Transcriptomic analysis of Caenorhabditis elegans treated with mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157 vs untreated control.
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE173646 | GEO | 2021/05/01
REPOSITORIES: GEO
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Project description:Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.
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Project description:The binding of Ca(2+) to two adjacent Ca(2+)-binding domains, CBD1 and CBD2, regulates ion transport in the Na(+)/Ca(2+) exchanger. As sensors for intracellular Ca(2+), the CBDs form electrostatic switches that induce the conformational changes required to initiate and sustain Na(+)/Ca(2+) exchange. Depending on the presence of a few key residues in the Ca(2+)-binding sites, zero to four Ca(2+) ions can bind with affinities between 0.1 to 20 ?m. Importantly, variability in CBD2 as a consequence of alternative splicing modulates not only the number and affinities of the Ca(2+)-binding sites in CBD2 but also the Ca(2+) affinities in CBD1.
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Project description:The cardiac Na(+)/Ca(2+) exchanger (NCX) regulates cellular [Ca(2+)](i) and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C(2) regulatory domains, Ca(2+) binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I(NCX), whereas [H(+)](i) was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pH(i) = 7.2 and [Ca(2+)](i) < 120 nM, I(NCX) was less than 4% that of its maximally Ca(2+)-activated value. I(NCX) increases steeply at [Ca(2+)](i) between 130-150 nM with a Hill coefficient (n(H)) of 8.0 ± 0.7 and K(0.5) = 310 ± 5 nM. At pH(i) = 6.87, the threshold of Ca(2+)-dependent activation of I(NCX) was shifted to much higher [Ca(2+)](i) (600-700 nM), and the relationship was similarly steep (n(H) = 8.0±0.8) with K(0.5) = 1042 ± 15 nM. The V(max) of Ca(2+)-dependent activation of I(NCX) was not significantly altered by low pH(i). The Ca(2+) affinities for CBD1 (0.39 ± 0.06 μM) and CBD2 (K(d) = 18.4 ± 6 μM) were exquisitely sensitive to [H(+)], decreasing 1.3-2.3-fold as pH(i) decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C(2) regulatory domains CBD1 and CBD2.
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Project description:Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.
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Project description:The calcium carbonate skeletons of corals provide the underlying structure of coral reefs; however, the cellular mechanisms responsible for coral calcification remain poorly understood. In osteoblasts from vertebrate animals, a Na+/Ca2+ exchanger (NCX) present in the plasma membrane transports Ca2+ to the site of bone formation. The aims of this study were to establish whether NCX exists in corals and its localization within coral cells, which are essential first steps to investigate its potential involvement in calcification. Data mining identified genes encoding for NCX proteins in multiple coral species, a subset of which were more closely related to NCXs from vertebrates (NCXA). We cloned NCXA from Acropora yongei (AyNCXA), which, unexpectedly, contained a peptide signal that targets proteins to vesicles from the secretory pathway. AyNCXA subcellular localization was confirmed by heterologous expression of fluorescently tagged AyNCXA protein in sea urchin embryos, which localized together with known markers of intracellular vesicles. Finally, immunolabeling of coral tissues with specific antibodies revealed AyNCXA was present throughout coral tissue. AyNCXA was especially abundant in calcifying cells, where it exhibited a subcellular localization pattern consistent with intracellular vesicles. Altogether, our results demonstrate AyNCXA is present in vesicles in coral calcifying cells, where potential functions include intracellular Ca2+ homeostasis and Ca2+ transport to the growing skeleton as part of an intracellular calcification mechanism.
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Project description:Regulation of ion-transport in the Na(+)/Ca(2+) exchanger (NCX) occurs via its cytoplasmic Ca(2+)-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca(2+)-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca(2+)-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca(2+). Striking differences in the electrostatic potential between the Ca(2+)-bound and -free forms strongly suggest that Ca(2+)-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C(2)-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca(2+)-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca(2+) is influenced by the closely located cassette exons. We further propose that Ca(2+)-binding to CBD2 induces a second electrostatic switch, required to alleviate Na(+)-dependent inactivation of Na(+)/Ca(2+) exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.
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Project description:BACKGROUND: The Na+-Ca2+ exchanger (NCX) is an important regulator of cytosolic Ca2+ levels. Many of its structural features are highly conserved across a wide range of species. Invertebrates have a single NCX gene, whereas vertebrate species have multiple NCX genes as a result of at least two duplication events. To examine the molecular evolution of NCX genes and understand the role of duplicated genes in the evolution of the vertebrate NCX gene family, we carried out phylogenetic analyses of NCX genes and compared NCX gene structures from sequenced genomes and individual clones. RESULTS: A single NCX in invertebrates and the protochordate Ciona, and the presence of at least four NCX genes in the genomes of teleosts, an amphibian, and a reptile suggest that a four member gene family arose in a basal vertebrate. Extensive examination of mammalian and avian genomes and synteny analysis argue that NCX4 may be lost in these lineages. Duplicates for NCX1, NCX2, and NCX4 were found in all sequenced teleost genomes. The presence of seven genes encoding NCX homologs may provide teleosts with the functional specialization analogous to the alternate splicing strategy seen with the three NCX mammalian homologs. CONCLUSION: We have demonstrated that NCX4 is present in teleost, amphibian and reptilian species but has been secondarily and independently lost in mammals and birds. Comparative studies on conserved vertebrate homologs have provided a possible evolutionary route taken by gene duplicates subfunctionalization by minimizing homolog number.
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Project description:Prokaryotic and eukaryotic Na+/Ca2+ exchangers (NCX) control Ca2+ homeostasis. NCX orthologs exhibit up to 104-fold differences in their turnover rates (kcat), whereas the ratios between the cytosolic (cyt) and extracellular (ext) Km values (Kint = KmCyt/KmExt) are highly asymmetric and alike (Kint ? 0.1) among NCXs. The structural determinants controlling a huge divergence in kcat at comparable Kint remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on kcat and Kint. Among 55 tested residues, 26 mutations affect either kcat or Kint, where two major groups can be distinguished. The first group of mutations (14 residues) affect kcat rather than Kint. The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect Kint rather than kcat, whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca2+ and Na+ signaling.
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Project description:We expressed full-length Na(+)-Ca(2+) exchangers (NCXs) with mutations in two Ca(2+)-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca(2+)-dependent regulation of NCX. CBD1 has four Ca(2+)-binding sites, and mutation of residues Asp(421) and Glu(451), which primarily coordinate Ca(2+) at sites 1 and 2, had little effect on regulation of NCX by Ca(2+). In contrast, mutations at residues Glu(385), Asp(446), Asp(447), and Asp(500), which coordinate Ca(2+) at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca(2+). Another mutant, M7, with 7 key residues of CBD1 replaced, showed a further decrease in apparent Ca(2+) affinity but retained regulation, confirming a contribution of CBD2 to Ca(2+) regulation. Addition of the mutation K585E (located in CBD2) into the M7 background induced a marked increase in Ca(2+) affinity for both steady-state and peak currents. Also, we have shown previously that the CBD2 mutations E516L and E683V have no Ca(2+)-dependent regulation. We now demonstrate that introduction of a positive charge at these locations rescues Ca(2+)-dependent regulation. Finally, our data demonstrate that deletion of the unstructured loops between beta-strands F and G of both CBDs does not alter the regulation of the exchanger by Ca(2+), indicating that these segments are not important in regulation. Thus, CBD1 and CBD2 have distinct roles in Ca(2+)-dependent regulation of NCX. CBD1 determines the affinity of NCX for regulatory Ca(2+), although CBD2 is also necessary for Ca(2+)-dependent regulation.
| S-EPMC2781690 | biostudies-literature
Project description:The mammalian Na(+)/Ca(2+) exchanger, NCX1.1, serves as the main mechanism for Ca(2+) efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca(2+), NCX1.1 activity is also strongly regulated by Ca(2+) binding to two intracellular regulatory domains, CBD1 and CBD2. The structures of both of these domains have been solved by NMR spectroscopy and x-ray crystallography, greatly enhancing our understanding of Ca(2+) regulation. Nevertheless, the mechanisms by which Ca(2+) regulates the exchanger remain incompletely understood. The initial NMR study showed that the first regulatory domain, CBD1, unfolds in the absence of regulatory Ca(2+). It was further demonstrated that a mutation of an acidic residue involved in Ca(2+) binding, E454K, prevents this structural unfolding. A contradictory result was recently obtained in a second NMR study in which Ca(2+) removal merely triggered local rearrangements of CBD1. To address this issue, we solved the crystal structure of the E454K-CBD1 mutant and performed electrophysiological analyses of the full-length exchanger with mutations at position 454. We show that the lysine substitution replaces the Ca(2+) ion at position 1 of the CBD1 Ca(2+) binding site and participates in a charge compensation mechanism. Electrophysiological analyses show that mutations of residue Glu-454 have no impact on Ca(2+) regulation of NCX1.1. Together, structural and mutational analyses indicate that only two of the four Ca(2+) ions that bind to CBD1 are important for regulating exchanger activity.
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