Cytosolic Ca2+ buffers

"Ca(2+) buffers," a class of cytosolic Ca(2+)-binding proteins, act as modulators of short-lived intracellular Ca(2+) signals; they affect both the temporal and spatial aspects of these transient increases in [Ca(2+)](i). Examples of Ca(2+) buffers include parvalbumins (α and β isoforms), calbindin-D9k, calbindin-D28k, and calretinin. Besides their proven Ca(2+) buffer function, some might additionally have Ca(2+) sensor functions. Ca(2+) buffers have to be viewed as one of the components implicated in the precise regulation of Ca(2+) signaling and Ca(2+) homeostasis. Each cell is equipped with proteins, including Ca(2+) channels, transporters, and pumps that, together with the Ca(2+) buffers, shape the intracellular Ca(2+) signals. All of these molecules are not only functionally coupled, but their expression is likely to be regulated in a Ca(2+)-dependent manner to maintain normal Ca(2+) signaling, even in the absence or malfunctioning of one of the components.     

In silico analysis of expression data for identification of genes involved in spatial accumulation of calcium in developing seeds of rice

The calcium (Ca(2+)) transporters, like Ca(2+) channels, Ca(2+) ATPases, and Ca(2+) exchangers, are instrumental for signaling and transport. However, the mechanism by which they orchestrate the accumulation of Ca(2+) in grain filling has not yet been investigated. Hence the present study was designed to identify the potential calcium transporter genes that may be responsible for the spatial accumulation of calcium during grain filling. In silico expression analyses were performed to identify Ca(2+) transporters that predominantly express during the different developmental stages of Oryza sativa. A total of 13 unique calcium transporters (7 from massively parallel signature sequencing [MPSS] data analysis, and 9 from microarray analysis) were identified. Analysis of variance (ANOVA) revealed differential expression of the transporters across tissues, and principal component analysis (PCA) exhibited their seed-specific distinctive expression profile. Interestingly, Ca(2+) exchanger genes are highly expressed in the initial stages, whereas some Ca(2+) ATPase genes are highly expressed throughout seed development. Furthermore, analysis of the cis-elements located in the promoter region of the subset of 13 genes suggested that D of proteins play essential roles in regulating the expression of Ca(2+) transporter genes during rice seed development. Based on these results, we developed a hypothetical model explaining the transport and tissue specific distribution of calcium in developing cereal seeds. The model may be extrapolated to understand the mechanism behind the exceptionally high level of calcium accumulation seen in grains like finger millet.     

Pharmacological modulation of ion channels and transporters

Ion channels and transporter proteins are prerequisites for formation and conduction of cardiac electrical impulses. Acting in concert, these proteins maintain cellular Na(+) and Ca(2+) homeostasis. Since intracellular Ca(2+) concentration determines contractile activation, we expect the majority of agents that modulate activity of ion channels and transporters not only to influence cellular action potentials but also contractile force. Drugs which block ion channels usually possess antiarrhythmic properties, those inhibiting the Na(+) pump have predominantly inotropic effects and those affecting Na(+),Ca(2+)- or Na(+),H(+)-exchanger protect against ischaemic cell damage. However, irrespective of their primary indication, all compounds targeted against ion channels and transporter proteins possess potential proarrhythmic activity.     

Evolutionary origins of STIM1 and STIM2 within ancient Ca2+ signaling systems

Human stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca(2+) signaling systems that include numerous plasma membrane (PM), endoplasmic reticulum (ER), and mitochondrial Ca(2+) transporters, channels and regulators. STIM2 and STIM1 function as Ca(2+) sensors with different sensitivities for ER Ca(2+). They translocate to ER-PM junctions and open PM Orai Ca(2+) influx channels when receptor-mediated Ca(2+) release lowers ER Ca(2+) levels. The resulting increase in cytosolic Ca(2+) leads to the activation of numerous Ca(2+) effector proteins that in turn regulate differentiation, cell contraction, secretion and other cell functions. In this review, we use an evolutionary perspective to survey molecular activation mechanisms in the Ca(2+) signaling system, with a particular focus on regulatory motifs and functions of the two STIM proteins. We discuss the presence and absence of STIM genes in different species, the order of appearance of STIM versus Orai, and the evolutionary addition of new signaling domains to STIM proteins.     

Simulating calcium influx and free calcium concentrations in yeast

Yeast can proliferate in environments containing very high Ca(2+) primarily due to the activity of vacuolar Ca(2+) transporters Pmc1 and Vcx1. Yeast mutants lacking these transporters fail to grow in high Ca(2+) environments, but growth can be restored by small increases in environmental Mg(2+). Low extracellular Mg(2+) appeared to competitively inhibit novel Ca(2+) influx pathways and to diminish the concentration of free Ca(2+) in the cytoplasm, as judged from the luminescence of the photoprotein aequorin. These Mg(2+)-sensitive Ca(2+) influx pathways persisted in yvc1 cch1 double mutants. Based on mathematical models of the aequorin luminescence traces, we propose the existence in yeast of at least two Ca(2+) transporters that undergo rapid feedback inhibition in response to elevated cytosolic free Ca(2+) concentration. Finally, we show that Vcx1 helps return cytosolic Ca(2+) toward resting levels after shock with high extracellular Ca(2+) much more effectively than Pmc1 and that calcineurin, a protein phosphatase regulator of Vcx1 and Pmc1, had no detectable effects on these factors within the first few minutes of its activation. Therefore, computational modeling of Ca(2+) transport and signaling in yeast can provide important insights into the dynamics of this complex system.     

The ins and outs of cellular Ca(2+) transport

The cytoplasmic Ca(2+) signals that participate in nearly all aspects of plant growth and development encode information as binary switches or information-rich signatures. They are the result of influx (thermodynamically passive) and efflux (thermodynamically active) activities mediated by membrane transport proteins. On the influx side, confirming the molecular identities of Ca(2+)-permeable channels is still a major research topic. Cyclic nucleotide-gated channels and glutamate receptor-like channels are candidates well supported by evidence. On the efflux side, CAX antiporters and P-type ATPase pumps are the principal molecular entities. Both of these active transporters load Ca(2+) into specific compartments and have the potential to reduce the magnitude and duration of a Ca(2+) transient. Recent studies indicate calmodulin-activated Ca(2+) pumps in endomembrane systems can dampen the magnitude and duration of a Ca(2+) transient that could otherwise grow into a Ca(2+) cell death signature. An important challenge following molecular characterization of the influx and efflux pathways is to understand how they are coordinately regulated to produce a Ca(2+) switch or encode specific information into a Ca(2+) signature.     

Abnormal intracellular ca(2+)homeostasis and disease

A whole range of cell functions are regulated by the free cytosolic Ca(2+)concentration. Activator Ca(2+)from the extracellular space enters the cell through various types of Ca(2+)channels and sometimes the Na(+)/Ca(2+)-exchanger, and is actively extruded from the cell by Ca(2+)pumps and Na(+)/Ca(2+)-exchangers. Activator Ca(2+)can also be released from internal Ca(2+)stores through inositol trisphosphate or ryanodine receptors and is taken up into these organelles by means of Ca(2+)pumps. The resulting Ca(2+)signal is highly organized in space, frequency and amplitude because the localization and the integrated free cytosolic Ca(2+)concentration over time contain specific information. Mutations or functional abnormalities in the various Ca(2+)transporters, which in vitro seem to induce trivial functional alterations, therefore, often lead to a plethora of diseases. Skeletal-muscle pathology can be caused by mutations in ryanodine receptors (malignant hyperthermia, porcine stress syndrome, central-core disease), dihydropyridine receptors (familial hypokalemic periodic paralysis, malignant hyperthermia, muscular dysgenesis) or Ca(2+)pumps (Brody disease). Ca(2+)-pump mutations in cutaneous epidermal keratinocytes and cochlear hair cells lead to, skin diseases (Darier and Hailey-Hailey) and hearing/vestibular problems respectively. Mutated Ca(2+)channels in the photoreceptor plasma membrane cause vision problems. Hemiplegic migraine, spinocerebellar ataxia type-6, one form of episodic ataxia and some forms of epilepsy can be due to mutations in plasma-membrane Ca(2+)channels, while antibodies against these channels play a pathogenic role in all patients with the Lambert-Eaton myasthenic syndrome and may be of significance in sporadic amyotrophic lateral sclerosis. Brain inositol trisphosphate receptors have been hypothesized to contribute to the pathology in opisthotonos mice, manic-depressive illness and perhaps Alzheimer's disease. Various abnormalities in Ca(2+)-handling proteins have been described in heart during aging, hypertrophy, heart failure and during treatment with immunosuppressive drugs and in diabetes mellitus. In some instances, disease-causing mutations or abnormalities provide us with new insights into the cell biology of the various Ca(2+)transporters.     

Molecular identity and functional properties of the mitochondrial na+/ca2+ exchanger

The mitochondrial membrane potential that powers the generation of ATP also facilitates mitochondrial Ca(2+) shuttling. This process is fundamental to a wide range of cellular activities, as it regulates ATP production, shapes cytosolic and endoplasmic recticulum Ca(2+) signaling, and determines cell fate. Mitochondrial Ca(2+) transport is mediated primarily by two major transporters: a Ca(2+) uniporter that mediates Ca(2+) uptake and a Na(+)/Ca(2+) exchanger that subsequently extrudes mitochondrial Ca(2+). In this minireview, we focus on the specific role of the mitochondrial Na(+)/Ca(2+) exchanger and describe its ion exchange mechanism, regulation by ions, and putative partner proteins. We discuss the recent molecular identification of the mitochondrial exchanger and how its activity is linked to physiological and pathophysiological processes.     

Genome-wide comparative in silico analysis of calcium transporters of rice and sorghum

The mechanism of calcium uptake, translocation and accumulation in Poaceae has not yet been fully understood. To address this issue, we conducted genome-wide comparative in silico analysis of the calcium (Ca(2+)) transporter gene family of two crop species, rice and sorghum. Gene annotation, identification of upstream cis-acting elements, phylogenetic tree construction and syntenic mapping of the gene family were performed using several bioinformatics tools. A total of 31 Ca(2+) transporters, distributed on 9 out of 12 chromosomes, were predicted from rice genome, while 28 Ca(2+) transporters predicted from sorghum are distributed on all the chromosomes except chromosome 10 (Chr 10). Interestingly, most of the genes on Chr 1 and Chr 3 show an inverse syntenic relationship between rice and sorghum. Multiple sequence alignment and motif analysis of these transporter proteins revealed high conservation between the two species. Phylogenetic tree could very well identify the subclasses of channels, ATPases and exchangers among the gene family. The in silico cis-regulatory element analysis suggested diverse functions associated with light, stress and hormone responsiveness as well as endosperm- and meristem-specific gene expression. Further experiments are warranted to validate the in silico analysis of the predicted transporter gene family and elucidate the functions of Ca(2+) transporters in various biological processes.     

Manganese specificity determinants in the Arabidopsis metal/H+ antiporter CAX2

In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol. Metal/H(+) antiporters are involved in metal sequestration into the vacuole. However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood. The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca(2+) into the vacuole. There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn(2+) transport when expressed in yeast. To determine the domains within CAX2 that mediate Mn(2+) specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter. One class displayed no alterations in Mn(2+) or Ca(2+) transport, the second class showed a reduction in Ca(2+) transport and no measurable Mn(2+) transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca(2+) transport and a complete loss of Mn(2+) transport. The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn(2+) specificity of CAX2. This study provides evidence for the feasibility of altering substrate specificity in a metal/H(+) antiporter, an important family of transporters found in a variety of organisms.     

Role of regulator of G protein signaling 2 (RGS2) in Ca(2+) oscillations and adaptation of Ca(2+) signaling to reduce excitability of RGS2-/- cells

Regulators of G protein signaling (RGS) proteins accelerate the GTPase activity of Galpha subunits to determine the duration of the stimulated state and control G protein-coupled receptor-mediated cell signaling. RGS2 is an RGS protein that shows preference toward Galpha(q).To better understand the role of RGS2 in Ca(2+) signaling and Ca(2+) oscillations, we characterized Ca(2+) signaling in cells derived from RGS2(-/-) mice. Deletion of RGS2 modified the kinetic of inositol 1,4,5-trisphosphate (IP(3)) production without affecting the peak level of IP(3), but rather increased the steady-state level of IP(3) at all agonist concentrations. The increased steady-state level of IP(3) led to an increased frequency of [Ca(2+)](i) oscillations. The cells were adapted to deletion of RGS2 by reducing Ca(2+) signaling excitability. Reduced excitability was achieved by adaptation of all transporters to reduce Ca(2+) influx into the cytosol. Thus, IP(3) receptor 1 was down-regulated and IP(3) receptor 3 was up-regulated in RGS2(-/-) cells to reduce the sensitivity for IP(3) to release Ca(2+) from the endoplasmic reticulum to the cytosol. Sarco/endoplasmic reticulum Ca(2+) ATPase 2b was up-regulated to more rapidly remove Ca(2+) from the cytosol of RGS2(-/-) cells. Agonist-stimulated Ca(2+) influx was reduced, and Ca(2+) efflux by plasma membrane Ca(2+) was up-regulated in RGS2(-/-) cells. The result of these adaptive mechanisms was the reduced excitability of Ca(2+) signaling, as reflected by the markedly reduced response of RGS2(-/-) cells to changes in the endoplasmic reticulum Ca(2+) load and to an increase in extracellular Ca(2+). These findings highlight the central role of RGS proteins in [Ca(2+)](i) oscillations and reveal a prominent plasticity and adaptability of the Ca(2+) signaling apparatus.     

The grateful dead: calcium and cell death in plant innate immunity

Plant cells sensing pathogenic microorganisms evoke defence systems that can confer resistance to infection. This innate immune reaction can include triggering of basal defence responses as well as programmed cell death, or hypersensitive response (HR). In both cases (basal defence and HR), pathogen perception is translated into elevated cytosolic Ca(2+) (mediated by plasma membrane and intracellular channels) as an early step in a signalling cascade. Cyclic nucleotide-gated channels contribute to this influx of Ca(2+) into the cell. The molecular nature of other transport proteins contributing to the Ca(2+) elevation is unclear. Pathogen recognition occurs at two levels: the perception of pathogen-associated molecular pattern (PAMP) molecules widely present in microorganisms, and an interaction between pathogen avirulence gene products (if present) and corresponding plant R (resistance) gene products. The Ca(2+) elevation occurring in response to PAMP perception or R gene interactions could occur due to phosphorylation events, G-protein signalling and/or an increase in cyclic nucleotides. Downstream from the initial Ca(2+) rise, the signalling cascade includes: activation of calmodulin and protein kinases, and nitric oxide and reactive oxygen species generation. Some of these downstream events amplify the Ca(2+) signal by further activation of Ca(2+) transporters.     

The protein kinase SOS2 activates the Arabidopsis H(+)/Ca(2+) antiporter CAX1 to integrate calcium transport and salt tolerance

The regulation of ions within cells is an indispensable component of growth and adaptation. The plant SOS2 protein kinase and its associated Ca(2+) sensor, SOS3, have been demonstrated to modulate the plasma membrane H(+)/Na(+) antiporter SOS1; however, how these regulators modulate Ca(2+) levels within cells is poorly understood. Here we demonstrate that SOS2 regulates the vacuolar H(+)/Ca(2+) antiporter CAX1. Using a yeast growth assay, co-expression of SOS2 specifically activated CAX1, whereas SOS3 did not. CAX1-like chimeric transporters were activated by SOS2 if the chimeric proteins contained the N terminus of CAX1. Vacuolar membranes from CAX1-expressing cells were made to be H(+)/Ca(2+)-competent by the addition of SOS2 protein in a dose-dependent manner. Using a yeast two-hybrid assay, SOS2 interacted with the N terminus of CAX1. In each of these yeast assays, the activation of CAX1 by SOS2 was SOS3-independent. In planta, the high level of expression of a deregulated version of CAX1 caused salt sensitivity. These findings suggest multiple functions for SOS2 and provide a mechanistic link between Ca(2+) and Na(+) homeostasis in plants.     

Cyclic ADP ribose is a novel regulator of intracellular Ca2+ oscillations in human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine. However, the cellular biology of these cells is not fully understood. The present study characterizes the cyclic ADP-ribose (cADPR)-mediated Ca(2+) signals in human MSCs and finds that externally applied cADPR can increase the frequency of spontaneous intracellular Ca(2+) (Ca(2+) (i) ) oscillations. The increase was abrogated by a specific cADPR antagonist or an inositol trisphosphate receptor (IP3R) inhibitor, but not by ryanodine. In addition, the cADPR-induced increase of Ca(2+) (i) oscillation frequency was prevented by inhibitors of nucleoside transporter or by inhibitors of the transient receptor potential cation melastatin-2 (TRPM2) channel. RT-PCR revealed mRNAs for the nucleoside transporters, concentrative nucleoside transporters 1/2 and equilibrative nucleoside transporters 1/3, IP3R1/2/3 and the TRPM2 channel, but not those for ryanodine receptors and CD38 in human MSCs. Knockdown of the TRPM2 channel by specific short interference RNA abolished the effect of cADPR on the Ca(2+) (i) oscillation frequency, and prevented the stimulation of proliferation by cADPR. Moreover, cADPR remarkably increased phosphorylated extracellular-signal-regulated kinases 1/2 (ERK1/2), but not Akt or p38 mitogen-activated protein kinase (MAPK). However, cADPR had no effect on adipogenesis or osteogenesis in human MSCs. Our results indicate that cADPR is a novel regulator of Ca(2+) (i) oscillations in human MSCs. It permeates the cell membrane through the nucleoside transporters and increases Ca(2+) oscillation via activation of the TRPM2 channel, resulting in enhanced phosphorylation of ERK1/2 and, thereby, stimulation of human MSC proliferation. This study delineates an alternate signalling pathway of cADPR that is distinct from its well-established role of serving as a Ca(2+) messenger for mobilizing the internal Ca(2+) stores. Whether cADPR can be used clinically for stimulating marrow function in patients with marrow disorders remains to be further studied.     

Responses of Ca2+-binding proteins to localized,transient changes in intracellular [Ca2+

In smooth muscle cells, various transient, localized [Ca(2+)] changes have been observed that are thought to regulate cell function without necessarily inducing contraction. Although a great deal of effort has been put into detecting these transients and elucidating the mechanisms involved in their generation, the extent to which these transient Ca(2+) signals interact with intracellular Ca(2+)-binding molecules remains relatively unknown. To understand how the spatial and temporal characteristics of an intracellular Ca(2+) signal influence its interaction with Ca(2+)-binding proteins, mathematical models of Ca(2+) diffusion and regulation in smooth muscle cells were used to study Ca(2+) binding to prototypical proteins with one or two Ca(2+)-binding sites. Simulations with the models: (1) demonstrate the extent to which the rate constants for Ca(2+)-binding to proteins and the spatial and temporal characteristics of different Ca(2+) transients influence the magnitude and time course of the responses of these proteins to the transients; (2) predict significant differences in the responses of proteins with one or two Ca(2+)-binding sites to individual Ca(2+) transients and to trains of transients; (3) demonstrate how the kinetic characteristics determine the fidelity with which the responses of Ca(2+)-sensitive molecules reflect the magnitude and time course of transient Ca(2+) signals. Overall, this work demonstrates the clear need for complete information about the kinetics of Ca(2+) binding for determining how well Ca(2+)-binding molecules respond to different types of Ca(2+) signals. These results have important implications when considering the possible modulation of Ca(2+)- and Ca(2+)/calmodulin-dependent proteins by localized intracellular Ca(2+) transients in smooth muscle cells and, more generally, in other cell types.     

Calcium signalling and cell-fate choice in B cells

Alterations in the cytosolic concentration of calcium ions (Ca2+) transmit information that is crucial for the development and function of B cells. Cytosolic Ca2+ concentration is determined by a balance of active transport and gradient-driven Ca2+ fluxes, both of which are subject to the influence of multiple receptors and environmental sensing pathways. Recent advances in genomics have allowed for the compilation of an increasingly comprehensive list of Ca2+ transporters and channels expressed by B cells. The increasing understanding of the function and regulation of these proteins has begun to shift the frontier of Ca2+ physiology in B cells from molecular analysis to determining how diverse inputs to cytosolic Ca2+ concentration are integrated in specific immunological contexts.     

Structural determinants of Ca2+ transport in the Arabidopsis H+/Ca2+ antiporter CAX1

Ca(2+) levels in plants, fungi, and bacteria are controlled in part by H(+)/Ca(2+) exchangers; however, the relationship between primary sequence and biological activity of these transporters has not been reported. The Arabidopsis H(+)/cation exchangers, CAX1 and CAX2, were identified by their ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. CAX1 has a much higher capacity for Ca(2+) transport than CAX2. An Arabidopsis thaliana homolog of CAX1, CAX3, is 77% identical (93% similar) and, when expressed in yeast, localized to the vacuole but did not suppress yeast mutants defective in vacuolar Ca(2+) transport. Chimeric constructs and site-directed mutagenesis showed that CAX3 could suppress yeast vacuolar Ca(2+) transport mutants if a nine-amino acid region of CAX1 was inserted into CAX3 (CAX3-9). Biochemical analysis in yeast showed CAX3-9 had 36% of the H(+)/Ca(2+) exchange activity as compared with CAX1; however, CAX3-9 and CAX1 appear to differ in their transport of other ions. Exchanging the nine-amino acid region of CAX1 into CAX2 doubled yeast vacuolar Ca(2+) transport but did not appear to alter the transport of other ions. This nine-amino acid region is highly variable among the plant CAX-like transporters. These findings suggest that this region is involved in CAX-mediated Ca(2+) specificity.