STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.     

Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.     

Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure

The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca(2+) ([Ca(2+)](ext))-induced [Ca(2+)](cyt) transients and stomatal closure in Arabidopsis. CAS (Ca(2+) sensing receptor) is a plant-specific putative Ca(2+)-binding protein that was originally proposed to be a plasma membrane-localized external Ca(2+) sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca(2+)-binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca(2+). In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca(2+). Furthermore, using the transgenic aequorin system, we showed that [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients and stomatal closure.     

Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons

Changes in cytosolic free Ca2+ concentration [( Ca2+]i) due to Ca2+ entry or Ca2+ release from internal stores were spatially resolved by digital imaging with the Ca2+ indicator fura-2 in frog sympathetic neurons. Electrical stimulation evoked a rise in [Ca2+]i spreading radially from the periphery to the center of the soma. Elevated [K+]o also increased [Ca2+]i, but only in the presence of external Ca2+, indicating that Ca2+ influx through Ca2+ channels is the primary event in the depolarization response. Ca2+ release or uptake from caffeine-sensitive internal stores was able to amplify or attenuate the effects of Ca2+ influx, to generate continued oscillations in [Ca2+]i, and to persistently elevate [Ca2+]i above basal levels after the stores had been Ca2(+)-loaded.     

Ca2+ transients control CNS neuronal migration

In the developing CNS, postmitotic neurons exhibit dynamic changes in the mode, direction and rate of migration as they traverse different cortical layers, but the mechanisms underlying this process is largely unknown. Recent studies show that the changes in Ca2+ transient frequency play a central role in controlling the neuronal cell migration in a cortical layer-specific manner. In this article, we will first describe how granule cells migrate through different terrains of the developing cerebellar cortex. We will then present how such migration of granule cells is controlled by altering the Ca2+ transient frequency in their somata. Finally, we will discuss how the loss of Ca2+ transients triggers the completion of granule cell migration at their final destination.     

A model-independent algorithm to derive Ca2+ fluxes underlying local cytosolic Ca2+ transients

Local intracellular Ca(2+) signals result from Ca(2+) flux into the cytosol through individual channels or clusters of channels. To gain a mechanistic understanding of these events we need to know the magnitude and spatial distribution of the underlying Ca(2+) flux. However, this is difficult to infer from fluorescence Ca(2+) images because the distribution of Ca(2+)-bound dye is affected by poorly characterized processes including diffusion of Ca(2+) ions, their binding to mobile and immobile buffers, and sequestration by Ca(2+) pumps. Several methods have previously been proposed to derive Ca(2+) flux from fluorescence images, but all require explicit knowledge or assumptions regarding these processes. We now present a novel algorithm that requires few assumptions and is largely model-independent. By testing the algorithm with both numerically generated image data and experimental images of sparklets resulting from Ca(2+) flux through individual voltage-gated channels, we show that it satisfactorily reconstructs the magnitude and time course of the underlying Ca(2+) currents.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators.

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Beta 1-subunits are required for regulation of coupling between Ca2+ transients and Ca2+-activated K+ (BK) channels by protein kinase C

Colonic myocytes have spontaneous, localized, Ins (1,4,5) trisphosphate (IP3) receptor-dependent Ca2+ transients that couple to the activation of Ca2+-dependent K+ channels and spontaneous transient outward currents (STOCs). We previously reported that the coupling strength between spontaneous Ca2+ transients and large conductance Ca2+ activated K+ (BK) channels is regulated by Ca2+ influx through nonselective cation channels and activation of protein kinase C (PKC). Here, we used confocal microscopy and the patch-clamp technique to further investigate the coupling between localized Ca2+ transients and STOCs in colonic myocytes from animals lacking the regulatory beta1-subunit of BK channels. Myocytes from beta1-knockout (beta1-/-) animals loaded with fluo 4 showed typical localized Ca2+ transients, but the STOCs coupled to these events were of abnormally low amplitude. Reduction in external Ca2+ or application of inhibitors of nonselective cation channels (SKF-96365) caused no significant change in the amplitude or frequency of STOCs. Likewise, an inhibitor of PKC, GF 109203X, had no significant effect on STOCs. Single-channel recording from BK channels showed that application of an activator (PMA) and an inhibitor (GF 109203X) of PKC did not affect BK channel openings in myocytes of beta1-/- mice. These data show that PKC-dependent regulation of coupling strength between Ca2+ transients and STOCs in colonic myocytes depends upon the interaction between alpha- and beta1-subunits.     

Doc2 is a Ca2+ sensor required for asynchronous neurotransmitter release

Synaptic transmission involves a fast synchronous phase and a slower asynchronous phase of neurotransmitter release that are regulated by distinct Ca(2+) sensors. Though the Ca(2+) sensor for rapid exocytosis, synaptotagmin I, has been studied in depth, the sensor for asynchronous release remains unknown. In a screen for neuronal Ca(2+) sensors that respond to changes in [Ca(2+)] with markedly slower kinetics than synaptotagmin I, we observed that Doc2--another Ca(2+), SNARE, and lipid-binding protein--operates on timescales consistent with asynchronous release. Moreover, up- and downregulation of Doc2 expression levels in hippocampal neurons increased or decreased, respectively, the slow phase of synaptic transmission. Synchronous release, when triggered by single action potentials, was unaffected by manipulation of Doc2 but was enhanced during repetitive stimulation in Doc2 knockdown neurons, potentially due to greater vesicle availability. In summary, we propose that Doc2 is a Ca(2+) sensor that is kinetically tuned to regulate asynchronous neurotransmitter release.     

Ca2+ transients and Mn2+ entry in human neutrophils induced by thapsigargin

Human neutrophils, preloaded with the fluorescent probe, Fura-2, were exposed to Ca2+-releasing agents. The monitored traces of fluorescence were transformed by computer to cytosolic Ca2+ concentration ([ Ca2+]i). Due to quenching of Fura-2, the addition of Mn2+ enabled us to compute the cytosolic concentration of total manganese ([Mn]i). The agents used were the novel Ca2+-mobilizing agent, thapsigargin (Tg), the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), and the divalent cation ionophore, A23187. The agents caused transient rises of [Ca2+]i and monotonous rises of [Mn]i, suggesting influx but no efflux of Mn2+. The rise time of [Ca2+]i and the time constants and magnitude of the apparent Mn2+ influx were strongly dependent on the sequence of addition of the agonist and Ca2+. Contrary to FMLP, Tg needed several minutes to exert its full effect on the rise of [Ca2+]i and on the influx of Mn2+, the latter being dependent on two phases, activation and partial inactivation. Pretreatment with phorbol 12-myristate 13-acetate (PMA) inhibited the responses of Tg, FMLP and A23187. For comparison, human red blood cells were tested. Contrary to A23187, Tg did not induce Ca2+ uptake in ATP-depleted red cells but increased the Ca2+ pump flux in intact red cells by 10%. The experimental data and computer simulations of the granulocyte data suggest that time-dependent changes of both passive Ca2+ flux into the cytosol and Ca2+ flux of the plasma membrane pump are involved in the transient [Ca2+]i response.     

Transformation of local Ca2+ spikes to global Ca2+ transients: the combinatorial roles of multiple Ca2+ releasing messengers

In pancreatic acinar cells, low, threshold concentrations of acetylcholine (ACh) or cholecystokinin (CCK) induce repetitive local cytosolic Ca2+ spikes in the apical pole, while higher concentrations elicit global signals. We have investigated the process that transforms local Ca2+ spikes to global Ca2+ transients, focusing on the interactions of multiple intracellular messengers. ACh-elicited local Ca2+ spikes were transformed into a global sustained Ca2+ response by cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), whereas inositol 1,4,5-trisphosphate (IP3) had a much weaker effect. In contrast, the response elicited by a low CCK concentration was strongly potentiated by IP3, whereas cADPR and NAADP had little effect. Experiments with messenger mixtures revealed a local interaction between IP3 and NAADP and a stronger global potentiating interaction between cADPR and NAADP. NAADP strongly amplified the local Ca2+ release evoked by a cADPR/IP3 mixture eliciting a vigorous global Ca2+ response. Different combinations of Ca2+ releasing messengers can shape the spatio-temporal patterns of cytosolic Ca2+ signals. NAADP and cADPR are emerging as key messengers in the globalization of Ca2+ signals.     

Synaptotagmin-1 is a Ca2+ sensor for somatodendritic dopamine release

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Interaction of ATP sensor, cAMP sensor, Ca2+ sensor, and voltage-dependent Ca2+ channel in insulin granule exocytosis

ATP, cAMP, and Ca(2+) are the major signals in the regulation of insulin granule exocytosis in pancreatic beta cells. The sensors and regulators of these signals have been characterized individually. The ATP-sensitive K(+) channel, acting as the ATP sensor, couples cell metabolism to membrane potential. cAMP-GEFII, acting as a cAMP sensor, mediates cAMP-dependent, protein kinase A-independent exocytosis, which requires interaction with both Piccolo as a Ca(2+) sensor and Rim2 as a Rab3 effector. l-type voltage-dependent Ca(2+) channels (VDCCs) regulate Ca(2+) influx. In the present study, we demonstrate interactions of these molecules. Sulfonylurea receptor 1, a subunit of ATP-sensitive K(+) channels, interacts specifically with cAMP-GEFII through nucleotide-binding fold 1, and the interaction is decreased by a high concentration of cAMP. Localization of cAMP-GEFII overlaps with that of Rim2 in plasma membrane of insulin-secreting MIN6 cells. Localization of Rab3 co-incides with that of Rim2. Rim2 mutant lacking the Rab3 binding region, when overexpressed in MIN6 cells, is localized exclusively in cytoplasm, and impairs cAMP-dependent exocytosis in MIN6 cells. In addition, Rim2 and Piccolo bind directly to the alpha(1)1.2-subunit of VDCC. These results indicate that ATP sensor, cAMP sensor, Ca(2+) sensor, and VDCC interact with each other, which further suggests that ATP, cAMP, and Ca(2+) signals in insulin granule exocytosis are integrated in a specialized domain of pancreatic beta cells to facilitate stimulus-secretion coupling.     

A novel Ca2+ indicator protein using FRET and calpain-sensitive linker

Here, we report the properties of a FRET-based calcium indicator protein. We constructed a tandem fusion protein, named F2C, of ECFP and EYFP combined with calpain-sensitive sequences of alpha-spectrin, with N-terminal palmitoylation signal of GAP-43. It was previously reported that calpain cleaved a similar ECFP-EYFP fusion protein linked by a calpain-sensitive sequence of alpha-spectrin (fodrin). Unexpectedly, F2C was not cleaved by calpain, but demonstrated properties of a Ca(2+) indicator when transiently infected in Purkinje cells of rat primary cerebellar culture or in the brainstem neurons infected in vivo using Sindbis virus encoding F2C. The emission ratio of 480nm/535nm was repeatedly increased when the intracellular Ca(2+) concentration ([Ca(2+)](i)) was raised. F2C had a Ca(2+) sensitivity with an apparent dissociation constant (K(d) for Ca(2+)) of 150nM, and demonstrated kinetics that paralleled Fura-2 when [Ca(2+)](i) was measured simultaneously. These properties of F2C are useful to be a Ca(2+) indicator.     

Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Here, we find that overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.