Plasticity and adaptation of Ca2+ signaling and Ca2+-dependent exocytosis in SERCA2(+/-) mice

Darier's disease (DD) is a high penetrance, autosomal dominant mutation in the ATP2A2 gene, which encodes the SERCA2 Ca2+ pump. Here we have used a mouse model of DD, a SERCA2(+/-) mouse, to define the adaptation of Ca2+ signaling and Ca2+-dependent exocytosis to a deletion of one copy of the SERCA2 gene. The [Ca2+]i transient evoked by maximal agonist stimulation was shorter in cells from SERCA2(+/-) mice, due to an up-regulation of specific plasma membrane Ca2+ pump isoforms. The change in cellular Ca2+ handling caused approximately 50% reduction in [Ca2+]i oscillation frequency. Nonetheless, agonist-stimulated exocytosis was identical in cells from wild-type and SERCA2(+/-) mice. This was due to adaptation in the levels of the Ca2+ sensors for exocytosis synaptotagmins I and III in cells from SERCA2(+/-) mice. Accordingly, exocytosis was approximately 10-fold more sensitive to Ca2+ in cells from SERCA2(+/-) mice. These findings reveal a remarkable plasticity and adaptability of Ca2+ signaling and Ca2+-dependent cellular functions in vivo, and can explain the normal function of most physiological systems in DD patients.     

PKC-induced sensitization of Ca2+-dependent exocytosis is mediated by reducing the Ca2+ cooperativity in pituitary gonadotropes

The highly cooperative nature of Ca2+-dependent exocytosis is very important for the precise regulation of transmitter release. It is not known whether the number of binding sites on the Ca2+ sensor can be modulated or not. We have previously reported that protein kinase C (PKC) activation sensitizes the Ca2+ sensor for exocytosis in pituitary gonadotropes. To further unravel the underlying mechanism of how the Ca2+ sensor is modulated by protein phosphorylation, we have performed kinetic modeling of the exocytotic burst and investigated how the kinetic parameters of Ca2+-triggered fusion are affected by PKC activation. We propose that PKC sensitizes exocytosis by reducing the number of calcium binding sites on the Ca2+ sensor (from three to two) without significantly altering the Ca2+-binding kinetics. The reduction in the number of Ca2+-binding steps lowers the threshold for release and up-regulates release of fusion-competent vesicles distant from Ca2+ channels.     

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis

Aim

Neurotransmitter release is elicited by an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The action potential triggers Ca²⁺ influx through Ca²⁺ channels which causes local changes of [Ca²⁺]_i for vesicle release. However, any direct role of extracellular Ca²⁺ (besides Ca²⁺ influx) on Ca²⁺-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.

Results

Using photolysis of caged Ca²⁺ and caffeine-induced release of stored Ca²⁺, we found that extracellular Ca²⁺ inhibited exocytosis following moderate [Ca²⁺]_i rises (2–3 µM). The IC₅₀ for extracellular Ca²⁺ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca²⁺ concentration ([Ca²⁺]_o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca²⁺]_o. The calcimimetics Mg²⁺, Cd²⁺, G418, and neomycin all inhibited exocytosis. The extracellular Ca²⁺-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.

Conclusion/Significance

As an extension of the classic Ca²⁺ hypothesis of synaptic release, physiological levels of extracellular Ca²⁺ play dual roles in evoked exocytosis by providing a source of Ca²⁺ influx, and by directly regulating quantal size and release probability in neuronal cells.     

Ca2+ signaling in prokaryotes

The role of Ca²⁺ ions in the regulation of motility, cell cycle, and division of prokaryotes is discussed, as well as their involvement in the pathogenesis of some infectious diseases. The structural and functional organization of the prokaryotic Ca²⁺ signaling system and the mechanisms of Ca²⁺ membrane transport and homeostasis are described. Special attention is paid to the role of Ca²⁺ cation channels, Ca²⁺ transporters, and Ca²⁺-binding proteins in the regulation of the intercellular Ca²⁺ concentration.     

Altered Ca2+ signaling in enamelopathies

Biomineralization requires the controlled movement of ions across cell barriers to reach the sites of crystal growth. Mineral precipitation occurs in aqueous phases as fluids become supersaturated with specific ionic compositions. In the biological world, biomineralization is dominated by the presence of calcium (Ca²⁺) in crystal lattices. Ca²⁺ channels are intrinsic modulators of this process, facilitating the availability of Ca²⁺ within cells in a tightly regulated manner in time and space. Unequivocally, the most mineralized tissue produced by vertebrates, past and present, is dental enamel. With some of the longest carbonated hydroxyapatite (Hap) crystals known, dental enamel formation is fully coordinated by specialized epithelial cells of ectodermal origin known as ameloblasts. These cells form enamel in two main developmental stages: a) secretory; and b) maturation. The secretory stage is marked by volumetric growth of the tissue with limited mineralization, and the opposite is found in the maturation stage, as enamel crystals expand in width concomitant with increased ion transport. Disruptions in the formation and/or mineralization stages result, in most cases, in permanent alterations in the crystal assembly. This introduces weaknesses in the material properties affecting enamel's hardness and durability, thus limiting its efficacy as a biting, chewing tool and increasing the possibility of pathology. Here, we briefly review enamel development and discuss key properties of ameloblasts and their Ca²⁺-handling machinery, and how alterations in this toolkit result in enamelopathies.     

Timing in cellular Ca2+ signaling

Calcium (Ca2+) signals are generated across a broad time range. Kinetic considerations impact how information is processed to encode and decode Ca2+ signals, the choreography of responses that ensure specific and efficient signaling and the overall temporal amplification such that ephemeral Ca2+ signals have lasting physiological value. The reciprocal importance of timing for Ca2+ signaling, and Ca2+ signaling for timing is exemplified by the altered kinetic profiles of Ca2+ signals in certain diseases and the likely role of basal Ca2+ fluctuations in the perception of time itself.     

Mitochondrial dynamics and Ca2+ signaling

Recent data shed light on two novel aspects of the mitochondria-Ca2+ liaison. First, it was extensively investigated how Ca2+ handling is controlled by mitochondrial shape, and positioning; a playground also of cell death and survival regulation. On the other hand, significant progress has been made to explore how intra- and near-mitochondrial Ca2+ signals modify mitochondrial morphology and cellular distribution. Here, we shortly summarize these advances and provide a model of Ca2+-mitochondria interactions.     

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.     

Ca2+ signaling and spinocerebellar ataxia

Spinocerebellar ataxia (SCA) is a neural disorder, which is caused by degenerative changes in the cerebellum. SCA is primarily characterized by gait ataxia, and additional clinical features include nystagmus, dysarthria, tremors and cerebellar atrophy. Forty-four hereditary SCAs have been identified to date, along with >35 SCA-associated genes. Despite the great diversity and distinct functionalities of the SCA-related genes, accumulating evidence supports the occurrence of a common pathophysiological event among several hereditary SCAs. Altered calcium (Ca²⁺) homeostasis in the Purkinje cells (PCs) of the cerebellum has been proposed as a possible pathological SCA trigger. In support of this, signaling events that are initiated from or lead to aberrant Ca²⁺ release from the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1), which is highly expressed in cerebellar PCs, seem to be closely associated with the pathogenesis of several SCA types. In this review, we summarize the current research on pathological hereditary SCA events, which involve altered Ca²⁺ homeostasis in PCs, through IP₃R1 signaling.     

TRP channels and Ca2+ signaling

There is a rapidly growing interest in the family of transient receptor potential (TRP) channels because TRP channels are not only important for many sensory systems, but they are crucial components of the function of neurons, epithelial, blood and smooth muscle cells. These facts make TRP channels important targets for treatment of diseases arising from the malfunction of these channels in the above cells and for treatment of inflammatory pain. TRP channels are also important for a growing number of genetic diseases arising from mutations in various types of TRP channels. The Minerva-Gentner Symposium on TRP channels and Ca(2+) signaling, which took place in Eilat, Israel (February 24-28, 2006) has clearly demonstrated that the study of TRP channels is a newly emerging field of biomedicine with prime importance. In the Eilat symposium, investigators who have contributed seminal publications and insight into the TRP field presented their most recent, and in many cases still unpublished, studies. The excellent presentations and excitement generated by them demonstrated that much progress has been achieved. Nevertheless, it was also evident that the field of TRP channels is still in its infancy in comparison to other fields of ion channels, and even the fundamental knowledge of the gating mechanism of TRP channels is still unsolved. The beautiful location of the symposium, together with informal intensive discussions among the participants, contributed to the success of this meeting.     

Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.     

Multiple kinetic components and the Ca2+ requirements of exocytosis

The use of caged-Ca2+ compounds to stimulate Ca(2+)-dependent exocytosis has substantially increased our understanding of this complex process. By this approach, the existence of multiple kinetic components of exocytosis has been established. These components may correspond to a series of sequential steps that lead to a single fusion-ready state (sequential mechanism) or, alternatively, to heterogeneity in secretory vesicles or in fusion-ready states (parallel mechanism). It is suggested that both of these mechanisms can underlie exocytosis of a single type of vesicle (mixed sequential-parallel mechanism). Studies with caged-Ca2+ compounds have also indicated that the Ca2+ requirement for exocytosis is substantially greater than that suggested by conventional methodologies. This discrepancy is mainly attributable to the underestimation, by imaging studies with high-affinity Ca2+ indicators (due to dye saturation), of the local increases in cytosolic Ca2+ concentration that trigger the exocytosis of individual vesicles. The effects of local saturation of such indicators are explored by means of a simple theory.     

Ca2+ release via ryanodine receptors and Ca2+ entry: major mechanisms in NAADP-mediated Ca2+ signaling in T-lymphocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing nucleotide essentially involved in T cell activation. Using combined microinjection and single cell calcium imaging, we demonstrate that co-injection of NAADP and the D-myo-inositol 1,4,5-trisphosphate antagonist heparin did not inhibit Ca2+ mobilization. In contrast, co-injection of the ryanodine receptor antagonist ruthenium red efficiently blocked NAADP induced Ca2+ signalling. This pharmacological approach was confirmed using T cell clones stably transfected with plasmids expressing antisense mRNA targeted specifically against ryanodine receptors. NAADP induced Ca2+ signaling was strongly reduced in these clones. In addition, inhibition of Ca2+ entry by SK&F 96365 resulted in a dramatically decreased Ca2+ signal upon NAADP injection. Gd3+, a known blocker of Ca2+ release activated Ca2+ entry, only partially inhibited NAADP mediated Ca2+ signaling. These data indicate that in T cells (i) ryanodine receptor are the major intracellular Ca2+ release channels involved in NAADP induced Ca2+ signals, and that (ii) such Ca2+ release events are largely amplified by Ca2+ entry.     

Cytosolic Ca2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary

We have monitored cytosolic [Ca2+] with fura-2 and exocytosis by measuring the membrane capacitance, and we have studied the influence of cytosolic [Ca2+] on secretion in single endocrine cells. As in neurons, cytosolic Ca2+ is sufficient to trigger exocytosis. The rate of secretion grows with the fourth or fifth power of cytosolic [Ca2+], and paired stimuli reveal facilitation. Ca2+ influx through voltage-sensitive Ca2+ channels can stimulate secretion 1000-fold over the basal levels measured biochemically. Unlike neurons, however, melanotrophs continue to secrete for seconds afer a depolarizing pulse, while they extrude or sequester the Ca2+ that has entered through Ca2+ channels. Following episodes of secretion, pituitary cells can retrieve membrane with half-times around 30 s at 32 degrees C, even in the absence of cytosolic K+.     

Supralinear Ca2+ signaling by cooperative and mobile Ca2+ buffering in Purkinje neurons

Endogenous high-affinity Ca2+ buffering and its roles were investigated in mouse cerebellar Purkinje cells with the use of a low-affinity Ca2+ indicator and a high-affinity caged Ca2+ compound. Increases in the cytosolic Ca2+ concentration ([Ca2+]i) were markedly facilitated during repetitive depolarization, resulting in the generation of steep micromolar Ca2+ gradients along dendrites. Such supralinear Ca2+ responses were attributed to the saturation of a large concentration (0.36 mM) of a mobile, high-affinity (dissociation constant, 0.37 microM) Ca2+ buffer with cooperative Ca2+ binding sites, resembling calbindin-D28K, and to an immobile, low-affinity Ca2+ buffer. These data suggest that the high-affinity Ca2+ buffer operates as the neuronal computational element that enables efficient coincidence detection of the Ca2+ signal and that facilitates spatiotemporal integration of the Ca2+ signal at submicromolar [Ca2+]i.     

Akt and Ca2+ signaling in endothelial cells

The protein kinase Akt participates in such important functions of endothelial cells as nitric oxide production and angiogenesis, activities that involve changes in cytosolic Ca2+ concentration. However, it is not known if activation of Akt is itself involved in the regulation of Ca2+ signals produced in these cells. The objective of this study was to examine if Akt is involved in the regulation of Ca2+ signaling in endothelial cells. Agonist-stimulated Ca2+ signals, assessed using fura-2, were compared in porcine aortic endothelial cells under control conditions or conditions in which Akt was blocked either by different inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt or by transient expression of a dominant-negative form of Akt (dnAkt). We found that the release of intracellular Ca2+ stores stimulated by bradykinin or thapsigargin is not affected by the PI3 kinase inhibitors LY294002 and wortmannin, or by expression of dnAkt. LY294002 dose-dependently inhibits store-operated Ca2+ entry, an effect not seen with wortmannin. Expression of dnAkt has no effect on store-operated Ca2+ entry. We conclude that Akt is not involved in the regulation of agonist-stimulated Ca2+ signals in endothelial cells. The compound LY294002 inhibits store-operated Ca2+ entry in these cells by a mechanism independent of PI3 kinase/Akt inhibition.     

Synaptogyrins regulate Ca2+-dependent exocytosis in PC12 cells.

Synaptogyrins constitute a family of synaptic vesicle proteins of unknown function. With the full-length structure of a new brain synaptogyrin isoform, we now show that the synaptogyrin family in vertebrates includes two neuronal and one ubiquitous isoform. All of these synaptogyrins are composed of a short conserved N-terminal cytoplasmic sequence, four homologous transmembrane regions, and a variable cytoplasmic C-terminal tail that is tyrosine-phosphorylated. The localization, abundance, and conservation of synaptogyrins suggest a function in exocytosis. To test this, we employed a secretion assay in PC12 cells expressing transfected human growth hormone (hGH) as a reporter protein. When Ca2+-dependent hGH secretion from PC12 cells was triggered by high K+ or alpha-latrotoxin, co-transfection of all synaptogyrins with hGH inhibited hGH exocytosis as strongly as co-transfection of tetanus toxin light chain. Synaptophysin I, which is distantly related to synaptogyrins, was also inhibitory but less active. Inhibition was independent of the amount of hGH expressed but correlated with the amount of synaptogyrin transfected. Inhibition of exocytosis was not observed with several other synaptic proteins, suggesting specificity. Analysis of the regions of synaptogyrin required for inhibition revealed that the conserved N-terminal domain of synaptogyrin is essential for inhibition, whereas the long C-terminal cytoplasmic tail is largely dispensable. Our results suggest that synaptogyrins are conserved components of the exocytotic apparatus, which function as regulators of Ca2+-dependent exocytosis.