The ER and ageing II: Calcium homeostasis

Increase in intracellular Ca²⁺ concentration occurs by Ca²⁺ influx through the plasma membrane and by Ca²⁺ release from intracellular stores. The ER is the most important Ca²⁺ store. Its stress, characterized by the impairment of Ca²⁺ homeostasis and by the accumulation of misfolded proteins, can be induced by different factors. In turn, it induces defense mechanisms such as unfolded protein response, and when it is severe and prolonged, activation of the apoptotic pathway. Damage to the ER, impairment of its function, and a decreased level of its Ca²⁺-handling proteins might all play a role in physiological ageing by handicapping the ER stress response. Thus, healthy ageing is accompanied by subtle alterations of Ca²⁺ homeostasis and signaling, including alterations in the ER Ca²⁺ load and release. The expression and/or function of ryanodine receptors, IP3 receptors, and SERCA Ca²⁺ pumps located in the ER membrane, and Ca²⁺-binding proteins within ER lumen all seem to be affected in aged cells. Data are presented on age-dependent, tissue-specific changes in ER-related Ca²⁺ homeostasis in skeletal, cardiac and smooth muscles, as well as in the nervous and immune systems. Disturbances of Ca²⁺ homeostasis and of signaling are potential targets for intervention in aged humans.     

Differential STIM1 expression in T and B cell subsets suggests a role in determining antigen receptor signal amplitude

Ca²⁺ acts ubiquitously as a second messenger in transmembrane signal transduction. In lymphocytes, calcium mobilization is triggered by antigen and chemokine receptors, among others, and controls cell functions ranging from proliferation to migration. The primary mechanism of extracellular Ca²⁺ entry in lymphocytes is the CRAC influx. STIM1 is a crucial component of the CRAC influx mechanism in lymphocytes, acting as a sensor of low Ca²⁺ concentration in the ER and an activator of the Ca²⁺ selective channel ORAI1 in the plasma membrane. While STIM1 function has been studied extensively, little is known regarding whether it is differentially expressed and thereby affects the magnitude of calcium mobilization responses. We report here that STIM1 expression differs in murine T and B lymphocytes, and in respective subsets. For example, mature T cells express ∼4 times more STIM1 than mature B cells. Furthermore, we show that through the physiologic range of expression, STIM1 levels determine the magnitude of Ca²⁺ influx responses that follow BCR-induced intracellular store depletion. Considered in view of previous reports that differences in amplitude of lymphocyte Ca²⁺ mobilization determine alternate biological responses, these findings suggest that differential STIM1 expression may be important determinant of biological responses.     

Multifaceted roles of STIM proteins

Stromal interaction molecules (STIM1 and STIM2) are critical components of store-operated calcium entry. Sensing depletion of endoplasmic reticulum (ER) Ca²⁺ stores, STIM couples with plasma membrane Orai channels, resulting in the influx of Ca²⁺ across the PM into the cytosol. Although best recognized for their primary role as ER Ca²⁺ sensors, increasing evidence suggests that STIM proteins have a broader variety of sensory capabilities than first envisaged, reacting to cell stressors such as oxidative stress, temperature, and hypoxia. Further, the array of partners for STIM proteins is now understood to range far beyond the Orai channel family. Here we discuss the implications of STIM’s expanding role, both as a stress sensor and a general modulator of multiple physiological processes in the cell.     

γ-Secretase activity modulates store-operated Ca entry into rat sensory neurons

Presenilin-1 is required for γ-secretase activity, which participates in Notch receptor processing, the pathogenesis of Alzheimer's disease and the modulation of Ca²⁺ signaling. We tested the hypothesis that γ-secretase proteolytic activity modulates store-operated Ca²⁺ entry (SOCE) in rat dorsal root ganglion (DRG) neurons. Depletion of intracellular Ca²⁺ stores by blocking the endoplasmic reticulum (ER) Ca²⁺ pump with cyclopiazonic acid (CPA) evoked a transient increase in [Ca²⁺]_i but no sustained Ca²⁺ influx. However, in cells expressing a dominant negative presenilin-1 mutant (PS1-D257A), γ-secretase activity was inhibited and treatment with CPA evoked sustained Ca²⁺ influx. Similarly, pharmacologic inhibition of γ-secretase with DAPT for 48 h enhanced SOCE. SKF96365, an inhibitor of store-operated channels, blocked SOCE in cells expressing PS1-D257A. Thus, γ-secretase proteolytic activity regulates a SOCE pathway in sensory neurons.     

ATP depletion induces translocation of STIM1 to puncta and formation of STIM1–ORAI1 clusters: translocation and re-translocation of STIM1 does not require ATP

Depletion of the endoplasmic reticulum (ER) calcium store triggers translocation of stromal interacting molecule one (STIM1) to the sub-plasmalemmal region and formation of puncta—structures in which STIM1 interacts and activates calcium channels. ATP depletion induced the formation of STIM1 puncta in PANC1, RAMA37, and HeLa cells. The sequence of events triggered by inhibition of ATP production included a rapid decline of ATP, depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and a slow calcium leak from the ER followed by formation of STIM1 puncta. STIM1 puncta induced by ATP depletion were co-localized with clusters of ORAI1 channels. STIM1–ORAI1 clusters that developed as a result of ATP depletion were very poor mediators of Ca²⁺ influx. Re-translocation of STIM1 from puncta back to the ER was observed during total ATP depletion. We can therefore conclude that STIM1 translocation and re-translocation as well as formation of STIM1–ORAI1 clusters occur in an ATP-independent fashion and under conditions of PI(4,5)P₂ depletion. Michael Chvanov and Ciara M. Walsh are considered as equal first authors.     

Intracellular Ca2+ release via the ER translocon activates store-operated calcium entry

Store-operated Ca²⁺ entry (SOCE) is activated in response to depletion of intracellular Ca²⁺ from the endoplasmic reticulum (ER). A variety of agonists stimulate SOCE via IP₃-dependent Ca²⁺ depletion. SOCE is also activated by thapsigargin, an inhibitor of Ca²⁺ reuptake into the ER that induces a net Ca²⁺ loss from the ER by unmasking a Ca²⁺ “leak” pathway. The molecular identity of this Ca²⁺ leak channel and the physiological conditions under which such agonist-independent Ca²⁺ depletion might occur remain poorly characterized. In this study, we report that inhibition of the initiation step of protein synthesis (with pactamycin) resulted in detectable Ca²⁺ depletion in ER and activation of SOCE. This was completely prevented if the ribosome–nascent chain complexes were first stabilized with an irreversible inhibitor of translational elongation (emetine), suggesting that ER Ca²⁺ depletion had occurred through open translocons at the ER. Notably, emetine pretreatment also attenuated thapsigargin-mediated Ca²⁺ release and SOCE. Furthermore, both pactamycin and thapsigargin stimulated translocation of STIM1, a protein required for activation of SOCE, to the subplasma membrane region and activated the SOCE-associated current, I _SOC. In aggregate, these data reveal an agonist-independent mechanism for internal Ca²⁺ store depletion and activation of SOCE. We suggest that the functional coupling between SOCE and protein synthesis is likely to be critical for maintaining [Ca²⁺]_ER within a range that is required to prevent ER stress during changes in cellular translational activity.     

How Does the Ca2+-paradox Injury Induce Contracture in the Heart?—A Combined Study of the Intracellular Ca2+ Dynamics and Cell Structures in Perfused Rat Hearts—

The calcium (Ca²⁺)-paradox injury of the heart, induced by restoration of extracellular Ca²⁺ after its short-term depletion, is known to provoke cardiomyocyte contracture. However, undetermined is how the Ca²⁺-paradox provokes such a distinctive presentation of myocytes in the heart. To address this, we imaged sequential intracellular Ca²⁺ dynamics and concomitant structures of the subepicardial ventricular myocytes in fluo3-loaded, Langendorff-perfused rat hearts produced by the Ca²⁺ paradox. Under rapid-scanning confocal microscopy, repletion of Ca²⁺ following its depletion produced high-frequency Ca²⁺ waves in individual myocytes with asynchronous localized contractions, resulting in contracture within 10 min. Such alterations of myocytes were attenuated by 5-mM NiCl₂, but not by verapamil, SEA0400, or combination of ryanodine and thapsigargin, indicating a contribution of non-specific transmembrane Ca²⁺ influx in the injury. However, saponin-induced membrane permeabilization of Ca²⁺ showed no apparent contracture despite the emergence of high-frequency Ca²⁺ waves, indicating an essential role of myocyte-myocyte and myocyte-extracellular matrix (ECM) mechanical connections in the Ca²⁺ paradox. In immunohistochemistry Ca²⁺ depletion produced separation of the intercalated disc that expresses cadherin and dissipation of β-dystroglycan located along the sarcolemma. Taken together, along with the trans-sarcolemmal Ca²⁺ influx, disruption of cell-cell and cell-ECM connections is essential for contracture in the Ca²⁺-paradox injury.     

Store-operated Ca2+ entry in platelets occurs independently of transient receptor potential (TRP) C1

Changes in [Ca²⁺]_i are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca²⁺ stores triggers Ca²⁺ entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca²⁺ sensor that regulates store-operated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1. David Varga-Szabo and Kalwant S. Authi contributed equally to this article.     

The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1

Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most active antitumor agents used in human chemotherapy of non-small cell lung cancer. Cisplatin forms crosslinked DNA adducts and its cytotoxicity has been shown to be mediated by propagation of DNA damage recognition signals to downstream pathways prompting apoptosis. The steps involved in the process include changes in Ca²⁺ signaling with dysregulated tumor cell turn-over. Stromal interaction molecules 1 (STIM1), as one of the most potent tumor suppressor genes, are identified as the endoplasmic-reticulum (ER) Ca²⁺ sensor controlling store-operated Ca²⁺ entry (SOCE) in non-excitable cells, which is main pathway to extracellular Ca²⁺ influx. Its role in STIM1 cisplatin-induced apoptosis of non-small cell lung cancer was the focus of study with focus on SOCE inhibitors 2-APB- and SKF96365-cisplatin-induced apoptosis in the non-small cell lung cancer (NSCLC) cell lines A549 and H460. In this experimental model, cisplatin-induced apoptosis and decreased concentration of intracellular Ca²⁺ was demonstrated. The expression of STIM1 was significantly higher in carcinoma tissue than in the adjacent non-neoplastic lung tissue. These findings support the conclusion that STIM1 may play an important role in the development of NSCLC which makes drugs that repress the expression of STIM1 to be a potential target for lung cancer therapy.     

Initiation site of Ca(2+) entry evoked by endoplasmic reticulum Ca(2+) depletion in mouse parotid and pancreatic acinar cells

Purpose

In non-excitable cells, which include parotid and pancreatic acinar cells, Ca²⁺ entry is triggered via a mechanism known as capacitative Ca²⁺ entry, or store-operated Ca²⁺ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca²⁺ stores, which acts as an important factor triggering Ca²⁺ entry. However, both the mechanism of store-mediated Ca²⁺ entry and the molecular identity of store-operated Ca²⁺ channel (SOCC) remain uncertain.

Materials and Methods

In the present study we investigated the Ca²⁺ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.

Results

Treatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca²⁺-ATPase, in an extracellular Ca²⁺ free state, and subsequent exposure to a high external calcium state evoked Ca²⁺ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca²⁺ channel, completely blocked Tg-induced Ca²⁺ entry. Microfluorometric imaging showed that Tg-induced Ca²⁺ entry started at a basal membrane, not a apical membrane.

Conclusion

These results suggest that Ca²⁺ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.     

Arrhythmogenesis Toxicity of Aconitine Is Related to Intracellular Ca2+ Signals

Aconitine is a well-known arrhythmogenic toxin and induces triggered activities through cardiac voltage-gated Na⁺ channels. However, the effects of aconitine on intracellular Ca²⁺ signals were previously unknown. We investigated the effects of aconitine on intracellular Ca²⁺ signals in rat ventricular myocytes and explored the possible mechanism of arrhythmogenic toxicity induced by aconitine. Ca²⁺ signals were evaluated by measuring L-type Ca²⁺ currents, caffeine-induced Ca²⁺ release and the expression of NCX and SERCA2a. Action potential and triggered activities were recorded by whole-cell patch-clamp techniques. In rat ventricular myocytes, the action potential duration was significantly prolonged by 1 µM aconitine. At higher concentrations (5 µM and 10 µM), aconitine induced triggered activities and delayed after-depolarizations (6 of 8 cases), which were inhibited by verapamil. Aconitine (1 µM) significantly increased the I_Ca-L density from 12.77 ± 3.12 pA/pF to 18.98 ± 3.89 pA/pF (n=10, p<0.01). The activation curve was shifted towards more negative potential, while the inactivation curve was shifted towards more positive potential by 1 μM aconitine. The level of Ca²⁺ release induced by 10 mM caffeine was markedly increased. Aconitine (1 µM) increased the expression of NCX, while SERCA2a expression was reduced. In conclusion, aconitine increased the cytosolic [Ca²⁺]_i by accelerating I_Ca-L and changing the expression of NCX and SERCA2a. Then, the elevation of cytosolic [Ca²⁺]_i induced triggered activities and delayed after-depolarizations. Arrhythmogenesis toxicity of aconitine is related to intracellular Ca²⁺ signals.     

Histamine-activated,non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein

Permeation properties and modulation of an ionic current gated by histamine were measured in single endothelial cells from human umbilical cord veins by use of the patch-clamp technique in the ruptured-whole-cell mode or using perforated patches. We combined these current measurements with a microfluorimetric method to measure concomitantly free intracellular calcium concentration ([Ca²⁺]_i). Application of histamine induced an intracellular calcium transient and an ionic current that reversed near 0 mV. The amplitude of the current ranged from –0.2 to –2nA at –100mV. The tonic rise in [Ca²⁺]_i and the ionic current are partly due to Ca²⁺ influx. This Ca²⁺ entry pathway is also permeable for Ba²⁺ and Mn²⁺. The amplitude of the histamine-activated current was also closely correlated with the amplitude of the concomitant Ca²⁺ transient, suggesting that the latter is at least partially due to Ca²⁺ influx through histamine-activated channels. The reversal potential of the histamine-induced current was 7.6±4.1 mV (n=14) when the calcium concentration in the bath solution ([Ca²⁺]_o) was 1.5mmol/l. With 10 mmol/l [Ca²⁺]_o it was –13.7±4.7 mV and shifted to +13.0±1.5 mV in nominally Ca²⁺-free solution (n=3 cells). The amplitude of the current in Ca²⁺-free solution was enhanced compared to that in 10 mmol/l [Ca²⁺]_o. The shift of the reversal potential and the concomitant change of the current amplitude suggest that the channel is permeable for calcium but has a smaller permeability for calcium than for monovalent cations. The latency between the application of histamine and the appearance of the current was voltage dependent and was much smaller at more negative potentials. This effect is unlikely to be due to desensitization, but may suggest a voltage-dependent step in the signal transduction chain. Similar histamine-induced Ca²⁺ signals were observed if the currents were measured in patches perforated with nystatin. The onset of the agonist-activated current was, however, much more delayed and its amplitude significantly lower than in ruptured patches. The histamine-induced currents and intracellular Ca²⁺-transients were largely reduced after incubation of endothelial cells with the phorbol ester TPA. H7, a blocker of protein kinase C, induced membrane currents and Ca²⁺ signals in the absence of an agonist. It is concluded that the agonist-activated Ca²⁺-entry in endothelial cells occurs through non-selective cation channels which can be down-regulated by protein kinase C activation.     

Capacitative Ca2+ influx and a Ca2u+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells

We have investigated the effect of genistein on the hormone-stimulated Ca²⁺ influx and on a 28 pS nonselective cation channel in mouse pancreatic acinar cells using the Ca²⁺ indicator fluo-3 and the patch-clamp technique. The identity of the Ca^2u+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca²⁺-dependent nonselective cation channel as a potential pathway for Ca²⁺ influx. Capacitative Ca²⁺ entry was induced by depletion of intracellular Ca²⁺ stores with 500 nM acetylcholine or with the Ca²⁺ ATPase inhibitor 2,5-di-tert- butylhydroquinone. In the presence of 100 M genistein, Ca²⁺ release was unimpaired, whereas Ca²⁺ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca²⁺-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28 pS nonselective cation channel does not contribute to Ca²⁺ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca²⁺ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca²⁺ entry mechanism in mouse pancreatic acinar cells is unclear.     

Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca²⁺ oscillations from an inositol 1,4,5-trisphosphate-sensitive cytoplasmic Ca²⁺ store. In this study, Ca²⁺ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca²⁺ video imaging and the Fura-2/Mn²⁺ quenching technique. Oscillations were dependent on extracellular Ca²⁺ concentration and were inhibited by extracellularly applied La³⁺, Co²⁺ and Ni²⁺. Depolarization of the cell membrane with high K⁺ concentrations and the L-type Ca²⁺ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca²⁺ channels. Mn²⁺ quenching experiments disclosed significant Ca²⁺ influx into MDCK-F cells. The rate of this influx was constant between Ca²⁺ spikes, but markedly increased during the spontaneous Ca²⁺ spikes. Similar transient increases in Ca²⁺ entry could be mimicked by agents triggering intracellular Ca²⁺ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca²⁺ permeability permitting Ca²⁺ entry into the cytoplasm. The rate of Ca²⁺ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca²⁺ concentration.     

Neuropeptide Y2-type receptor-mediated activation of large-conductance Ca2+-sensitive K+ channels in a human neuroblastoma cell line

We have proposed recently that a pertussistoxin-insensitive Ca²⁺ influx stimulated by Y₂-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca²⁺ and independent of internal Ca²⁺ stores. We describe here the actions of NPY in these same cells, using the activity of Ca²⁺-activated K⁺ channels as an indicator of [Ca²⁺]_i. The elementary slope conductance of these channels was 110±3 pS (with an asymmetrical K⁺gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca²⁺, increased the channel open probability. ATP applied in the absence of external Ca²⁺ caused rises both in channel open probability and [Ca²⁺]_i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca²⁺ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5-triphosphate (GTP) by (guanosine 5-O-(2-thiodiphosphate) (GDP[S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y₂-type receptors to Ca²⁺ influx in CHP-234 cells.     

Endoplasmic reticulum protein BI-1 regulates Ca²⁺-mediated bioenergetics to promote autophagy

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²⁺ mediated by inositol triphosphate receptors (IP₃Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP₃R-dependent manner. By reducing steady-state levels of ER Ca²⁺ via IP₃Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1''s ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca²⁺ signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.     

Estimating intracellular Ca concentrations and buffering in a dendritic inhibitory hippocampal interneuron

Calcium is known to regulate several phenomena like neuronal excitability and plasticity. Interestingly, the spatiotemporal profile of dendritic calcium depends on several processes, specific to each neuronal type. In this study, we investigated Ca²⁺ buffering and action potential (AP)-evoked Ca²⁺ signaling in the dendrites of anatomically identified oriens lacunosum-moleculare (O-LM) cells, a major type of dendrite-targeting interneurons in the hippocampal CA1 region, using a combination of whole-cell patch-clamp recording and fast Ca²⁺ imaging in acute rat brain slices. Cells were loaded with fluorescent Ca²⁺ indicators fura-2 or Oregon Green BAPTA-1 (OGB-1) via patch-clamping electrode, and the effect of fura-2 on AP-evoked dendritic Ca²⁺ transients was determined by ratiometric Ca²⁺ imaging. To estimate intracellular Ca²⁺ concentrations ([Ca²⁺]_i) and endogenous Ca²⁺-binding ratio (κ_s) in the proximal dendrite, fluorescence signals were converted into [Ca²⁺]_i using the ratioing method and were analyzed on the basis of the “single compartment model.” Resting [Ca²⁺]_i was 22±5 nM and the build-up of [Ca²⁺]_i during a single AP was up to 656±226 nM. Analysis of Ca²⁺ transients revealed that O-LM cells have a relatively low endogenous Ca²⁺-binding ratio (κ_s): the κ_s was 20±8 estimated during fura-2 loading and 27 estimated under steady-state fura-2 concentrations, respectively. To further examine the spatial profile of dendritic Ca²⁺ transients, we measured somatic AP-evoked Ca²⁺ transients beyond proximal dendrites using OGB-1. Dendritic Ca²⁺ transients evoked by single APs or AP trains are not limited to regions close to the soma. The amplitude and decay of [Ca²⁺]_i associated with backpropagating APs are relatively independent of the distance from the soma. In sum, O-LM cells exhibit low endogenous Ca²⁺-binding ratios and relatively distance-independent Ca²⁺ dynamics in the dendrites. These special features of Ca²⁺ signaling in O-LM cells may have important functional implications for both normal and pathological conditions.     

Initiation sites for Ca2+ signals in endothelial cells

Intracellular Ca²⁺ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca²⁺ oscillations and propagating Ca²⁺ waves originating at discrete initiation sites. We studied the spatial organization of the Ca²⁺ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise in cytoplasmic Ca²⁺ concentration and rate of wave propagation than the cell body. Ca²⁺ waves originated preferentially in one of these processes and then invaded the cell body. Removal of external Ca²⁺ induced a progressive inhibition up to blockade of the response in the process but not in the cell body. These findings suggest that CPAE cells contain many individual store units, each of which has the inherent ability to set the stage for Ca²⁺ release. A diffusing messenger originating from the initiation zone then coordinates the events leading to Ca²⁺ release in the individual store units to produce a Ca²⁺ wave.     

Ca2+ signaling by T-type Ca2+ channels in neurons

Among the major families of voltage-gated Ca²⁺ channels, the low-voltage-activated channels formed by the Ca_v3 subunits, referred to as T-type Ca²⁺ channels, have recently gained increased interest in terms of the intracellular Ca²⁺ signals generated upon their activation. Here, we provide an overview of recent reports documenting that T-type Ca²⁺ channels act as an important Ca²⁺ source in a wide range of neuronal cell types. The work is focused on T-type Ca²⁺ channels in neurons, but refers to non-neuronal cells in cases where exemplary functions for Ca²⁺ entering through T-type Ca²⁺ channels have been described. Notably, Ca²⁺ influx through T-type Ca²⁺ channels is the predominant Ca²⁺ source in several neuronal cell types and carries out specific signaling roles. We also emphasize that Ca²⁺ signaling through T-type Ca²⁺ channels occurs often in select subcellular compartments, is mediated through strategically co-localized targets, and is exploited for unique physiological functions. Lucius Cueni and Marco Canepari contributed equally to this review.