Plant and animal type 2B Ca2+-ATPases: evidence for a common auto-inhibitory mechanism

Plant auto-inhibited Ca²⁺-ATPase 8 (ACA8) and animal plasma membrane Ca²⁺-ATPase 4b (PMCA4b) are representatives of plant and animal 2B P-type ATPases with a regulatory auto-inhibitory domain localized at the N- and C-terminus, respectively. To check whether the regulatory domain works independently of its terminal localization and if auto-inhibitory domains of different organisms are interchangeable, a mutant in which the N-terminus of ACA8 is repositioned at the C-terminus and chimeras in which PMCA4b C-terminus is fused to the N- or C-terminus of ACA8 were analysed in the yeast mutant K616 devoid of endogenous Ca²⁺-ATPases. Results show that the regulatory function of the terminal domain is independent from its position in ACA8 and that the regulatory domain belonging to PMCA4b is able to at least partially auto-inhibit ACA8.     

The plasma membrane-localised Ca2+-ATPase ACA8 plays a role in sucrose signalling involved in early seedling development in Arabidopsis

Key message

Arabidopsis Ca ²⁺ -ATPase ACA8 plays a role in sucrose signalling during early seedling development by integrating developmental signals with carbon source availability.

Abstract

Calcium (Ca²⁺) is an essential signal transduction element in eukaryotic organisms. Changes in the levels of intracellular Ca²⁺ affect multiple developmental processes in plants, including cell division, polar growth, and organogenesis. Here, we report that the plasma-membrane-localised Arabidopsis Ca²⁺-ATPase ACA8 plays a role in sucrose signalling during early seedling development. Disruption of the ACA8 gene elevated the expression of genes that encode transporters for Ca²⁺ efflux. The seedlings that carried a T-DNA insertion mutation in ACA8 experienced water stress during early development. This response was unrelated to inadequate osmoregulatory responses and was most likely caused by disruption of cell membrane integrity and severe ion leakage. In addition, aca8-1 seedlings displayed a significant decline in photosynthetic performance and arrested root growth after removal of sucrose from the growth medium. The two phenomena resulted from impaired photosynthesis, reduced cell proliferation in the root meristem and the sucrose control of cell-cycle events. All of the stress-response phenotypes were rescued when expression of ACA8 was restored in aca8-1 mutant. Taken together, our results indicate that ACA8-mediated Ca²⁺ signalling contributes to modulate early seedling development and coordinates root development with nutrient availability.     

Effects of differentiation on purinergic and neurotensin-mediated calcium signaling in human HT-29 colon cancer cells

Calcium signaling is a key regulator of processes important in differentiation. In colon cancer cells differentiation is associated with altered expression of specific isoforms of calcium pumps of the endoplasmic reticulum and the plasma membrane, suggesting that differentiation of colon cancer cells is associated with a major remodeling of calcium homeostasis. Purinergic and neurotensin receptor activation are known regulators of cytosolic free Ca²⁺ levels in colon cancer cells. This study aimed to assess changes in cytosolic free Ca²⁺ levels in response to ATP and neurotensin with differentiation induced by sodium butyrate or culturing post-confluence. Parameters assessed included peak cytosolic free Ca²⁺ level after activation; time to reach peak cytosolic free Ca²⁺ and the EC₅₀ of dose response curves. Our results demonstrate that differentiation of HT-29 colon cancer cells is associated with a remodeling of both ATP and neurotensin mediated Ca²⁺ signaling. Neurotensin-mediated calcium signaling appeared more sensitive to differentiation than ATP-mediated Ca²⁺ signaling.     

EGF Stimulates Growth by Enhancing Capacitative Calcium Entry in Corneal Epithelial Cells

In rabbit corneal epithelial cells (RCEC), we determined whether capacitative calcium entry (CCE) mediates the mitogenic response to epidermal growth factor, EGF. [Ca²⁺]_i was measured with single-cell fluorescence imaging of fura2-loaded RCEC. EGF (5 ng/ml) maximally increased [Ca²⁺]_i 4.4-fold. Following intracellular store (ICS) calcium depletion in calcium-free medium with 10 µM cyclopiazonic acid (CPA) (endoplasmic reticulum calcium ATPase inhibitor), calcium addback elicited plasma membrane Ca²⁺ influx as a result of activation of plasma membrane store operated channel (SOC) activity. Based on Mn²⁺ quench measurements of fura2 fluorescence, 5 ng/ml EGF enhanced such influx 2.3-fold, whereas with Rp-cAMPS (protein kinase A inhibitor) plus EGF it increased by 5.3-fold. In contrast, SOC activation was blocked with 100 µM 2-aminoethyldiphenylborate (2-APB, store-operated channel inhibitor). During exposure to either 50 µM UO126 (MEK-1/2 inhibitor) or 10 µM forskolin (adenylate cyclase activator), 5 ng/ml EGF failed to affect [Ca²⁺]_i. RT-PCR detected gene expression of: 1) transient receptor potential (TRP) protein isoforms 1, 3, 4, 6 and 7; 2) IP₃R isoforms 1–3. Immunocytochemistry, in conjunction with confocal and immunogold electron microscopy, detected plasma membrane localization of TRP4 expression. Inhibition of CCE with 2-APB and/or CPA, eliminated the 2.5-fold increase in intracellular [³H]-thymidine incorporation induced by EGF. Taken together, CCE in RCEC mediates the mitogenic response to EGF. EGF induces CCE through its stimulation of Erk1/2 activity, whereas PKA stimulation suppresses these effects of EGF. TRP4 may be a component of plasma membrane SOC activity, which is stimulated by ICS calcium depletion.     

Functional analysis of coiled-coil domains of MCU in mitochondrial calcium uptake

The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca²⁺-uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca²⁺-uptake function. Thus, it is essential for the Ca²⁺ uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca²⁺-uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU.     

Channel Formation by Yeast F-ATP Synthase and the Role of Dimerization in the Mitochondrial Permeability Transition

Purified F-ATP synthase dimers of yeast mitochondria display Ca²⁺-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca²⁺ ionophore ETH129, which allows electrophoretic Ca²⁺ uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains ΔTIM11 and ΔATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ.     

Arabidopsis Ca2+-ATPases 1, 2, and 7 in the endoplasmic reticulum contribute to growth and pollen fitness

Generating cellular Ca²⁺ signals requires coordinated transport activities from both Ca²⁺ influx and efflux pathways. In Arabidopsis (Arabidopsis thaliana), multiple efflux pathways exist, some of which involve Ca²⁺-pumps belonging to the Autoinhibited Ca²⁺-ATPase (ACA) family. Here, we show that ACA1, 2, and 7 localize to the endoplasmic reticulum (ER) and are important for plant growth and pollen fertility. While phenotypes for plants harboring single-gene knockouts (KOs) were weak or undetected, a triple KO of aca1/2/7 displayed a 2.6-fold decrease in pollen transmission efficiency, whereas inheritance through female gametes was normal. The triple KO also resulted in smaller rosettes showing a high frequency of lesions. Both vegetative and reproductive phenotypes were rescued by transgenes encoding either ACA1, 2, or 7, suggesting that all three isoforms are biochemically redundant. Lesions were suppressed by expression of a transgene encoding NahG, an enzyme that degrades salicylic acid (SA). Triple KO mutants showed elevated mRNA expression for two SA-inducible marker genes, Pathogenesis-related1 (PR1) and PR2. The aca1/2/7 lesion phenotype was similar but less severe than SA-dependent lesions associated with a double KO of vacuolar pumps aca4 and 11. Imaging of Ca²⁺ dynamics triggered by blue light or the pathogen elicitor flg22 revealed that aca1/2/7 mutants display Ca²⁺ transients with increased magnitudes and durations. Together, these results indicate that ER-localized ACAs play important roles in regulating Ca²⁺ signals, and that the loss of these pumps results in male fertility and vegetative growth deficiencies.

Autoinhibited Ca²⁺ pumps in the endoplasmic reticulum make important contributions to controlling the magnitude and duration of Ca²⁺ signals.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na⁺/H⁺ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1–3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

Cch1p Mediates Ca2+ Influx to Protect Saccharomyces cerevisiae against Eugenol Toxicity

Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast, Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca²⁺ elevations. We investigated the eugenol Ca²⁺ signature in further detail and show that exponentially growing cells exhibit Ca²⁺ elevation resulting exclusively from the influx of Ca²⁺ across the plasma membrane whereas in stationary growth phase cells Ca²⁺ influx from intracellular and extracellular sources contribute to the eugenol-induced Ca²⁺ elevation. Ca²⁺ channel deletion yeast mutants were used to identify the pathways mediating Ca²⁺ influx; intracellular Ca²⁺ release was mediated by the vacuolar Ca²⁺ channel, Yvc1p, whereas the Ca²⁺ influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca²⁺ channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca²⁺ elevations. Taken together, these results indicate that a cch1p-mediated Ca²⁺ influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies.     

Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.     

Regulation of calcium ion and its effect on growth and developmental behavior in wild type and ntcA mutant of Anabaena sp. PCC 7120 under varied levels of CaCl2

A study of calcium ion regulation in Anabaena 7120 and its derivative mutant (CSE2) strain impaired in ntcA gene were investigated in terms of altered morphological and physiological responses against various levels of calcium stress (0–100 mM). Calcium concentration of 10 mM was found to be inhibitory while 100 mM proved lethal for both wild type and mutant strain. The involvement of Ca²⁺ in the regulation of cellular processes has been described in terms of an influx or efflux of Ca²⁺ from the cytosol. A biphasic calcium uptake with difference in calcium influx and efflux rate was responsible for differential amount of remaining calcium which followed a decreasing trend both for wild type and mutant. Low K _{s 0.5} and high V _max in mutant suggest heavy and less restricted influx of calcium ion. Further, the interactive effect of calcium influx/efflux rate, remaining Ca²⁺ and intracellular levels of Na⁺ and K⁺ may be attributed for the degree of membrane damage and growth sustenance during exogenous supply of calcium salt. Widening in heterocyst spacing pattern, decreased heterocyst frequency and formation of abnormal cell structures at higher concentration (100 mM CaCl₂) suggest that calcium mediated regulatory process modulate heterocyst frequency and maintenance of cell structure. Further, poor regulation of calcium ion homeostasis in ntcA suggests that the calcium level and ntcA gene expression are inter-related.     

内质网上的蛋白质RCN2与STIM1-Orai1复合体相互作用

膜蛋白质Orail组成了一类被称为钙释放激活钙通道（CRAC）的离子通道,并且由相互作用的蛋白质STIM1作为其在内质网上的钙感受器．但是这类通道的调节机制还未研究透彻．通过串连亲和纯化STIM1-Orai1复合体,发现与之相互作用的内质网蛋白质RCN2．共聚焦显微术显示RCN2与STIM1在钙库排空前后完全共定位．对RCN2的EFhands结构突变体所作单细胞测钙,结果显示其对钙库操控通道电流特性有微弱影响．全内反射荧光显微术显示,RCN2以花环状围绕包围STIM1聚集堆,这提示RCN2在STIM1聚集中起到一种结构约束作用．     

Ca2+ Content and Expression of an Acidocalcisomal Calcium Pump Are Elevated in Intracellular Forms of Trypanosoma cruzi

The survival of a eukaryotic protozoan as an obligate parasite in the interior of a eukaryotic host cell implies its adaptation to an environment with a very different ionic composition from that of its extracellular habitat. This is particularly important in the case of Ca²⁺, the intracellular concentration of which is 3 orders of magnitude lower than the extracellular value. Ca²⁺ entry across the plasma membrane is a widely recognized mechanism for Ca²⁺ signaling, needed for a number of intracellular processes, and obviously, it would be restricted in the case of intracellular parasites. Here we show that Trypanosoma cruzi amastigotes possess a higher Ca²⁺ content than the extracellular stages of the parasite. This correlates with the higher expression of a calcium pump, the gene for which was cloned and sequenced. The deduced protein product (Tca1) of this gene has a calculated molecular mass of 121,141 Da and exhibits 34 to 38% identity with vacuolar Ca²⁺-ATPases of Saccharomyces cerevisiae and Dictyostelium discoideum, respectively. The tca1 gene suppresses the Ca²⁺ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca²⁺ accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that Tca1 colocalizes with the vacuolar H⁺-ATPase to the plasma membrane and to intracellular vacuoles of T. cruzi. These vacuoles were shown to have the same size and distribution as the calcium-containing vacuoles identified by the potassium pyroantimoniate-osmium technique and as the electron-dense vacuoles observed in whole unfixed parasites by transmission electron microscopy and identified in a previous work (D. A. Scott, R. Docampo, J. A. Dvorak, S. Shi, and R. D. Leapman, J. Biol. Chem. 272:28020–28029, 1997) as being acidic and possessing a high calcium content (i.e., acidocalcisomes). Together, these results suggest that acidocalcisomes are distinct from other previously recognized organelles present in these parasites and underscore the ability of intracellular parasites to adapt to the hostile environment of their hosts.