracellular calcium release, uptake, and signaling [18][19][20][21][22]. These micro/nanodomains enable the cell to use calcium in a specific part of the cell [18]. These micro/nanodomains were given names (sparklet, spark, blink, syntilla, and puff) depending on the type of transporter that created such domains [18] (Figure 1). ...
... These micro/nanodomains enable the cell to use calcium in a specific part of the cell [18]. These micro/nanodomains were given names (sparklet, spark, blink, syntilla, and puff) depending on the type of transporter that created such domains [18] (Figure 1). For example, sparklet originates from the calcium voltage-operated calcium channel (VOCC) opening, which creates a calcium microdomain by activating calcium-induced calcium release from the endoplasmic/sarcoplasmic reticulum (ER/SR) ryanodine type 2 receptor (RyR2). ...
Calcium is a highly positively charged ionic species. It regulates all cell types’ functions and is an important second messenger that controls and triggers several mechanisms, including membrane stabilization, permeability, contraction, secretion, mitosis, intercellular communications, and in the activation of kinases and gene expression. Therefore, controlling calcium transport and its intracellular homeostasis in physiology leads to the healthy functioning of the biological system. However, abnormal extracellular and intracellular calcium homeostasis leads to cardiovascular, skeletal, immune, secretory diseases, and cancer. Therefore, the pharmacological control of calcium influx directly via calcium channels and exchangers and its outflow via calcium pumps and uptake by the ER/SR are crucial in treating calcium transport remodeling in pathology. Here, we mainly focused on selective calcium transporters and blockers in the cardiovascular system.
... The concentration of free and bound [Ca 2+ ] cyt is reported to be approximately 100 nM and 10 μM, respectively [15] . While a localized increase in [Ca 2+ ] cyt has been evidenced in some studies, others show spatio-temporal calcium signaling restricted to nano and microdomains in neurons [16] and smooth muscle [17] [18] . ...
... In the development of a new model, consideration was given to the spatial structure of the soma and some key concepts related to the kinetics of Ca 2+ . The increase in Ca 2+ is located in the microdomain [16] formed mainly by the following factors [28] : the BK channels on the soma membrane [1] [44] and the Ca 2+and IP 3 -R-sensitive channels on the ER membrane [42] . ...
... Firstly, the influx of Ca 2+ to the cytosol depends on the rate of transport through the IP 3 -sensitive mechanism (r= V 1 *β) and the Ca 2+ -sensitive mechanism (V 3 ), the maximum rate of Ca 2+ pumping release from the ER store (V M3 ), the threshold constants for release and activation (K R and K A , respectively) (Figures 1 and 5 [53] . Khodakhah and Ogden [54] reported that IP 3 triggers a release of Ca 2+ from the ER with an initial well-defined delay, which decreases as the concentration of IP 3 rises from the ER takes place through sparklets, sparks, blink, scintilla, puffs, and other forms [16] . Since such elementary events are produced in microdomains, the multiple forms of Ca 2+ release confer intracellular Ca 2+ signaling with a broad architecture in space, time, and intensity, which in turn underlies signaling efficiency, stability, specificity, and diversity [55] . ...
Large conductance calcium-activated potassium (BK) channels carry out many functions in the central nervous system. These channels open in response to increased cytosolic calcium ([Ca2+]cyt) concentration. The influx of calcium ions to the cytosol can occur through voltage-gated calcium channels (VGCCs) on the plasma membrane and/ or through IP3 receptors (IP3-Rs) and ryanodine receptors (RyRs) on the endoplasmic reticulum membrane. The BK channel/IP3-R/RyR interaction has been widely reported in smooth muscle but scarcely investigated in relation to neurons. The aim of this study was to theoretically explore the function of the BK/IP3-R complex by means of a computational model of a neuron that replicates the interaction between the release of Ca2+ from the endoplasmic reticulum (through IP3-Rs) and the opening of the BK channels. The mathematical models are based on the Hodgkin-Huxley formalism and the Goldbeter model. These models were implemented on Visual Basic® and differential equations were solved numerically. Distinct conditions were contemplated for BK conductance and the efflux of endoplasmic Ca2+ to the cytosol. An abrupt rise in [Ca2+]cyt (≥ 5 μM) and short duration (spark) was found to activate BK channels and either pause or stop the action potential train.
... Traditional animal Ca V 1 and Ca V 2 channels possess high Ca 2+ selective over Na + (~1,000 to 1) on the condition that external Ca 2+ is present at low micromolar levels [63][64][65][66][67]. A tight regulation of selective Ca 2+ entry is of salient importance to cells as a key messenger of local, intracellular Ca 2+ signaling triggering a cascade of different functions within cells and to their intracellular compartments [68]. Most intracellular Ca 2+ ions are not free but are bound to a myriad of different intracellular Ca 2+ handling proteins, in affinities which differ within cells, and from cell to cell [68]. ...
... A tight regulation of selective Ca 2+ entry is of salient importance to cells as a key messenger of local, intracellular Ca 2+ signaling triggering a cascade of different functions within cells and to their intracellular compartments [68]. Most intracellular Ca 2+ ions are not free but are bound to a myriad of different intracellular Ca 2+ handling proteins, in affinities which differ within cells, and from cell to cell [68]. Requirement for the titration of microtargeted doses of Ca 2+ influx, at precise time and space along the plasma membrane, necessitates a high Ca 2+ selectivity within Ca V 1 and Ca V 2 channels, and less so for Ca V 3 channels [49,69] which have a more electrogenic, pace-making function in the nervous and cardiovascular systems. ...
One of nature’s exceptions was discovered when a Cav3 T-type channel was observed to switch phenotype from a calcium channel into a sodium channel by neutralizing an aspartate residue in the high field strength (HFS) +1 position within the ion selectivity filter. The HFS+1 site is dubbed a “beacon” for its location at the entryway just above the constricted, minimum radius of the HFS site’s electronegative ring. A classification is proposed based on the occupancy of the HFS+1 “beacon” which correlates with the calcium- or sodium-selectivity phenotype. If the beacon is a glycine, or neutral, non-glycine residue, then the cation channel is calcium-selective or sodium-permeable, respectively (Class I). Occupancy of a beacon aspartate are calcium-selective channels (Class II) or possessing a strong calcium block (Class III). A residue lacking in position of the sequence alignment for the beacon are sodium channels (Class IV). The extent to which animal channels are sodium-selective is dictated in the occupancy of the HFS site with a lysine residue (Class III/IV). Governance involving the beacon solves the quandary the HFS site as a basis for ion selectivity, where an electronegative ring of glutamates at the HFS site generates a sodium-selective channel in one-domain channels but generates a calcium-selective channel in four-domain channels. Discovery of a splice variant in an exceptional channel revealed nature’s exploits, highlighting the “beacon” as a principal determinant for calcium and sodium selectivity, encompassing known ion channels composed of one and four domains, from bacteria to animals.
... secretion, or long term, such as cell growth, proliferation, apoptosis and differentiation (Berridge, 2006;Carafoli, 2003;Dupont et al., 2011). The maintenance of Ca 2+ , in the absence of stimuli, at nanomolar concentrations in the cytoplasm of cells is an indispensable condition to avoid the activation of unwanted transduction signals (Berridge, 2006;Dupont et al., 2011;Ordenes et al., 2002;Urbina et al., 2006). ...
... secretion, or long term, such as cell growth, proliferation, apoptosis and differentiation (Berridge, 2006;Carafoli, 2003;Dupont et al., 2011). The maintenance of Ca 2+ , in the absence of stimuli, at nanomolar concentrations in the cytoplasm of cells is an indispensable condition to avoid the activation of unwanted transduction signals (Berridge, 2006;Dupont et al., 2011;Ordenes et al., 2002;Urbina et al., 2006). Allergens influence cellular signals including those of Ca 2+ , determining the elevation of Ca 2+ cytosolic, which triggers the production of cytokines as a response with an unknown mechanism (Jairaman et al., 2015;Matsuwaki et al., 2012). ...
Inhalation of olive pollen (Olea europaea L.) is one of the main causes of allergy in Mediterranean countries and some areas of North America. The response to allergens consists in the production of inflammatory cytokines which is mediated by the deregulation of Ca²⁺ signals. In this study, the biological activity of the material released in olive pollen hydration (PMR) was tested on Ca²⁺ cytosolic of PE/CA-PJ15 cells (PJ-15). Ca²⁺ cytosolic was determined by fluorometric assay with the cell line PE/CA-PJ15 (PJ-15) labeled with the fluorescent probe FURA 2 AM. The material released in olive pollen hydration (PMR) was analyzed by HPLC for the determination of phenolic acids. PMR was subjected to fractionation by gel filtration, and the fractions with Ca²⁺-chelating activity were tested with SDS-PAGE and the single bands characterized by proteomic analysis. PMR showed high Ca²⁺-chelating activity and is able of blocking the increase Ca²⁺-cytosolic produced by thapsigargin (TG). PMR then restored Ca²⁺ homeostasis in PJ-15 cells deregulated by the endoplasmic reticulum Ca²⁺-ATPases inhibitor. It is therefore possible that PMR can antagonize the effects of allergens on Ca²⁺ cytosolic. The analytical characterization of the material released by the pollen highlighted in the pollen allergen Ole e 3 and in the p-coumaric acid the possible culprits of the Ca²⁺-antagonist activity of PMR. Furthermore, the sequence of Ole e 3 could provide information for the possible construction of a synthetic peptide to be used in an allergy-targeted Ca²⁺-antagonist therapy.
... Due to the spatial distribution and temporal kinetics of different Ca 2+ permeable molecules, the actual elevation of intracellular Ca 2+ can vary widely between different cell types and sub-cellular compartments, and local concentrations have been estimated to reach levels as high as 10-100 μM during an induced Ca 2+ influx from the plasma membrane or Ca 2+ release from ER Ca 2+ store (Berridge, 2006;Matthews et al., 2013). ...
... Chelation of intracellular Ca 2+ with BAPTA-AM partially blocked this NaR reactivation. It is thus proposed that local Ca 2+ microdomains are underlying the quiescence exit of NaR cells under low [Ca 2+ ]i.In further support of this hypothesis, depletion of ER Ca 2+ store using SERCA inhibitors completely abolished low [Ca 2+ ]i -triggered NaR cell reactivation.Ca 2+ microdomains have long been recognized as critical regulators for Ca 2+ signaling(Berridge, 2006). The [Ca 2+ ]i a cell under resting conditions is around 100 nM, while ER [Ca 2+ ] ([Ca 2+ ]ER) is maintained around 100 µM. ...
Epithelial tissues renew rapidly and continuously by reactivating a pool of quiescent cells. How the quiescent cells are established, maintained, and reactivated is poorly defined. Recent studies suggest that the insulin-like growth factor (IGF)-PI3 kinase-AKT-mTOR signaling pathway plays a key role in regulating epithelial cell quiescence-proliferation decision but the underlying mechanism remains unclear. In my thesis work, I use a zebrafish model to investigate the IGF action in a group of Ca2+-transporting epithelial cells, known as Na+-K+-ATPase-rich (NaR) cells. When zebrafish are kept in normal and physiological [Ca2+] embryo rearing media, NaR cells are quiescent, characterized by a very slow division rate and undetectable Akt and Tor activity. When subjected to low [Ca2+] stress, the NaR cells exit the quiescent state and proliferate due to elevated IGF1 receptor-mediated Akt and Tor activity. To understand how the IGF signaling is activated exclusively in NaR cells under low [Ca2+] stress, I first investigated the role of Igfbp5a, a secreted protein belonging to the IGF binding protein (IGFBP) family. Zebrafish igfbp5a is specifically expressed in NaR cells and genetic deletion of igfbp5a blunted the low Ca2+ stress-induce IGF-Akt-Tor activity and NaR cell reactivation. Similarly, knockdown of IGFBP5 in human colon carcinoma cells resulted in reduced IGF-stimulated cell proliferation. Re-expression of zebrafish or human Igfbp5a/IGFBP5 in NaR cells restores NaR cell proliferation. Mechanistically, Igfbp5a acts by binding to IGFs using its ligand-binding domain and promoting IGF signaling in NaR cells. These results reveal a conserved mechanism by which a locally expressed Igfbp activates IGF signaling and promoting cell quiescence-proliferation transition under Ca2+-deficient states. NaR cells are functionally equivalent to human intestinal epithelial cells, and they contain all major molecular components of the transcellular Ca2+ transport machinery, including the epithelial calcium channel Trpv6. Ca2+ is a central intracellular second messenger controlling many aspects of cell biology. I next investigated the role of Trpv6 and intracellular [Ca2+]. I discovered that NaR cells are maintained in the quiescent state by Trpv6-mediated constitutive Ca2+ influx. Genetic deletion and pharmacological inhibition of Trpv6 promote NaR cell quiescence-proliferation transition. In zebrafish NaR cells and human colon carcinoma cells, Trpv6/TRPV6 elevated intracellular Ca2+ levels and activated PP2A, a group of conserved protein phosphatases, which down-regulates IGF signaling and promotes the quiescent state. Finally, chemical biology screens and genetic experiments identified CaMKK as a link between low Ca2+ stress and IGF signaling activation in NaR cells. Depletion of the ER Ca2+ store abolished NaR cell reactivation and IGF signaling. These results suggest that ER Ca2+ release in response to the low [Ca2+] stress activates CaMKK, which in turn increases IGF signaling and NaR cell reactivation. Taken together, the results of my thesis research provide new insights into the epithelial cell proliferation-quiescence regulation and have deepened our understanding of cellular quiescence regulation. These new findings may also contribute to the future development of strategies in improving wound healing and tissue regeneration.
... Ca 2+ signalling is indeed often restricted to local subcellular areas called micro-or nanodomains (Berridge, 2006;Parekh, 2008). Junctions between the plasma membrane (PM) and the adjacent endoplasmic reticulum (ER) are increasingly recognised as such Ca 2+ signalling nanodomains (Berridge, 2006;Stefan et al. 2013;Jin et al. 2016;Pacheco et al. 2016). ...
... Ca 2+ signalling is indeed often restricted to local subcellular areas called micro-or nanodomains (Berridge, 2006;Parekh, 2008). Junctions between the plasma membrane (PM) and the adjacent endoplasmic reticulum (ER) are increasingly recognised as such Ca 2+ signalling nanodomains (Berridge, 2006;Stefan et al. 2013;Jin et al. 2016;Pacheco et al. 2016). PM components of these ER-PM junctions often include GPCRs that induce Ca 2+ release from the ER via activation of phospholipase C (PLC), generation of inositol 1,4,5-trisphosphate (IP 3 ) and subsequent activation of the IP 3 receptors (IP 3 R) localised at the ER side of the junction (Jin et al. 2013(Jin et al. , 2016Stefan et al. 2013). ...
Key points
Rat somatosensory neurons express a junctional protein, junctophilin‐4 (JPH4)
JPH4 is necessary for the formation of store operated Ca²⁺ entry (SOCE) complex at the junctions between plasma membrane and endoplasmic reticulum in these neurons.
Knockdown of JPH4 impairs endoplasmic reticulum Ca²⁺ store refill and junctional Ca²⁺ signalling in sensory neurons.
In vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly attenuated experimentally induced inflammatory pain in rats.
Junctional nanodomain Ca²⁺ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.
Abstract
Junctions of endoplasmic reticulum and plasma membrane (ER‐PM junctions) form signalling nanodomains in eukaryotic cells. ER‐PM junctions are present in peripheral sensory neurons and are important for the fidelity of G protein coupled receptor (GPCR) signalling. Yet little is known about the assembly, maintenance and physiological role of these junctions in somatosensory transduction. Using fluorescence imaging, proximity ligation, super‐resolution microscopy, in vitro and in vivo gene knockdown we demonstrate that a member of the junctophilin protein family, junctophilin‐4 (JPH4), is necessary for the formation of store operated Ca²⁺ entry (SOCE) complex at the ER‐PM junctions in rat somatosensory neurons. Thus we show that JPH4 localises to the ER‐PM junctional areas and co‐clusters with SOCE proteins STIM1 and Orai1 upon ER Ca²⁺ store depletion. Knockdown of JPH4 impairs SOCE and ER Ca²⁺ store refill in sensory neurons. Furthermore, we demonstrate a key role of the JPH4 and junctional nanodomain Ca²⁺ signalling in the pain‐like response induced by the inflammatory mediator bradykinin. Indeed, an in vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly shortened the duration of nocifensive behaviour induced by hindpaw injection of bradykinin in rats. Since the ER supplies Ca²⁺ for the excitatory action of multiple inflammatory mediators, we suggest that junctional nanodomain Ca²⁺ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.
... These sequester calcium ions at high levels near the plasma membrane, a configuration called a "mitochondrial firewall." 70 We speculate that the RPE's distinctive mitochondrial layer also serves this purpose for the basolaterally located chloride channels important in fluid balance. 64,71 Our demonstration of abundant endoplasmic reticulum converges with the previously reported strong signal for the calcium-binding protein calreticulin in an RPE cell line, 24 to underscore the importance of calcium signaling. ...
Purpose
Despite the centrality of the retinal pigment epithelium (RPE) in vision and retinopathy our picture of RPE morphology is incomplete. With a volumetric reconstruction of human RPE ultrastructure, we aim to characterize major membranous features including apical processes and their interactions with photoreceptor outer segments, basolateral infoldings, and the distribution of intracellular organelles.
Methods
A parafoveal retinal sample was acquired from a 21-year-old male organ donor. With serial block-face scanning electron microscopy, a tissue volume from the inner-outer segment junction to basal RPE was captured. Surface membranes and complete internal ultrastructure of an individual RPE cell were achieved with a combination of manual and automated segmentation methods.
Results
In one RPE cell, apical processes constitute 69% of the total cell surface area, through a dense network of over 3000 terminal branches. Single processes contact several photoreceptors. Basolateral infoldings facing the choriocapillaris resemble elongated filopodia and comprise 22% of the cell surface area. Membranous tubules and sacs of endoplasmic reticulum represent 20% of the cell body volume. A dense basal layer of mitochondria extends apically to partly overlap electron-dense pigment granules. Pores in the nuclear envelope form a distinct pattern of rows aligned with chromatin.
Conclusions
Specialized membranes at the apical and basal side of the RPE cell body involved in intercellular uptake and transport represent over 90% of the total surface area. Together with the polarized distribution of organelles within the cell body, these findings are relevant for retinal clinical imaging, therapeutic approaches, and disease pathomechanisms.
... Apoptosis can be triggered by increased intracellular calcium ([Ca 2+ ] i ) and ROS levels. Therefore, transient receptor potential (TRP) channels responsible for Ca 2+ movements between extracellular and cytosolic media have important functions in RPE cell physiology [6][7][8]. Caspase-3 and -9 proteins have been found to be effective in the regulation of apoptosis [9]. Vascular endothelial growth factor (VEGF) secreted by RPE cells is needed for the physiological function of the choriocapillaris. ...
Diabetic retinopathy (DR), a complication of diabetes mellitus (DM), can cause severe visual loss. The retinal pigment epithelium (RPE) plays a crucial role in retinal physiology but is vulnerable to oxidative damage. We investigated the protective effects of selenium (Se) on retinal pigment epithelium (ARPE-19) and primary human retinal microvascular endothelial (ACBRI 181) cells against high glucose (HG)-induced oxidative stress and apoptotic cascade. To achieve this objective, we utilized varying concentrations of D-glucose (ranging from 5 to 80 mM) to induce the HG model. HG-induced oxidative stress in ARPE-19 and ACBRI 181 cells and the apoptotic cascade were evaluated by determining Ca2+ overload, mitochondrial membrane depolarization, caspase-3/-9 activation, intracellular reactive oxygen species (ROS), lipid peroxidation (LP), glutathione (GSH), glutathione peroxidase (GSH-Px), vascular endothelial growth factor (VEGF) and apoptosis levels. A cell viability assay utilizing MTT was conducted to ascertain the optimal concentration of Se to be employed. The quantification of MTT, ROS, VEGF levels, and caspase-3 and -9 activation was accomplished using a plate reader. To quantitatively assess LP and GSH levels, GSH-Px activities were utilized by spectrophotometer and apoptosis, mitochondrial membrane depolarization, and the release of Ca2+ from intracellular stores were evaluated by spectrofluorometer. Our investigation revealed a significant augmentation in oxidative stress induced by HG, leading to cellular damage through modulation of mitochondrial membrane potential, ROS levels, and intracellular Ca2+ release. Incubation with Se resulted in a notable reduction in ROS production induced by HG, as well as a reduction in apoptosis and the activation of caspase-3 and -9. Additionally, Se incubation led to decreased levels of VEGF and LP while concurrently increasing levels of GSH and GSH-Px. The findings from this study strongly suggest that Se exerts a protective effect on ARPE-19 and ACBRI 181 cells against HG-induced oxidative stress and apoptosis. This protective mechanism is partially mediated through the intracellular Ca2+ signaling pathway.
... Second messengers, including cyclic nucleotides (cAMP and cGMP) and calcium (Ca 2+ ), are key signaling molecules involved in a wide range of cellular pathways. Although diffusing freely in aqueous buffers, the mechanisms enabling them to achieve specificity for their many downstream cellular processes rely on the compartmentation of these signaling molecules 1,2 . The compartmentation of Ca 2+ has been identified in a range of cell types with a variety of subcellular locations. ...
Second messengers, including cAMP, cGMP and Ca²⁺ are often placed in an integrating position to combine the extracellular cues that orient growing axons in the developing brain. This view suggests that axon repellents share the same set of cellular messenger signals and that axon attractants evoke opposite cAMP, cGMP and Ca²⁺ changes. Investigating the confinement of these second messengers in cellular nanodomains, we instead demonstrate that two repellent cues, ephrin-A5 and Slit1, induce spatially segregated signals. These guidance molecules activate subcellular-specific second messenger crosstalk, each signaling network controlling distinct axonal morphology changes in vitro and pathfinding decisions in vivo.
... R. Soc. B 378: 20220162 cell excitability [114,115]. The latter in turn may constitute a subcategory of a broader group of Ca 2+ -mediated actions including Ca 2+ -enhanced slow RyR inactivation [116]. ...
Skeletal and cardiac muscle excitation–contraction coupling commences with Nav1.4/Nav1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward, allosteric or Ca²⁺-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca²⁺ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca²⁺]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca²⁺ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na⁺ currents (INa) following SR Ca²⁺ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca²⁺-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1β−/− cardiomyocytes also showed reduced INa accompanying [Ca²⁺]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca²⁺ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca²⁺-mediated effects to further, Ca²⁺, K⁺ and Cl⁻, channel types.
This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’.
... Calcium pulses were not observed in any WG (n = 7 larvae; Fig. 8D; Movie 3) and the knock-down of Inx2 had no effect (Nrv2.GCaMP6S, Inx2-RNAi; n = 7 larvae; Fig. 8E; Movie 4). We did observe microdomain calcium oscillations (Berridge, 2006) in the WG of both the CNS and PNS suggesting the GCaMP6S was expressed at sufficient levels for Ca 21 detection (Movies 3, 4). Thus, while there were robust Inx2-dependent Ca 21 pulses observed in the SPG, Ca 21 pulses were absent from the neighboring WG suggesting that functional gap junctions do not form between the SPG and WG. ...
Glia are essential to protecting and enabling nervous system function and a key glial function is the formation of the glial sheath around peripheral axons. Each peripheral nerve in the Drosophila larva is ensheathed by three glial layers, which structurally support and insulate the peripheral axons. How peripheral glia communicate with each other and between layers is not well established and we investigated the role of Innexins in mediating glial function in the Drosophila periphery. Of the eight Drosophila Innexins, we found two (Inx1 and Inx2) are important for peripheral glia development. In particular loss of Inx1 and Inx2 resulted in defects in the wrapping glia leading to disruption of the glia wrap. Of interest loss of Inx2 in the subperineurial glia also resulted in defects in the neighbouring wrapping glia. Inx plaques were observed between the subperineurial glia and the wrapping glia suggesting that gap junctions link these two glial cell types. We found Inx2 is key to Ca+2 pulses in the peripheral subperineurial glia but not in the wrapping glia, and we found no evidence of gap junction communication between subperineurial and wrapping glia. Rather we have clear evidence that Inx2 plays an adhesive and channel-independent role between the subperineurial and wrapping glia to ensure the integrity of the glial wrap.
... Ca 2+ -mediated signal transduction is highly localized (81). Compartmentalized Ca 2+ permeable channels are found in neuronal and immunological synapses. ...
The flagellar-specific Ca ²⁺ channel CatSper is the predominant Ca ²⁺ entry site in mammalian sperm. CatSper-mediated Ca ²⁺ signaling impacts nearly every event that regulates sperm to acquire fertilizing capability. In this review, we summarize some of the main findings from 20 years of CatSper research and highlight recent progress and prospects.
... Dysfunctions can originate from (excessive) post-translational modifications, protease-mediated cleavages or mutations. Ca 2+ signaling events such as Ca 2+ puffs and sparks can also be considered to result from Ca 2+ -leak activity, although the channels involved (IP 3 R and RyR, respectively) are not dysfunctional, and their activity strongly depend on the local environment, especially [Ca 2+ ] cyt and [Ca 2+ ] ER (Berridge, 2006;Cheng and Lederer, 2008;Konieczny et al., 2012). Even SERCA pumps can participate in Ca 2+ leakage from the ER either by increased slippage of the pump (Inesi and de Meis, 1989) or when truncated (Chami et al., 2008). ...
The heterotrimeric Sec61 protein complex forms the functional core of the so-called translocon that forms an aqueous channel in the endoplasmic reticulum (ER). The primary role of the Sec61 complex is to allow protein import in the ER during translation. Surprisingly, a completely different function in intracellular Ca2+ homeostasis has emerged for the Sec61 complex, and the latter is now accepted as one of the major Ca2+-leak pathways of the ER. In this review, we first discuss the structure of the Sec61 complex and focus on the pharmacology and regulation of the Sec61 complex as a Ca2+-leak channel. Subsequently, we will pay particular attention to pathologies that are linked to Sec61 mutations, such as plasma cell deficiency and congenital neutropenia. Finally, we will explore the relevance of the Sec61 complex as a Ca2+-leak channel in various pathophysiological (ER stress, apoptosis, ischemia-reperfusion) and pathological (type 2 diabetes, cancer) settings.
... Free Calcium (Ca 2+ ) is an important and universal second messenger in all cells [1,2], red blood cells (RBCs) included [3][4][5]. This results in the abundance of Ca 2+binding proteins in RBCs with differing Ca 2+ sensitivities as outlined in Fig. 25.1. ...
... Although RyRs and IP 3 Rs display different physiological and pharmacological properties [89][90][91], their functions are tightly interconnected, as localized Ca 2+ signals, termed Ca 2+ "sparks" and "puffs", generated by clusters of RyRs and IP 3 Rs, respectively, can diffuse to neighboring receptors to trigger global Ca 2+ waves and oscillations that spread within cells and mediate neuronal excitability and synaptic plasticity [45][46][47][92][93][94][95][96]. Irregular expression or activation of RyRs and IP 3 Rs produces excessive ER Ca 2+ release, which may, in turn, lead to cytosolic Ca 2+ overload, as well as increased mitochondrial Ca 2+ uptake, implicated in the pathology of a multitude of disorders of Ca 2+ homeostasis, including neurodegenerative diseases [97][98][99][100][101]. ...
Ca²⁺ is a critical mediator of neurotransmitter release, synaptic plasticity, and gene expression, but also excitotoxicity. Ca²⁺ signaling and homeostasis are coordinated by an intricate network of channels, pumps, and calcium-binding proteins, which must be rapidly regulated at all expression levels. Τhe role of neuronal miRNAs in regulating ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs) was investigated to understand the underlying mechanisms that modulate ER Ca²⁺ release. RyRs and IP3Rs are critical in mounting and propagating cytosolic Ca²⁺ signals by functionally linking the ER Ca²⁺ content, while excessive ER Ca²⁺ release via these receptors is central to the pathophysiology of a wide range of neurological diseases. Herein, two brain-restricted microRNAs, miR-124-3p and miR-153-3p, were found to bind to RyR1-3 and IP3R3 3′UTRs, and suppress their expression at both the mRNA and protein level. Ca²⁺ imaging studies revealed that overexpression of these miRNAs reduced ER Ca²⁺ release upon RyR/IP3R activation, but had no effect on [Ca²⁺]i under resting conditions. Interestingly, treatments that cause excessive ER Ca²⁺ release decreased expression of these miRNAs and increased expression of their target ER Ca²⁺ channels, indicating interdependence of miRNAs, RyRs, and IP3Rs in Ca²⁺ homeostasis. Furthermore, by maintaining the ER Ca²⁺ content, miR-124 and miR-153 reduced cytosolic Ca²⁺ overload and preserved protein-folding capacity by attenuating PERK signaling. Overall, this study shows that miR-124-3p and miR-153-3p fine-tune ER Ca²⁺ homeostasis and alleviate ER stress responses.
... Calcium (Ca 2+ ) mediated downstream processes are one of the most widespread transducing systems (1), regulating a wide range of cardiovascular muscle functions, including contraction, relaxation, and growth responses (2,3). Although the prevalence of essential hypertension is higher than diabetes mellitus (4,5), the association of both disorders represents one of the five principal risk factors for mortality worldwide (5). ...
Aims:
This raised the issue of whether in vivo long-term red wine treatment can act as a modulator of these targets.
Main methods:
We monitored SBP, glucose tolerance, oxidative stress, and cardiovascular function. Aortic and atrial tissues from normotensive-WKY, hypertensive-SHR, and diabetic-STZ animals, chronically exposed to red wine (3.715 ml/kg/v.o/day) or alcohol (12%) for 21-days, were used to measure contractile/relaxation responses by force transducers. Key findings: red wine, but not alcohol, prevented the increase of SBP and hyperglycemic peak. Additionally, was observed prevention of oxidative stress metabolites formation and an improvement in ROS scavenging antioxidant capacity of SHR. We also revealed that red wine intake enhances the endothelium-dependent relaxation, decreases the hypercontractile mediated by angiotensin-II in the aorta, and via β1-adrenoceptors in the atrium.
Significance:
The long-term consumption of red wine can improve oxidative stress and the functionality of angiotensin-II and β1-adrenoceptors, inspiring new pharmacologic and dietetic therapeutic approaches for the treatment of hypertension and diabetes.Abbreviation Acronyms and/or abbreviations: [Ca2+]cyt = Cytosolic Ca2+ Concentration; ACh = Acetylcholine; ANG II = Angiotensin II; AT1 = ANG II type 1 receptor; AUC = Area Under the Curve; Ca2+ = Calcium; Endo + = Endothelium Intact; Fen = Phenylephrine (1 μM); GTT = Glucose Tolerance Test; ISO = Isoprenaline (isoproterenol); KHN = Krebs-Henseleit Nutrient; LA = Left Atria; LH = Lipid Hydroperoxide; NO = Nitric Oxide; RA = Right Atria; RAS = Renin-Angiotensin System; ROS = Reactive Oxygen Species; SBP = Systolic Blood Pressure; SHR = Spontaneously Hypertensive Rats; STZ = Streptozotocin; WKY = Normotensive Wistar Kyoto Rats.
... Indeed, as one example, calcium signaling in eukaryotes relies on many of the principles germane to c-di-GMP signaling in bacteria: Calcium signal transduction lies at the core of cell physiology and function, there exist multiple sources of the calcium messenger molecule, it is diffusible, and a large set of effectors respond to changes in its levels [35]. A rich body of literature demonstrates that calcium signaling fidelity is achieved via formation of local microdomains that promote directed signal transmission [36]. Thus, in different guises, evolution has solved the same issues associated with generically used, diffusible second messenger signaling by devising mechanisms for locally restricting signal transduction. ...
Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae . Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which, in turn, drives alterations specifically to biofilm gene expression. A long-standing question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V . cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input–output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies.
... The long-and short-range calcium waves observed in epithelial cells are primarily mediated by IP 3dependent release of calcium from the ER (Balaji et al., 2017;Jaffe, 2008;Soto et al., 2013;Wallingford et al., 2001;Webb and Miller, 2006). In contrast, localized calcium transients are generally mediated by PM-localized calcium channels, thereby creating a local calcium increase near the opening of the channel, called a "calcium microdomain" (Berridge, 2006;Tsai et al., 2015;Wei et al., 2009). In our study, local calcium flashes associated with TJ remodeling originate close to the junctional PM, consistent with forming a calcium microdomain near the PM, suggesting a role for PM-localized calcium channels. ...
Epithelial cell-cell junctions remodel in response to mechanical stimuli to maintain barrier function. Previously, we found that local leaks in tight junctions (TJs) are rapidly repaired by local, transient RhoA activation, termed "Rho flares," but how Rho flares are regulated is unknown. Here, we discovered that intracellular calcium flashes and junction elongation are early events in the Rho flare pathway. Both laser-induced and naturally occurring TJ breaks lead to local calcium flashes at the site of leaks. Additionally, junction elongation induced by optogenetics increases Rho flare frequency, suggesting that Rho flares are mechanically triggered. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSCs) reduces the amplitude of calcium flashes and diminishes the sustained activation of Rho flares. MSC-dependent calcium influx is necessary to maintain global barrier function by regulating reinforcement of local TJ proteins via junction contraction. In all, we uncovered a novel role for MSC-dependent calcium flashes in TJ remodeling, allowing epithelial cells to repair local leaks induced by mechanical stimuli.
... Activation of the cell by various stimuli, such as changes in the membrane potential or stimulation of receptors in the membrane, can induce an increase in Ca 2+ levels across Ca 2+ channels that respond to the particular stimulus. Overall, Ca 2+ signals are translated into diverse biological output signals, ranging from short-term events, such as secretion or contraction, to long-term processes, such as gene transcription or proliferation [1,7,8]. Their versatility is ensured by diverse patterns of Ca 2+ signals, including sustained and oscillatory Ca 2+ signals, which are controlled by various proteins within the cell [9]. ...
Calcium ion channels are involved in numerous biological functions such as lymphocyte activation, muscle contraction, neurotransmission, excitation, hormone secretion, gene expression, cell migration, memory, and aging. Therefore, their dysfunction can lead to a wide range of cellular abnormalities and, subsequently, to diseases. To date various conventional techniques have provided valuable insights into the roles of Ca2+ signaling. However, their limited spatiotemporal resolution and lack of reversibility pose significant obstacles in the detailed understanding of the structure–function relationship of ion channels. These drawbacks could be partially overcome by the use of optogenetics, which allows for the remote and well-defined manipulation of Ca²⁺-signaling. Here, we review the various optogenetic tools that have been used to achieve precise control over different Ca2+-permeable ion channels and receptors and associated downstream signaling cascades. We highlight the achievements of optogenetics as well as the still-open questions regarding the resolution of ion channel working mechanisms. In addition, we summarize the successes of optogenetics in manipulating many Ca2+-dependent biological processes both in vitro and in vivo. In summary, optogenetics has significantly advanced our understanding of Ca2+ signaling proteins and the used tools provide an essential basis for potential future therapeutic application.
... The effects of calcium occur either through direct binding of the ion to a target proteins, or the stimulation of calcium sensors which detect a change in its concentration and thus regulate different downstream effectors [150]. Calcium is especially important for muscle contraction by binding to proteins such as calmodulin and troponin C [151] and the fast release of neurotransmitters at nerve terminals [146,152]. ...
Cyclic adenosine monophosphate (cAMP), the ubiquitous second messenger produced upon stimulation of GPCRs which couple to the stimulatory GS protein, orchestrates an array of physiological processes including cardiac function, neuronal plasticity, immune responses, cellular proliferation and apoptosis. By interacting with various effector proteins, among others protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), it triggers signaling cascades for the cellular response. Although the functional outcomes of GSPCR-activation are very diverse depending on the extracellular stimulus, they are all mediated exclusively by this single second messenger. Thus, the question arises how specificity in such responses may be attained. A hypothesis to explain signaling specificity is that cellular signaling architecture, and thus precise operation of cAMP in space and time would appear to be essential to achieve signaling specificity. Compartments with elevated cAMP levels would allow specific signal relay from receptors to effectors within a micro- or nanometer range, setting the molecular basis for signaling specificity. Although the paradigm of signaling compartmentation gains continuous recognition and is thoroughly being investigated, the molecular composition of such compartments and how they are maintained remains to be elucidated. In addition, such compartments would require very restricted diffusion of cAMP, but all direct measurements have indicated that it can diffuse in cells almost freely. In this work, we present the identification and characterize of a cAMP signaling compartment at a GSPCR. We created a Förster resonance energy transfer (FRET)-based receptor-sensor conjugate, allowing us to study cAMP dynamics in direct vicinity of the human glucagone-like peptide 1 receptor (hGLP1R). Additional targeting of analogous sensors to the plasma membrane and the cytosol enables assessment of cAMP dynamics in different subcellular regions. We compare both basal and stimulated cAMP levels and study cAMP crosstalk of different receptors. With the design of novel receptor nanorulers up to 60nm in length, which allow mapping cAMP levels in nanometer distance from the hGLP1R, we identify a cAMP nanodomain surrounding it. Further, we show that phosphodiesterases (PDEs), the only enzymes known to degrade cAMP, are decisive in constraining cAMP diffusion into the cytosol thereby maintaining a cAMP gradient. Following the discovery of this nanodomain, we sought to investigate whether downstream effectors such as PKA are present and active within the domain, additionally studying the role of A-kinase anchoring proteins (AKAPs) in targeting PKA to the receptor compartment. We demonstrate that GLP1-produced cAMP signals translate into local nanodomain-restricted PKA phosphorylation and determine that AKAP-tethering is essential for nanodomain PKA. Taken together, our results provide evidence for the existence of a dynamic, receptor associated cAMP nanodomain and give prospect for which key proteins are likely to be involved in its formation. These conditions would allow cAMP to exert its function in a spatially and temporally restricted manner, setting the basis for a cell to achieve signaling specificity. Understanding the molecular mechanism of cAMP signaling would allow modulation and thus regulation of GPCR signaling, taking advantage of it for pharmacological treatment.
... 80 Both CRU and SOCE channels can induce the formation of CCMs in cardiomyocytes. 81 CRU-elicited CCMs are located in the deep cytosol, far from the PM-localised SOCE channels ( Figure 2). Therefore, sub-PM-localised CCMs (induced by SOCE activation) contribute to SOCE inhibition. ...
Store‐operated Ca2+ entry (SOCE) machinery, including Orai channels, TRPCs, and STIM1, is key to cellular calcium homeostasis. The following characteristics of mitochondria are involved in the physiological and pathological regulation of cells: mitochondria mediate calcium uptake through calcium uniporters; mitochondria are regulated by mitochondrial dynamic related proteins (OPA1, MFN1/2, and DRP1) and form mitochondrial networks through continuous fission and fusion; mitochondria supply NADH to the electron transport chain through the Krebs cycle to produce ATP; under stress, mitochondria will produce excessive reactive oxygen species to regulate mitochondria‐endoplasmic reticulum interactions and the related signalling pathways. Both SOCE and mitochondria play critical roles in mediating cardiac hypertrophy, diabetic cardiomyopathy, and cardiac ischaemia‐reperfusion injury. All the mitochondrial characteristics mentioned above are determinants of SOCE activity, and vice versa. Ca2+ signalling dictates the reciprocal regulation between mitochondria and SOCE under the specific pathological conditions of cardiomyocytes. The coupling of mitochondria and SOCE is essential for various pathophysiological processes in the heart. Herein, we review the research focussing on the reciprocal regulation between mitochondria and SOCE and provide potential interplay patterns in cardiac diseases.
... Upon further investigation of the original residual Ca 2+ model, novel studies confirmed the necessity of expanding it to incorporate both spatial and temporal characteristics of Ca 2+ ion concentrations; dubbed the spatiotemporal model. Numerous studies have implicated the existence of transient microdomains of elevated Ca 2+ upon AP arrival at the presynaptic terminal (Llinás et al., 1992;Sugimori et al., 1994;Berridge, 2006), and that there is a dramatic drop off in concentration of the surrounding area within the nerve terminal. Additionally, even a single open VGCC generates a discrete influx of Ca 2+ centered on its pore (Chad & Eckert, 1984). ...
Synaptic scaling is a form of homeostatic plasticity that initiates compensatory adaptations in synaptic strength to buffer chronic aberrant levels of activity within neural circuits. L-type voltage-gated Ca2+- channels (LTCCs) play a key role in the induction of this process as evident in the regulation of scaling mediated by LTCC signaling blockade with dihydropyridine antagonists. These agents, however, do not distinguish between the two LTCC subtypes CaV1.2 and CaV1.3 expressed in the brain. Outlined in Chapter 2, we investigated the unique roles of these LTCC subtypes and found that the deletion of CaV1.2 in excitatory neurons induced a significant increase in basal synaptic strength and surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) whilst occluding TTX-induced synaptic upscaling. By contrast, TTX-induced upscaling of miniature excitatory postsynaptic currents (mEPSCs) is lost in CaV1.3 deficient neurons, with no alterations in basal synaptic properties accompanying CaV1.3 deletion. In addition to mechanisms that induce synaptic scaling, we investigated whether previous homeostatic functional alterations reverse upon activity renormalization and whether a previous history of homeostatic scaling in networks altered subsequent homeostatic responses to chronic activity manipulations. We identified a novel “resetting” phase of synaptic scaling whereby homeostatic changes in synaptic strength revert to basal levels after activity renormalization. Furthermore, future synaptic scaling in response to the same, and even opposite, activity challenges is robustly suppressed by a prior history of scaling in hippocampal neurons. This history-dependent suppression is specific to homeostatic plasticity as networks with prior scaling history showed no deficits in Hebbian forms of synaptic potentiation (cLTP). We further demonstrated that hippocampal neurons with a prior history of synaptic scaling exhibited widespread alterations in activity-dependent transcriptional regulation despite normal engagement of activity-dependent signaling through the ERK/MAPK signaling pathway. Taken together, our data suggests that LTCC subtypes, CaV1.2 and CaV1.3, play nonredundant roles in the induction of synaptic scaling and that the history of homeostatic signaling in neural circuits plays a key role in shaping future compensatory adaptations to chronic changes in network activity.
... The 148-amino acid CaM is an evolutionarily conserved protein across all vertebrates (Davis and Thorner, 1989;Tripathi et al., 2015a), while it is capable of binding more than 300 variations of CaMBTs (Yamniuk and Vogel, 2004). Such structural variability permits its regulation upon the stimulation of calcium ions processes (Clapham, 2007;Carafoli, 2002;Bootman, 2012) in a wide range of biological activities (Chin and Means, 2000;Hoeflich and Ikura, 2002) including cellular motility, neurogenesis, memory formation, muscle contraction, and neuronal transmission (Bootman, 2012;Marambaud et al., 2009;Berridge, 2006). A large and growing body of literature has investigated the structures and dynamics of CaM upon calcium binding (Wriggers et al., 1998;Mehler et al., 1991;Her et al., 2018;Anthis et al., 2011). ...
Calmodulin (CaM) is a calcium-binding protein that transduces signals to downstream proteins through target binding upon calcium binding in a time-dependent manner. Understanding the target binding process that tunes CaM’s affinity for the calcium ions (Ca ²⁺ ), or vice versa, may provide insight into how Ca ²⁺ -CaM selects its target binding proteins. However, modeling of Ca ²⁺ -CaM in molecular simulations is challenging because of the gross structural changes in its central linker regions while the two lobes are relatively rigid due to tight binding of the Ca ²⁺ to the calcium-binding loops where the loop forms a pentagonal bipyramidal coordination geometry with Ca ²⁺ . This feature that underlies the reciprocal relation between Ca ²⁺ binding and target binding of CaM, however, has yet to be considered in the structural modeling. Here, we presented a coarse-grained model based on the Associative memory, Water mediated, Structure, and Energy Model (AWSEM) protein force field, to investigate the salient features of CaM. Particularly, we optimized the force field of CaM and that of Ca ²⁺ ions by using its coordination chemistry in the calcium-binding loops to match with experimental observations. We presented a “community model” of CaM that is capable of sampling various conformations of CaM, incorporating various calcium-binding states, and carrying the memory of binding with various targets, which sets the foundation of the reciprocal relation of target binding and Ca ²⁺ binding in future studies.
... In neurons, the diversity of the responses to Ca 2+ signal depends on the ability to activate or suppress specific intracellular signal transduction pathways. Given the complexity of the nervous system, the intensity and duration of Ca 2+ transients usually depend on the coordinated influx through voltage-gated calcium channels (VGCCs) and calcium-permeable-channels, both located in the plasma membrane, the release from internal stores and the efficiency of the re-uptake mechanisms to restore resting membrane potential [1,2]. To date, the contribution of numerous regulators of Ca 2+ signaling located in the plasma membrane and intracellular organelles has been documented, and various chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified. ...
The activity of specific populations of neurons in different brain areas makes decisions regarding proper synaptic transmission, the ability to make adaptations in response to different external signals, as well as the triggering of specific regulatory pathways to sustain neural function. The endocannabinoid system (ECS) appears to be a very important, highly expressed, and active system of control in the central nervous system (CNS). Functionally, it allows the cells to respond quickly to processes that occur during synaptic transmission, but can also induce long-term changes. The endocannabinoids (eCBs) belong to a large family of bioactive lipid mediators that includes amides, esters, and ethers of long-chain polyunsaturated fatty acids. They are produced “on demand” from the precursors located in the membranes, exhibit a short half-life, and play a key role as retrograde messengers. eCBs act mainly through two receptors, CB1R and CB2R, which belong to the G-protein coupled receptor superfamily (GPCRs), but can also exert their action via multiple non-receptor pathways. The action of eCBs depends on Ca2+, but eCBs can also regulate downstream Ca2+ signaling. In this short review, we focus on the regulation of neuronal calcium channels by the most effective members of eCBs-2-arachidonoylglycerol (2-AG), anandamide (AEA) and originating from AEA-N-arachidonoylglycine (NAGly), to better understand the contribution of ECS to brain function under physiological conditions.
... This ensures the versatility of the Ca 2+ ion in regulating a wide variety of biological processes, from the short term, such as secretion, to the long term, such as gene transcription or proliferation. This diversity of Ca 2+ signaling mechanisms is further established by a toolbox of Ca 2+ sensing, Ca 2+ buffering, Ca 2+ binding and Ca 2+ transporting proteins, which can act in a cell type-specific manner [1][2][3][4]. ...
Ca2+ ion channels are critical in a variety of physiological events, including cell growth, differentiation, gene transcription and apoptosis. One such essential entry pathway for calcium into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel. It consists of the Ca2+ sensing protein, stromal interaction molecule 1 (STIM1) located in the endoplasmic reticulum (ER) and a Ca2+ ion channel Orai in the plasma membrane. The Orai channel family includes three homologues Orai1, Orai2 and Orai3. While Orai1 is the “classical” Ca2+ ion channel within the CRAC channel complex and plays a universal role in the human body, there is increasing evidence that Orai2 and Orai3 are important in specific physiological and pathophysiological processes. This makes them an attractive target in drug discovery, but requires a detailed understanding of the three Orai channels and, in particular, their differences. Orai channel activation is initiated via Ca2+ store depletion, which is sensed by STIM1 proteins, and induces their conformational change and oligomerization. Upon STIM1 coupling, Orai channels activate to allow Ca2+ permeation into the cell. While this activation mechanism is comparable among the isoforms, they differ by a number of functional and structural properties due to non-conserved regions in their sequences. In this review, we summarize the knowledge as well as open questions in our current understanding of the three isoforms in terms of their structure/function relationship, downstream signaling and physiology as well as pathophysiology.
... Microdomains of elevated [Ca 2+ ] occur in all mammalian cells, where they play a functional role at least in part through selective effects on colocalized Ca 2+ effectors [4][5][6][7][8]. A well studied class of such microdomains is produced in ER/PM junctions during influx through store-operated Ca 2+ channels [6,9,10]. ...
The Ca²⁺ sensor protein calmodulin interacts in a Ca²⁺-dependent manner with a large number of proteins that among them encompass a diverse assortment of functions and subcellular localizations. A method for monitoring calmodulin-protein interactions as they occur throughout a living cell would thus uniquely enable investigations of the intracellular landscape of [Ca²⁺] and its relationship to cell function. We have developed such a method based on capture of calmodulin-protein interactions by rapid photoactivated cross-linking (t1/2 ∼7s) in cells stably expressing a tandem affinity tagged calmodulin that have been metabolically labeled with a photoreactive methionine analog. Tagged adducts are stringently enriched, and captured calmodulin interactors are identified and quantified based on tandem mass spectrometry data for their tryptic peptides. In this paper we show that the capture behaviors of interactors in cells are consistent with the presence of basal microdomains of elevated [Ca²⁺]. Ca²⁺ sensitivities for capture were determined, and these suggest that [Ca²⁺] levels are above ∼1 μM in these regions. Although the microdomains appear to affect capture of most proteins, capture of some is at an apparent Ca²⁺-dependent maximum, suggesting they are targeted to the domains. Removal of extracellular Ca²⁺ has both immediate (5 min) and delayed (30 min) effects on capture, implying that the microdomains are supported by a combination of Ca²⁺ influx across the cell membrane and efflux from internal stores. The known properties of the presumptive microdomain targeted proteins suggest that they play roles in a variety of Ca²⁺-dependent basal metabolism and in formation and maintenance of the microdomains.
... The precise control of intracellular calcium is critical for neuronal development (Berridge, 2006), and the formation of dendritic arbors and synapses (Konur and Ghosh, 2005;Lohmann and Wong, 2005). One converging mechanism by which POPs interfere with dendritic and spine plasticity is the modulation of calcium signaling within developing neurons Pessah et al., 2010;Stamou et al., 2013). ...
The developing nervous system is sensitive to environmental and physiological perturbations in part due to its protracted period of prenatal and postnatal development. Epidemiological and experimental studies link developmental exposures to persistent organic pollutants (POPs) including polychlorinated biphenyls, polychlorinated dibenzo‐p‐dioxins, polybrominated diphenyl ethers, and benzo(a)pyrene to increased risk for neurodevelopmental disorders in children. Mechanistic studies reveal that many of the complex cellular processes that occur during sensitive periods of rapid brain development are cellular targets for developmental neurotoxicants. One area of research interest has focused on synapse formation and plasticity, processes that involve the growth and retraction of dendrites and dendritic spines. For each chemical discussed in this review, we summarize the morphological and electrophysiological data that provide evidence that developmental POP exposure produces long‐lasting effects on dendritic morphology, spine formation, glutamatergic and GABAergic signaling systems, and synaptic transmission. We also discuss shared intracellular mechanisms, with a focus on calcium and thyroid hormone homeostasis, by which these chemicals act to modify synapses. We conclude our review highlighting research gaps that merit consideration when characterizing synaptic pathology elicited by chemical exposure. These gaps include low‐dose and non‐monotonic dose‐response effects, revealing the temporal relationship between dendritic growth, spine formation, and synaptic activity, excitation‐inhibition balance, hormonal effects, and the critical need for more studies in females to identify sex differences. By identifying converging pathological mechanisms elicited by POP exposure at the synapse, we can define future research directions that will advance our understanding of these chemicals on synapse structure and function.
Calcium ions serve as key intracellular signals. Local, transient increases in calcium concentrations can activate calcium sensor proteins that in turn trigger downstream effectors. In neurons, calcium transients play a central role in regulating neurotransmitter release and synaptic plasticity. However, it is challenging to capture the molecular events associated with these localized and ephemeral calcium signals. Here we present an engineered biotin ligase that generates permanent molecular traces in a calcium-dependent manner. The enzyme, calcium-dependent BioID (Cal-ID), biotinylates nearby proteins within minutes in response to elevated local calcium levels. The biotinylated proteins can be identified via mass spectrometry and visualized using microscopy. In neurons, Cal-ID labeling is triggered by neuronal activity, leading to prominent protein biotinylation that enables transcription-independent activity labeling in the brain. In summary, Cal-ID produces a biochemical record of calcium signals and neuronal activity with high spatial resolution and molecular specificity.
Brain-derived neurotrophic factor (BDNF) plays a critical role in synaptic physiology, as well as mechanisms underlying various neuropsychiatric diseases and their treatment. Despite its clear physiological role and disease relevance, BDNF’s function at the presynaptic terminal, a fundamental unit of neurotransmission, remains poorly understood. In this study, we evaluated single synapse dynamics using optical imaging techniques in hippocampal cell cultures. We find that exogenous BDNF selectively increases evoked excitatory neurotransmission without affecting spontaneous neurotransmission. However, acutely blocking endogenous BDNF has no effect on evoked or spontaneous release, demonstrating that different approaches to studying BDNF may yield different results. When we suppressed BDNF-Tropomyosin receptor kinase B (TrkB) activity chronically over a period of days to weeks using a mouse line enabling conditional knockout of TrkB, we found that evoked glutamate release was significantly decreased while spontaneous release remained unchanged. Moreover, chronic blockade of BDNF-TrkB activity selectively downscales evoked calcium transients without affecting spontaneous calcium events. Via pharmacological blockade by voltage-gated calcium channel (VGCC) selective blockers, we found that the changes in evoked calcium transients are mediated by the P/Q subtype of VGCCs. These results suggest that BDNF-TrkB activity increases presynaptic VGCC activity to selectively increase evoked glutamate release.
How do neurons that implement cell-autonomous self-regulation of calcium react to knockout of individual ion-channel conductances? To address this question, we used a heterogeneous population of 78 conductance-based models of hippocampal pyramidal neurons that maintained cell-autonomous calcium homeostasis while receiving theta-frequency inputs. At calcium steady-state, we individually deleted each of the 11 active ion-channel conductances from each model. We measured the acute impact of deleting each conductance (one at a time) by comparing intrinsic electrophysiological properties before and immediately after channel deletion. The acute impact of deleting individual conductances on physiological properties (including calcium homeostasis) was heterogeneous, depending on the property, the specific model, and the deleted channel. The underlying many-to-many mapping between ion channels and properties pointed to ion-channel degeneracy. Next, we allowed the other conductances (barring the deleted conductance) to evolve towards achieving calcium homeostasis during theta-frequency activity. When calcium homeostasis was perturbed by ion-channel deletion, post-knockout plasticity in other conductances ensured resilience of calcium homeostasis to ion-channel deletion. These results demonstrate degeneracy in calcium homeostasis, as calcium homeostasis in knockout models was implemented in the absence of a channel that was earlier involved in the homeostatic process. Importantly, in reacquiring homeostasis, ion-channel conductances and physiological properties underwent heterogenous plasticity (dependent on the model, the property, and the deleted channel), even introducing changes in properties that were not directly connected to the deleted channel. Together, post-knockout plasticity geared towards maintaining homeostasis introduced heterogenous off-target effects on several channels and properties, suggesting that extreme caution be exercised in interpreting experimental outcomes involving channel knockouts.
Calcium (Ca²⁺) signaling is tightly regulated within a presynaptic bouton. Here, we visualize Ca²⁺ signals within hippocampal presynaptic boutons using GCaMP8s tagged to synaptobrevin, a synaptic vesicle protein. We identify evoked presynaptic Ca²⁺ transients (ePreCTs) that derive from synchronized voltage-gated Ca²⁺ channel openings, spontaneous presynaptic Ca²⁺ transients (sPreCTs) that originate from ryanodine sensitive Ca²⁺ stores, and a baseline Ca²⁺ signal that arises from stochastic voltage-gated Ca²⁺ channel openings. We find that baseline Ca²⁺, but not sPreCTs, contributes to spontaneous glutamate release. We employ photobleaching as a use-dependent tool to probe nano-organization of Ca²⁺ signals and observe that all three occur in non-overlapping domains within the synapse at near-resting conditions. However, increased depolarization induces intermixing of these Ca²⁺ domains via both local and non-local synaptic vesicle turnover. Our findings reveal nanosegregation of Ca²⁺ signals within a presynaptic terminal that derive from multiple sources and in turn drive specific modes of neurotransmission.
The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGFRa+ cells. Together these cells form the SIP syncytium. ICC and PDGFRa+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRa+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transientsoccur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarization (STDs). Neurotransmission increases or decreases Ca2+transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract, suggest the term myogenic should be replaced by the term, SIPgenic, as this review discusses.
The balance between the degeneration and regeneration of damaged neurons depends on intrinsic and environmental variables. In nematodes, neuronal degeneration can be reversed by intestinal GABA and lactate-producing bacteria, or by hibernation driven by food deprivation. However, it is not known whether these neuroprotective interventions share common pathways to drive regenerative outcomes. Using a well established neuronal degeneration model in the touch circuit of the bacterivore nematode Caenorhabditis elegans , we investigate the mechanistic commonalities between neuroprotection offered by the gut microbiota and hunger-induced diapause. Using transcriptomics approaches coupled to reverse genetics, we identify genes that are necessary for neuroprotection conferred by the microbiota. Some of these genes establish links between the microbiota and calcium homeostasis, diapause entry, and neuronal function and development. We find that extracellular calcium as well as mitochondrial MCU-1 and reticular SCA-1 calcium transporters are needed for neuroprotection by bacteria and by diapause entry. While the benefits exerted by neuroprotective bacteria require mitochondrial function, the diet itself does not affect mitochondrial size. In contrast, diapause increases both the number and length of mitochondria. These results suggest that metabolically induced neuronal protection may occur via multiple mechanisms.
Consisting of three signaling pathways, the unfolded protein response (UPR) can be either protective or detrimental to cells that undergo ER stress. Elaborate regulation of the UPR is key to the cell-fate decision, but how it is achieved remains vague. Here, by studying cells deficient in vacuole membrane protein 1 (VMP1), a UPR regulator, we report a model of UPR regulation in which the three pathways are divergently controlled. Under basal conditions, calcium binding specifically activates PERK. Under ER stress, ER-mitochondria interaction-induced mitochondrial stress cooperates with PERK to suppress IRE1α and ATF6 by decelerating global protein synthesis. Such sophisticated regulation commits limited activation of the UPR yet refrains from UPR hyperactivation, protecting cells from chronic ER stress despite decreasing cell proliferation. Therefore, our study reveals interorganelle-interaction-dependent and calcium-dependent regulation of the UPR that dictates cell fate.
A bstract
Second messengers, including cAMP, cGMP and Ca ²⁺ are often placed in an integrating position to combine the extracellular cues that orient growing axons in the developing brain. This view suggests that axon repellents share the same set of cellular messenger signals and that axon attractants evoke opposite cAMP, cGMP and Ca ²⁺ changes. Investigating the confinement of these second messengers in cellular nanodomains, we instead demonstrate that two repellent cues, ephrin-A5 and Slit1, induce spatially segregated signals. These guidance molecules activate subcellular-specific second messenger crosstalks, each signaling network controlling distinct axonal morphology changes in vitro and pathfinding decisions in vivo .
Alzheimer’s disease (AD) is a hereditary and sporadic neurodegenerative illness defined by the gradual and cumulative loss of neurons in specific brain areas. The processes that cause AD are still under investigation and there are no available therapies to halt it. Current progress puts at the forefront the “calcium (Ca2+) hypothesis” as a key AD pathogenic pathway, impacting neuronal, astrocyte and microglial function. In this review, we focused on mitochondrial Ca2+ alterations in AD, their causes and bioenergetic consequences in neuronal and glial cells, summarizing the possible mechanisms linking detrimental mitochondrial Ca2+ signals to neuronal death in different experimental AD models.
It is well appreciated now without any second thought, the first impression of a word like potassium (K⁺) in the biological world is as more than an ion. K⁺ is well known for its essential functions for regulating cell turgor, pH maintenance, and an enzyme cofactor in living systems. In few organisms such as bacteria, surprisingly K⁺ has been shown to function as a cytoplasmic-signaling molecule. This newer piece of information raised the question of existence of this function of K⁺ in the higher organisms. In plants, few reports suggest that K⁺ has a potential to act as a signal or signal mediator. But solid experimental proofs are required to materialize this concept in plants. In this chapter, we discuss the possibility of K⁺ qualifying as a signaling molecule and a signal mediator that can potentially crosstalk with other signaling pathways.
Neurogenesis plays a critical role in brain physiology and behavioral performance, and defective neurogenesis leads to neurological and psychiatric disorders. Here, we show that PLCβ4 expression is markedly reduced in SENP2-deficient cells and mice, resulting in decreased IP3 formation and altered intracellular calcium homeostasis. PLCβ4 stability is regulated by the SUMO-dependent ubiquitin-mediated proteolytic pathway, which is catalyzed by PIAS2α and RNF4. SUMOylated PLCβ4 is transported to the nucleus through Nup205- and RanBP2-dependent pathways and regulates nuclear signaling. Furthermore, dysregulated calcium homeostasis induced defects in neurogenesis and neuronal viability in SENP2-deficient mice. Finally, SENP2 and PLCβ4 are stimulated by starvation and oxidative stress, which maintain calcium homeostasis regulated neurogenesis. Our findings provide mechanistic insight into the critical roles of SENP2 in the regulation of PLCβ4 SUMOylation, and the involvement of SENP2-PLCβ4 axis in calcium homeostasis regulated neurogenesis under stress.
Osteocytes are embedded dendritic bone cells; by virtue of their position in bone tissue, ability to coordinate bone building osteoblasts and resorbing osteoclasts, and sensitivity to tissue level mechanical loading, they serve as the resident bone mechanosensor. The mechanisms osteocytes use to change mechanical loading into biological signals that drive tissue level changes has been well studied over the last 30 years, however the ways loading parameters are encoded at the cellular level are still not fully understood. Calcium signaling is a first messenger signal exhibited by osteocytes in response to mechanical forces. A body of work interrogating the mechanisms of osteocyte calcium signaling exists and is presently expanding, presenting the opportunity to better understand the relationship between calcium signaling characteristics and tuned osteocyte responses to tissue level strain features (e.g. magnitude, duration, frequency). This review covers the history of osteocyte load induced calcium signaling and highlights potential cellular mechanisms used by osteocytes to turn details about loading parameters into biological events.
Cinobufagin, a bufadienolide of toad venom of Bufo bufo gargarizans, is used as a cardiotonic, central nervous system (CNS) respiratory agent, as well as an analgesic and anesthetic. However, several research showed that bufadienolide has a few side effects on the CNS, such as breathlessness or coma. Although cinobufagin was shown to display pharmacological effects in various models, the toxic effect of cinobufagin is elusive in brain cell models. The aim of this study was to explore whether cinobufagin affected viability, Ca2+ homeostasis, and reactive oxygen species (ROS) production in Gibco® Human Astrocyte (GHA) and HCN‐2 neuronal cell line. In GHA cells but not in HCN‐2 cells, cinobufagin (20–60 μM) induced [Ca2+]i rises. In terms of cell viability, chelation of cytosolic Ca2+ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N'N'‐tetraacetic acid reduced cinobufagin‐induced cytotoxicity in GHA cells. In GHA cells, cinobufagin‐induced Ca2+ entry was inhibited by 2‐aminoethoxydiphenyl borate or SKF96365. In a Ca2+‐free medium, treatment with thapsigargin or U73122 abolished cinobufagin‐evoked [Ca2+]i rises. Furthermore, treatment with N‐acetylcysteine reversed ROS production and cytotoxicity in cinobufagin‐treated GHA cells. Together, in GHA cells but not in HCN‐2 cells cinobufagin caused cytotoxicity that was linked to preceding [Ca2+]i rises by Ca2+ influx via store‐operated Ca2+ entry and phospholipase C‐dependent Ca2+ release from the endoplasmic reticulum. Moreover, cinobufagin induced ROS‐associated cytotoxicity.
Transient Receptor Potential (TRP) channels participate in calcium ion (Ca ²⁺ ) influx and intracellular Ca ²⁺ release. TRP channels have not been studied in Toxoplasma gondii or any other apicomplexan parasite. In this work we characterize TgGT1_310560, a protein predicted to possess a TRP domain (TgTRPPL-2) and determined its role in Ca ²⁺ signaling in T. gondii , the causative agent of toxoplasmosis. TgTRPPL-2 localizes to the plasma membrane and the endoplasmic reticulum (ER) of T. gondii . The ΔTgTRPPL-2 mutant was defective in growth and cytosolic Ca ²⁺ influx from both extracellular and intracellular sources. Heterologous expression of TgTRPPL-2 in HEK-3KO cells allowed its functional characterization. Patching of ER-nuclear membranes demonstrates that TgTRPPL-2 is a non-selective cation channel that conducts Ca ²⁺ . Pharmacological blockers of TgTRPPL-2 inhibit Ca ²⁺ influx and parasite growth. This is the first report of an apicomplexan ion channel that conducts Ca ²⁺ and may initiate a Ca ²⁺ signaling cascade that leads to the stimulation of motility, invasion and egress. TgTRPPL-2 is a potential target for combating Toxoplasmosis.
Ca²⁺ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca²⁺. Intracellular Ca²⁺ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca²⁺ handling properties by combining the ‘added buffer’ approach [1] with perforated patch-clamp recordings [2]. Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca²⁺ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca²⁺ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca²⁺ handling properties, including immobile as well as mobile Ca²⁺ buffers.
Compartmentalization of cells and their organelles with protein-studded lipid bilayer membranes enables the cells to use ions for intracellular communication. Owing to the intrinsic tendency of bilipid membranes to exclude ions and pumps to actively transport specific ions across the membrane, ions such as Na⁺, K⁺, Cl–, and Ca²⁺ are selectively concentrated on one side of the organelle or plasma membrane. The concentration of Ca²⁺ in cytosol is maintained extremely low at 10–7 M, which is three orders of magnitude lower than those in the extracellular space and in the lumen of the endoplasmic reticulum. Like a repressed spring, this steep electrochemical gradient across the membrane awaits a stimulus that opens ion channels to let out specific ions. Since the activities of Ca²⁺ channels are delicately modulated by the local level of Ca²⁺ itself or by the changes of membrane potentials, Ca²⁺ can constitute a positive feedback loop that amplifies the decaying signal and lets it propagate or oscillate inside the cell. Under a constant ionic tension, the cytoplasm thus serves as an excitable medium that can conduct Ca²⁺ signals across the intracellular space. This phenomenon of calcium wave has been found in many different cell types in as many diverse physiological contexts. In this article, we summarize some of the key examples of the intracellular calcium waves and discuss the underlying mechanisms and their physiological significance.
Hypaconitine, a neuromuscular blocker, is a diterpene alkaloid found in the root of Aconitum carmichaelii. Although hypaconitine was shown to affect various physiological responses in neurological models, the effect of hypaconitine on cell viability and the mechanism of its action of Ca2+ handling is elusive in cortical neurons. This study examined whether hypaconitine altered viability and Ca2+ signalling in HCN‐2 neuronal cell lines. Cell viability was measured by the cell proliferation reagent (WST‐1). Cytosolic Ca2+ concentrations [Ca2+]i was measured by the Ca2+‐sensitive fluorescent dye fura‐2. In HCN‐2 cells, hypaconitine (10–50 μmol/L) induced cytotoxicity and [Ca2+]i rises in a concentration‐dependent manner. Removal of extracellular Ca2+ partially reduced the hypaconitine's effect on [Ca2+]i rises. Furthermore, chelation of cytosolic Ca2+ with BAPTA‐AM reduced hypaconitine's cytotoxicity. In Ca2+‐containing medium, hypaconitine‐induced Ca2+ entry was inhibited by modulators (2‐APB and SKF96365) of store‐operated Ca2+ channels and a protein kinase C (PKC) inhibitor (GF109203X). Hypaconitine induced Mn2+ influx indirectly suggesting that hypaconitine evoked Ca2+ entry. In Ca2+‐free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished hypaconitine‐induced [Ca2+]i rises. Conversely, treatment with hypaconitine inhibited thapsigargin‐induced [Ca2+]i rises. However, inhibition of phospholipase C (PLC) with U73122 did not inhibit hypaconitine‐induced [Ca2+]i rises. Together, hypaconitine caused cytotoxicity that was linked to preceding [Ca2+]i rises by Ca2+ influx via store‐operated Ca2+ entry involved PKC regulation and evoking PLC‐independent Ca2+ release from the endoplasmic reticulum. Because BAPTA‐AM loading only partially reversed hypaconitine‐induced cell death, it suggests that hypaconitine induced a second Ca2+‐independent cytotoxicity in HCN‐2 cells.
The primary function of inositol 1,4,5-trisphosphate (InsP3) is to function as a second messenger to release Ca²⁺ from internal stores. Many cell stimuli act on receptors that are coupled to phospholipase C that hydrolyzes phosphatidylinosol 4,5-bisphosphate (PIP2) to release InsP3 to the cytosol. InsP3 receptors located on the endoplasmic reticulum respond to this elevation of InsP3 by releasing Ca²⁺, which is often organized into characteristic spatial (elementary events and waves) and temporal (Ca²⁺ oscillations) patterns. This InsP3/Ca²⁺ pathway has been adapted to control processes as diverse as fertilization, proliferation, contraction, cell metabolism, vesicle and fluid secretion, and information processing in neuronal cells.
Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These ‘calcium sparks’ activate calcium-dependent potassium (BK) channels comprised of α and β1 subunits. Here we show that targeted deletion of the gene for the β1 subunit leads to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The β1 subunit of the BK channel, by tuning the channel's calcium sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.
Of fundamental importance in understanding neuronal function is the unambiguous determination of the smallest unit of neuronal integration. It was recently suggested that a whole dendritic branchlet, including tens of spines, acts as the fundamental unit in terms of dendritic calcium dynamics in Purkinje cells. By contrast, we demonstrate that the smallest such unit is the single spine. The results show, by two-photon excited fluorescence laser scanning microscopy, that individual spines are capable of independent calcium activation. Moreover, two distinct spine populations were distinguished by their opposite response to membrane hyperpolarization. Indeed, in a subpopulation of spines calcium entry can also occur through a pathway other than voltage-gated channels. These findings challenge the assumption of a unique parallel fiber activation mode and prompt a reevaluation of the level of functional complexity ascribed to single neurons.
Spontaneous local increases in the concentration of intracellular calcium, called "calcium sparks," were detected in quiescent
rat heart cells with a laser scanning confocal microscope and the fluorescent calcium indicator fluo-3. Estimates of calcium
flux associated with the sparks suggest that calcium sparks result from spontaneous openings of single sarcoplasmic reticulum
(SR) calcium-release channels, a finding supported by ryanodine-dependent changes of spark kinetics. At resting intracellular
calcium concentrations, these SR calcium-release channels had a low rate of opening (approximately 0.0001 per second). An
increase in the calcium content of the SR, however, was associated with a fourfold increase in opening rate and resulted in
some sparks triggering propagating waves of increased intracellular calcium concentration. The calcium spark is the consequence
of elementary events underlying excitation-contraction coupling and provides an explanation for both spontaneous and triggered
changes in the intracellular calcium concentration in the mammalian heart.
Previous studies of (InsP3)-evoked elementary Ca2+ events suggested a hierarchy of signals; fundamental events (“Ca2+ blips”) arising from single InsP3receptors (InsP3Rs), and intermediate events (“Ca2+ puffs”) reflecting the coordinated opening of a cluster of InsP3Rs. The characteristics of such elementary Ca2+ release signals provide insights into the functional interaction and distribution of InsP3Rs in living cells. Therefore we investigated whether elementary Ca2+ signaling is truly represented by such stereotypic release events. A histogram of >900 events revealed a wide spread of signal
amplitudes (20–600 nm; mean 216 ± 4 nm; n = 206 cells), which cannot be explained by stochastic variation of a stereotypic Ca2+ release site. We identified elementary Ca2+ release sites with consistent amplitudes (<20% difference) and locations with variable amplitudes (∼500% difference). Importantly,
within single cells, distinct sites displayed events with significantly different mean amplitudes. Additional determinants
affecting the magnitude of elementary Ca2+release were identified to be (i) hormone concentration, (ii) day-to-day variability, and (iii) a progressively decreasing
Ca2+ release during prolonged stimulation. We therefore suggest that elementary Ca2+ events are not stereotypic, instead a continuum of signals can be achieved by either recruitment of entire clusters with
different numbers of InsP3Rs or by a graded recruitment of InsP3Rs within a cluster.
Astrocytes are considered a reticulate network of cells, through which calcium signals can spread easily. In Bergmann glia, astrocytic cells of the cerebellum, we identified subcellular compartments termed 'glial microdomains'. These elements have a complex surface consisting of thin membrane sheets, contain few mitochondria and wrap around synapses. To test for neuronal interaction with these structures, we electrically stimulated parallel fibers. This stimulation increased intracellular calcium concentration ([Ca2+]i) in small compartments within Bergmann glial cell processes similar in size to glial microdomains. Thus, a Bergmann glial cell may consist of hundreds of independent compartments capable of autonomous interactions with the particular group of synapses that they ensheath.
Ca2+-induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals. In heart cells, this mechanism is operated between voltage-gated L-type Ca2+ channels (LCCs) in the plasma membrane and Ca2+ release channels, commonly known as ryanodine receptors, in the sarcoplasmic reticulum. The Ca2+ influx through LCCs traverses a cleft of roughly 12 nm formed by the cell surface and the sarcoplasmic reticulum membrane, and activates adjacent ryanodine receptors to release Ca2+ in the form of Ca2+ sparks. Here we determine the kinetics, fidelity and stoichiometry of coupling between LCCs and ryanodine receptors. We show that the local Ca2+ signal produced by a single opening of an LCC, named a 'Ca2+ sparklet', can trigger about 4-6 ryanodine receptors to generate a Ca2+ spark. The coupling between LCCs and ryanodine receptors is stochastic, as judged by the exponential distribution of the coupling latency. The fraction of sparklets that successfully triggers a spark is less than unity and declines in a use-dependent manner. This optical analysis of single-channel communication affords a powerful means for elucidating Ca2+-signalling mechanisms at the molecular level.
Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca(2+) to play a pivotal role in cell biology results from the facility that cells have to shape Ca(2+) signals in space, time and amplitude. To generate and interpret the variety of observed Ca(2+) signals, different cell types employ components selected from a Ca(2+) signalling 'toolkit', which comprises an array of homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca(2+) signals that suit their physiology. Recent studies have demonstrated the importance of local Ca(2+) signals in defining the specificity of the interaction of Ca(2+) with its targets. Furthermore, local Ca(2+) signals are the triggers and building blocks for larger global signals that propagate throughout cells.
Ca2+ is a highly versatile intracellular signal that operates over a wide temporal range to regulate many different cellular processes. An extensive Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics. Rapid highly localized Ca2+ spikes regulate fast responses, whereas slower responses are controlled by repetitive global Ca2+ transients or intracellular Ca2+ waves. Ca2+ has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease.
Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.
The existence of spontaneous calcium transients (SCaTs) dependent on intracellular store activation has been reported in putative axonal terminals of cerebellar basket interneurons. We used the two-photon imaging technique to optically identify basket terminals in acute cerebellar slices of young rats (11-16 d old) and study the properties of SCaTs unambiguously localized in these regions. The whole-cell recording configuration and preloading technique were alternatively used to load the calcium-dependent dye in the interneuron and compare SCaTs with action potential evoked calcium transients. SCaTs were observed in the basket terminals at frequencies that were significantly increased after bath application of 10 microm ryanodine and did not depend on P/Q- or N-type voltage-dependent calcium channel activation. They originated at specific sites where bursts of events with temporal separation as small as 200 msec could be generated. Their sites of origin were spaced on average 6 microm apart and were preferentially located near axonal endings. SCaTs had amplitudes comparable with those of Ca2+ rises evoked by single action potentials that lead to release of neurotransmitter, as confirmed by parallel recordings of preloaded terminals and evoked IPSCs in the postsynaptic Purkinje cells. These results support the hypothesis that SCaTs at basket terminals underlie the large miniature IPSCs characteristic of Purkinje cells.
We examined the regulation of calcium signalling in atrial cardiomyocytes during excitation-contraction coupling, and how changes in the distribution of calcium impacts on contractility. Under control conditions, calcium transients originated in subsarcolemmal locations and showed local regeneration through activation of calcium-induced calcium release from ryanodine receptors. Despite functional ryanodine receptors being expressed at regular (approximately 2 microm) intervals throughout atrial myocytes, the subsarcolemmal calcium signal did not spread in a fully regenerative manner through the interior of a cell. Rather, there was a diminishing centripetal propagation of calcium. The lack of regeneration was due to mitochondria and SERCA pumps preventing the inward movement of calcium. Inhibiting these calcium buffering mechanisms allowed the globalisation of action potential-evoked responses. In addition, physiological positive inotropic agents, such as endothelin-1 and beta-adrenergic agonists, as well as enhanced calcium current, calcium store loading and inositol 1,4,5-trisphosphate infusion also led to regenerative global responses. The consequence of globalising calcium signals was a significant increase in cellular contraction. These data indicate how calcium signals and their consequences are determined by the interplay of multiple subcellular calcium management systems.
Previous work showed that calmodulin (CaM) and Ca2+-CaM-dependent protein kinase II (CaMKII) are somehow involved in cardiac hypertrophic signaling, that inositol 1,4,5-trisphosphate receptors (InsP3Rs) in ventricular myocytes are mainly in the nuclear envelope, where they associate with CaMKII, and that class II histone deacetylases (e.g., HDAC5) suppress hypertrophic gene transcription. Furthermore, HDAC phosphorylation in response to neurohumoral stimuli that induce hypertrophy, such as endothelin-1 (ET-1), activates HDAC nuclear export, thereby regulating cardiac myocyte transcription. Here we demonstrate a detailed mechanistic convergence of these 3 issues in adult ventricular myocytes. We show that ET-1, which activates plasmalemmal G protein-coupled receptors and InsP3 production, elicits local nuclear envelope Ca2+ release via InsP3R. This local Ca2+ release activates nuclear CaMKII, which triggers HDAC5 phosphorylation and nuclear export (derepressing transcription). Remarkably, this Ca2+-dependent pathway cannot be activated by the global Ca2+ transients that cause contraction at each heartbeat. This novel local Ca2+ signaling in excitation-transcription coupling is analogous to but separate (and insulated) from that involved in excitation-contraction coupling. Thus, myocytes can distinguish simultaneous local and global Ca2+ signals involved in contractile activation from those targeting gene expression.
In cardiac cells, Ca2+ signals appear as brief transients responsible for controlling both contraction and transcription. Information may be encoded in these digital signals through changes in both frequency and shape. An increase in Ca2+ signalling contributes to a process of phenotypic remodelling during hypertrophy. The increase in Ca2+ that drives the larger contractions may be responsible for switching on a second process of signalosome remodelling to down-regulate the Ca2+ signalling pathway. It is a change in the properties of the Ca2+ transient that seems to carry the information responsible for the remodelling of the cardiac gene transcription programme that leads first to hypertrophy and then to congestive heart failure.
1. Confocal microscopy was used to investigate hormone-induced subcellular Ca2+ release signals from the endoplasmic reticulum (ER) in a prototype non-excitable cell line (HeLa cells). 2. Histamine application evoked two types of elementary Ca2+ signals: (i) Ca2+ blips arising from single ER Ca2+ release channels (amplitude, 30 nM; lateral spreading, 1.3 microns); (ii) Ca2+ puffs resulting from the concerted activation of several Ca2+ blips (amplitude, 170 nM; spreading, 4 microns). 3. Ca2+ waves in the HeLa cells arose from a variable number of initiation sites, but for individual cells, the number and subcellular location of the initiation sites were constant. The kinetics and amplitude of global Ca2+ signals were directly proportional to the number of initiation sites recruited. 4. Reduction of the feedback inherent in intracellular Ca2+ release caused saltatoric Ca2+ waves, revealing the two principal steps underlying wave propagation: diffusion and regeneration. Threshold stimulation evoked abortive Ca2+ waves, caused by the limited recruitment of Ca2+ puffs. 5. The hierarchy of Ca2+ signalling events, from fundamental levels (blips) to intermediate levels (puffs) to Ca2+ waves, is a prototype for Ca2+ signal transduction for non-excitable cells, and is also analogous to the Ca2+ quarks, Ca2+ sparks and Ca2+ waves in cardiac muscle cells.
Calcium is a ubiquitous second messenger used to regulate a wide range of cellular processes. This role in signalling has to be conducted against the rigid homeostatic mechanisms that ensure that the resting level of Ca2+ is kept low (i.e. between 20 and 100 nmol l-1) in order to avoid the cytotoxic effects of a prolonged elevation of [Ca2+]. Cells have evolved a sophisticated signalling system based on the generation of brief pulses of Ca2+ which enables this ion to be used as a messenger, thus avoiding its toxic effects. Such Ca2+ spikes usually result from the coordinated release of Ca2+ from internal stores using either inositol 1,4,5-trisphosphate or ryanodine receptors. Using Ca2+ imaging techniques, the opening of individual channels has now been visualized and models have been proposed to explain how these elementary events are coordinated to generate the global Ca2+ signals that regulate cellular activity.
Recent studies have suggested that global intracellular Ca2+ signals arise from the summation and coordination of subcellular elementary release events (e.g., "Ca2+ puffs"), although the modes of recruitment of such signals are unknown. In order to understand how cells utilize elementary Ca2+ release events, we imaged Ca2+ transients evoked through the phosphoinositide pathway in HeLa cells using confocal microscopy. During the pacemaker phase leading to the global Ca2+ signal, elementary Ca2+ release events were recruited in (1) frequency, (2) amplitude, and (3) spatial domains. Since each digital elementary event contributes to a small change of the analog cytosolic Ca2+ concentration, the net effect of the advancement in the three domains is to drive the ambient Ca2+ concentration toward a threshold where the signal becomes regenerative, resulting in a global Ca2+ wave.
The concept of second messenger signalling originated from the discovery of the role of cyclic AMP, although it is now known that cytosolic calcium [Ca2+]i mediates numerous signalling pathways and plays an equally vital role in many cellular events. In the last few years there has been a great deal of interest in the substantial molecular and functional diversity of mammalian adenylyl cyclases (ACs). Although AC was viewed as a generic activity, which was either stimulated or inhibited by stimulatory or inhibitory receptors, respectively, acting via alpha-subunits of trimeric GTP-regulatory proteins, the recent cloning of nine full-length isoforms, which significantly differ in their regulatory properties and tissue distributions, has revealed an unexpected level of complex regulation. In fact, each AC may integrate convergent inputs from many distinct signal-generating pathways. The nine isoforms can be divided into four distinct families, which reflect their distinct patterns of regulation by betagamma subunits of G-proteins, protein kinase C (PKC) and Ca2+. The mechanisms of regulation are often highly synergistic or conditional, suggesting a function of ACs as coincident detectors. Since all nine isoforms can be regulated either directly or indirectly by Ca2+ or PKC, a complex range of responses is possible. The Ca2+ concentration that stimulates the major ACs in brain has been found to inhibit AC activity in a number of peripheral tissues and cell lines. The purpose of this article is to review many of the important aspects about the distinct regulatory properties and cellular distribution of Ca2+-regulated ACs. Indeed, the notion that Ca2+ and cAMP are "synarchic" messengers acting in concert to regulate cellular activity was formally proposed some time ago. Here, we will focus on acute interactions between Ca2+ and cAMP and attempt to understand how AC activities can be regulated by discrete, physiological [Ca2+]i rises in intact cells. All Ca2+-regulated isoforms have characteristic distribution patterns in the brain. Also discussed are emerging insights on the temporal and spatial regulation of Ca2+- and cAMP-regulated pathways which may enable cell stimuli to elicit specific responses.
By affecting the activity of the adaptation motor, Ca2+ entering a hair bundle through mechanoelectrical transduction channels regulates the sensitivity of the bundle to stimulation. For adaptation to set the position of mechanosensitivity of the bundle accurately, the free Ca2+ concentration in stereocilia must be tightly controlled. To define the roles of Ca2+-regulatory mechanisms and thus the factors influencing adaptation motor activity, we used confocal microscopy to detect Ca2+ entry into and clearance from individual stereocilia of hair cells dialyzed with the Ca2+ indicator fluo-3. We also developed a model of stereociliary Ca2+ homeostasis that incorporates four regulatory mechanisms: Ca2+ clearance from the bundle by free diffusion in one dimension, Ca2+ extrusion by pumps, Ca2+ binding to fixed stereociliary buffers, and Ca2+ binding to mobile buffers. To test the success of the model, we compared the predicted profiles of fluo-3 fluorescence during the response to mechanical stimulation with the fluorescence patterns measured in individual stereocilia. The results indicate that all four of the Ca2+ regulatory mechanisms must be included in the model to account for the observed rate of clearance of the ion from the hair bundle. The best fit of the model suggests that a free Ca2+ concentration of a few micromolar is attained near the adaptation motor after transduction-channel opening. The free Ca2+ concentration substantially rises only in the upper portion of the stereocilium and quickly falls toward the resting level as adaptation proceeds.
Excitation-contraction coupling (E-C coupling) was studied in isolated fluo-3-loaded rat atrial myocytes at 22 and 37 degrees C using rapid confocal microscopy. Within a few milliseconds of electrical excitation, spatially discrete subsarcolemmal Ca2+ signals were initiated. Twenty to forty milliseconds after stimulation the spatial overlap of these Ca2+ signals gave a 'ring' of elevated Ca2+ around the periphery of the cells. However, this ring was not continuous and substantial Ca2+ gradients were observed. The discrete subsarcolemmal Ca2+-release sites, which responded in a reproducible sequence to repetitive depolarisations and displayed the highest frequencies of spontaneous Ca2+ sparks in resting cells, were denoted 'eager sites'. Immunostaining atrial myocytes for type II ryanodine receptors (RyRs) revealed both subsarcolemmal 'junctional' RyRs, and also 'non-junctional' RyRs in the central bulk of the cells. A subset of the junctional RyRs comprises the eager sites. For cells paced in the presence of 1 mM extracellular Ca2+, the response was largely restricted to a subsarcolemmal 'ring', while the central bulk of the cell displayed a approximately 5-fold lower Ca2+ signal. Under these conditions the non-junctional RyRs were only weakly activated during E-C coupling. However, these channels are functional and the Ca2+ stores were at least partially loaded, since substantial homogeneous Ca2+ signals could be stimulated in the central regions of atrial myocytes by application of 2.5 mM caffeine. Neither the location nor activation order of the eager sites was affected by increasing the trigger Ca2+ current (by increasing extracellular Ca2+ to 10 mM) or the sarcoplasmic reticulum (SR) Ca2+ load (following 1 min incubation in 10 mM extracellular Ca2+), although with increased SR Ca2+ load, but not greater Ca2+ influx, the delay between the sequential activation of eager sites was reduced. In addition, increasing the trigger Ca2+ current or the SR Ca2+ load changed the spatial pattern of the Ca2+ response, in that the Ca2+ signal propagated more reliably from the subsarcolemmal initiation sites into the centre of the cell. Due to the greater spatial spread of the Ca2+ signals, the averaged global Ca2+ transients increased by approximately 500 %. We conclude that rat atrial myocytes display a predetermined spatiotemporal pattern of Ca2+ signalling during early E-C coupling. A consistent set of eager Ca2+ release sites with a fixed location and activation order on the junctional SR serve to initiate the cellular response. The short latency for activation of these eager sites suggests that they reflect clusters of RyRs closely coupled to voltage-operated Ca2+ channels in the sarcolemma. Furthermore, their propensity to show spontaneous Ca2+ sparks is consistent with an intrinsically higher sensitivity to Ca2+-induced Ca2+ release. While the subsarcolemmal Ca2+ response can be considered as stereotypic, the central bulk of the cell grades its response in direct proportion to cellular Ca2+ load and Ca2+ influx.
Loading slices of rat barrel cortex with 50 microM BAPTA-AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca(2+) stores with 20 microM cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca(2+) stores, results in a similar reduction of mEPSCs to that observed with BAPTA-AM, indicating that the source for intracellular Ca(2+) is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 microM ryanodine or 10 mM caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca(2+) release from ryanodine stores. Blocking IP(3) receptors with 14 microM 2-aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP(3) stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 microM (RS)-3,5-dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP(3) receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca(2+)-induced Ca(2+) release (CICR) from ryanodine stores after the spontaneous opening of IP(3) stores.
Action potential-independent transmitter release is random and produces small depolarizations in the postsynaptic neuron. This process is, therefore, not thought to play a significant role in impulse propagation across synapses. Here we show that calcium flux through presynaptic neuronal nicotinic receptors leads to mobilization of store calcium by calcium-induced calcium release. Recruitment of store calcium induces vesicular release of glutamate in a manner consistent with synchronization across multiple active zones in the CA3 region of the rat hippocampus. This modulation of action potential-independent release of glutamate is sufficient to drive the postsynaptic pyramidal cell above its firing threshold, thus providing a mechanism for impulse propagation.
Dendritic spines receive excitatory synapses and serve as calcium compartments, which appear to be necessary for input-specific synaptic plasticity. Dendrites of GABAergic interneurons have few or no spines and thus do not possess a clear morphological basis for synapse-specific compartmentalization. We demonstrate using two-photon calcium imaging that activation of single synapses on aspiny dendrites of neocortical fast spiking (FS) interneurons creates highly localized calcium microdomains, often restricted to less than 1 microm of dendritic space. We confirm using ultrastructural reconstruction of imaged dendrites the absence of any morphological basis for this compartmentalization and show that it is dependent on the fast kinetics of calcium-permeable (CP) AMPA receptors and fast local extrusion via the Na+/Ca2+ exchanger. Because aspiny dendrites throughout the CNS express CP-AMPA receptors, we propose that CP-AMPA receptors mediate a spine-free mechanism of input-specific calcium compartmentalization.
Luminal Ca²⁺ in the endoplasmic and sarcoplasmic reticulum (ER/SR) plays an important role in regulating vital biological processes, including store-operated capacitative Ca²⁺ entry, Ca²⁺-induced Ca²⁺ release, and ER/SR stress-mediated cell death. We report rapid and substantial decreases in luminal [Ca²⁺], called “Ca²⁺ blinks,” within nanometer-sized stores (the junctional cisternae of the SR) during elementary Ca²⁺ release events in heart cells. Blinks mirror small local increases in cytoplasmic Ca²⁺,orCa²⁺ sparks, but changes of [Ca²⁺] in the connected free SR network were below detection. Store microanatomy suggests that diffusional strictures may account for this paradox. Surprisingly, the nadir of the store depletion trails the peak of the spark by about 10 ms, and the refilling of local store occurs with a rate constant of 35 s⁻¹, which is ≈6-fold faster than the recovery of local Ca²⁺ release after a spark. These data suggest that both local store depletion and some time-dependent inhibitory mechanism contribute to spark termination and refractoriness. Visualization of local store Ca²⁺ signaling thus broadens our understanding of cardiac store Ca²⁺ regulation and function and opens the possibility for local regulation of diverse store-dependent functions.
• calcium-induced calcium release
• calcium spark
• cardiac myocytes
• endoplasmic reticulum
• sarcoplasmic reticulum
For decades, astrocytes have been considered to be non-excitable support cells of the brain. However, this view has changed radically during the past twenty years. The recent recognition that they are organized in separate territories and possess active properties--notably a competence for the regulated release of 'gliotransmitters', including glutamate--has enabled us to develop an understanding of previously unknown functions for astrocytes. Today, astrocytes are seen as local communication elements of the brain that can generate various regulatory signals and bridge structures (from neuronal to vascular) and networks that are otherwise disconnected from each other. Examples of their specific and essential roles in normal physiological processes have begun to accumulate, and the number of diseases known to involve defective astrocytes is increasing.
Calcium ions are ubiquitous and versatile signaling molecules, capable of decoding a variety of extracellular stimuli (hormones, neurotransmitters, growth factors, etc.) into markedly different intracellular actions, ranging from contraction to secretion, from proliferation to cell death. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca(2+)] rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be initiated. In this review, we discuss the structural and functional bases that generate the subcellular heterogeneity in cellular Ca(2+) levels at rest and under stimulation. This complex choreography requires the concerted action of many different players; the central role is, of course, that of the calcium ion, with the main supporting characters being all the entities responsible for moving Ca(2+) between different compartments, while the cellular architecture provides a determining framework within which all the players have their exits and their entrances. In particular, we concentrate on the molecular mechanisms that lead to the generation of cytoplasmic Ca(2+) microdomains, focusing on their different subcellular location, mechanism of generation, and functional role.
Vasoregulation by the bold beta 1 subunit of the calcium-activated potassium channel
- A J Wiler
- M T Patterson
- R W Nelson
- Aldrich
Wiler, A.J. Patterson, M.T. Nelson, R.W. Aldrich, Vasoregulation by the bold beta 1 subunit of the calcium-activated potassium channel, Nature (London) 407 (2000) 870–876.
Microdomains for neuron-glia interaction: parallel fiber signalling to Bergmann glial cells
- J Grosche
- V Matyash
- T Möller
- A Verkhratsky
- A Reichenbach
- H Kettermann
J. Grosche, V. Matyash, T. Möller, A. Verkhratsky, A. Reichenbach, H.
Kettermann, Microdomains for neuron-glia interaction: parallel fiber
signalling to Bergmann glial cells, Nat. Neurosci. 2 (1999) 139-143.
Local InsP 3 -dependent perinuclear Ca 2+ signalling in cardiac myocyte excitation-transcription coupling
- X Wu
- T Zhang
- J Bossuyt
- X Li
- T A Mckinsey
- J R Dedman
- E N Olsen
- J Chen
- J H Brown
- D M Bers
X. Wu, T. Zhang, J. Bossuyt, X. Li, T.A. McKinsey, J.R. Dedman,
E.N. Olsen, J. Chen, J.H. Brown, D.M. Bers, Local InsP 3 -dependent
perinuclear Ca 2+ signalling in cardiac myocyte excitation-transcription
coupling, J. Clin. Invest. 116 (2006) 675-682.
Ca2+-sensitive adenylyl cyclases, key intergrators of cation channels
- Mons