Active Calcium Transport Research Articles

COX6A2 deficiency leads to cardiac remodeling in human pluripotent stem cell-derived cardiomyocytes

BackgroundCardiac remodeling is the initiating factor for the development of heart failure, which can result from various cardiomyopathies. Cytochrome c oxidase subunit 6A2 (COX6A2) is one of the components of cytochrome c oxidase that drives oxidative phosphorylation. The pathogenesis of myocardial remodeling caused by COX6A2 deficiency in humans remains unclear because there are no suitable research models. In this study, we established a COX6A2-deficient human cardiac myocyte (CM) model that mimics the human COX6A2 homozygous mutation and determined the effects of COX6A2 dysfunction and its underlying mechanism.MethodsA human COX6A2 homozygous knockout cardiomyocyte model was established by combining CRISPR/Cas9 gene editing technology and hiPSC-directed differentiation technology. Cell model phenotypic assays were done to characterize the pathological features of the resulting COX6A2-deficient cardiomyocytes.ResultsCOX6A2 gene knockout did not affect the pluripotency and differentiation efficiency of hiPSCs. Myocardial cells with a COX6A2 gene knockout showed abnormal energy metabolism, increased oxidative stress levels, abnormal calcium transport activity, and decreased contractility. In addition, L-carnitine and trimetazidine significantly improved energy metabolism in the COX6A2-deficient human myocardial model.ConclusionsWe have established a COX6A2-deficient human cardiomyocyte model that exhibits abnormal energy metabolism, elevated oxidative stress levels, abnormal calcium transport, and reduced contractility. This model represents an important tool to gain insight into the mechanism of action of energy metabolism disorders resulting in myocardial remodeling, elucidate the gene-phenotype relationship of COX6A2 deficiency, and facilitate drug screening.

Solubility and Intestinal Transit Time Limit Calcium Absorption in Rats

Six-week-old male rats were placed on two high calcium regimens: one with calcium carbonate and monobasic calcium phosphate, with calcium content increased via calcium carbonate; and another with calcium phosphate and calcium gluconate, with calcium gluconate the source of increased calcium. Animals fed the gluconate-containing diets absorbed 29% of the ingested calcium over the entire calcium intake range, whereas those fed the calcium carbonate diets absorbed 25% over an intake range of 225 to 450 mg Ca/d, but at calcium intakes above 450 mg Ca/d their absorption reached a plateau at approximately 109 mg/d. Active calcium transport decreased with increased calcium intake in both the calcium carbonate- and calcium gluconate-fed groups. Nonsaturable transport was unchanged as a result of increasing calcium intake and did not differ among the diet groups. Because the absorptive processes were unaffected by the calcium source, events in the lumen must have been responsible for the observed differences. Because phosphate is nearly 18 times more soluble than carbonate, very little calcium of calcium carbonate origin can have been solubilized in the presence of phosphate and this, we conclude, accounts for the limit on calcium absorption observed in diets high in calcium carbonate. Moreover, when intake is expressed as soluble calcium, absorption approaches 50%, the value expected when intestinal transit time (approximately 3 h) is multiplied by 16%/h, the experimental value of nonsaturable absorption.

Lipid dependence of BsYetJ calcium transport activity by fluorescence and ESR techniques.

Recent studies based on X-ray crystallography and pulsed electron spin resonance results have revealed substantial structural insights for the mechanistic understanding of the Ca2+ transport mechanism in the membrane protein YetJ from Bacillus subtilis (BsYetJ). One striking finding is that its structure-function relationship is sensitive to the difference between the surrounding environments, detergent versus POPC lipid. Here, we continue on the study to explore the molecular details of BsYetJ in response to changes in lipid composition, at various mixtures of PC, PE, PI, and cholesterol. Using mutagenesis and fluorescence-based functional assays, we investigate several important negatively charged residues in the BsYetJ and reveal how the interplay determines the calcium channel conductance. This study identifies the conformations of BsYetJ in a complex lipid environment using electron spin resonance and molecular dynamics. Together with the functional data, we propose a mechanism, composed of previously unidentified selectivity filters, for the calcium transport activity of BsYetJ. We show that the calcium transport activity of BsYetJ can be modulated by lipid compositions. Our study provides new insights into the structural impact of the mutations and the development of the mechanistic model of BsYetJ.

Complementary in vitro functional studies and in silico alchemical free energy simulations of the sarco-endoplasmic reticulum calcium pump (SERCA) and phospholamban (PLN) complex.

SERCA is ubiquitously expressed in all eukaryotic cells and is responsible for actively transporting calcium ions from the cytoplasm into the SR/ER lumen. PLN is a small transmembrane peptide that regulates SERCA's calcium transport activity in cardiac muscle. Human genetic variants of PLN have been implicated in cardiomyopathies. PLN is a 52 amino acid peptide with an N-terminal cytoplasmic helix (residues 1-17), a short linker region (residues 18-27) and a C-terminal transmembrane helix (residues 28-52). Previous structural studies have identified the binding groove for PLN formed by transmembrane segments M2, M6, and M9 of SERCA (PDB: 4KYT). The structure of the SERCA-PLN complex consists of SERCA in a calcium-free, E1-like state and includes the linker region and transmembrane helix of PLN (residues 20-52). Here, we carry out in vitro functional studies and in silico free energy calculations to examine the effects of alanine scanning mutations in PLN (residues 20-52) on SERCA regulation. From the functional studies, changes in kinetics parameters such as calcium affinity (KCa) and maximal activity (VMax) of SERCA are determined for each of the alanine substitutions. From the in silico alchemical free energy simulations (using Compute Canada supercomputing resources), each of the residues in PLN are alchemically transformed to alanine to determine the change in Gibbs free energy (Δ;Δ;G) relative to the wild-type SERCA-PLN complex. Together, the complementary in vitro and in silico mutational studies provide novel insights into a membrane protein-peptide complex. The PLN variants are quantified in terms of their physiological significance – gain-of-function, loss-of-function, or neutral – and the molecular determinants that control binding specificity.

A novel calcium-binding peptide from bovine bone collagen hydrolysate and chelation mechanism and calcium absorption activity of peptide-calcium chelate

A novel calcium-binding peptide from bovine bone collagen hydrolysate was screened based on a new target—the calcium-sensing receptor (CaSR), and its chelation mechanism and calcium absorption activity were investigated. Glu-Tyr-Gly exhibited superior binding affinities to CaSR because of its interaction with the active sites of the CaSR Venus Flytrap (VFT) domain. Glu-Tyr-Gly-Ca may exist in five potential chelation modes and its potential chelation mechanism was that calcium ions were located in the center and surrounded by ionic bonds (carboxyl group) and coordination bonds (carbonyl, amino, and carboxyl group). Glu-Tyr-Gly-Ca was slightly damaged in the intestinal fluid and at different temperatures, whereas it was severely damaged in the gastric fluid and acidic conditions. The results of the calcium dialysis percentage and Caco-2 cells experiments showed that Glu-Tyr-Gly-Ca possessed good calcium transport activity and bioavailability. The findings provided theoretical basis for Glu-Tyr-Gly-Ca as potential calcium supplement to improve intestinal calcium absorption.

Vasoactive intestinal peptide and cystic fibrosis transmembrane conductance regulator contribute to the transepithelial calcium transport across intestinal epithelium-like Caco-2 monolayer.

Vasoactive intestinal peptide (VIP) as a neurocrine factor released by enteric neurons has been postulated to participate in the regulation of transcellular active calcium transport across intestinal epithelium, but the preceding evidence is scant and inconclusive. Herein, transepithelial calcium flux and epithelial electrical parameters were determined by Ussing chamber technique with radioactive tracer in the intestinal epithelium-like Caco-2 monolayer grown on Snapwell. After 3-day culture, Caco-2 cells expressed mRNA of calcium transporters, i.e., TRPV6, calbindin-D9k, PMCA1b and NCX1, and exhibited transepithelial resistance of ~200 Ω cm2, a characteristic of leaky epithelium similar to the small intestine. VIP receptor agonist was able to enhance transcellular calcium flux, whereas VIP receptor antagonist totally abolished calcium fluxes induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Since the intestinal cystic fibrosis transmembrane conductance regulator (CFTR) could be activated by VIP and calciotropic hormones, particularly parathyroid hormone, we sought to determine whether CFTR also contributed to the 1,25(OH)2D3-induced calcium transport. A selective CFTR inhibitor (20-200 μM CFTRinh-172) appeared to diminish calcium fluxes as well as transepithelial potential difference and short-circuit current, both of which indicated a decrease in electrogenic ion transport. On the other hand, 50 μM genistein-a molecule that could rapidly activate CFTR-was found to increase calcium transport. Our in silico molecular docking analysis confirmed direct binding of CFTRinh-172 and genistein to CFTR channels. In conclusion, VIP and CFTR apparently contributed to the intestinal calcium transport, especially in the presence of 1,25(OH)2D3, thereby supporting the existence of the neurocrine control of intestinal calcium absorption.

Novel gain-of-function mutation of TRPC6 Q134P contributes to late onset focal segmental glomerulosclerosis in a Chinese pedigree.

Focal segmental glomerulosclerosis (FSGS, OMIM®#603 965) is an overriding cause that leads to end-stage renal disease (ESRD). As a member of TRP superfamily, mutations of TRPC6 gene are closely linked to FSGS. By now, 20 missense mutations have been reported, among them, nine gain-of-function (GOF), and five loss-of-function (LOF) mutations have been recognized according to the effect on TRPC6 channel activity. Systematic investigations of functional mutations will provide valuable evidences for understanding the pathophysiology of TRPC6 involved in FSGS. The aim of this study is to investigate the pathogenicity of a novel TRPC6 mutation p.Q134P in FSGS. High-throughput sequencing was performed to analyse 436 genes which are associated with hereditary kidney diseases in a Chinese pedigree. Then we constructed TRPC6 expression plasmids of wide type and variant. Immunofluorescence, cell-surface biotinylation assays and electrophysiology were used to analyse the localization, cell surface expression, and calcium transport activity of TRPC6. A novel variant c.401A>C (p.Q134P) in exon 2 of TRPC6 gene was found. There was no significant difference between the expression levels of p.Q134P mutant and WT TRPC6 protein in the whole cell lysate and cell-surface fraction. Q134P mutant-bearing TRPC6 elicited much higher Ca+ current amplitude than WT. We identified a novel GOF mutation p.Q134P of TRPC6 which contributed to late-onset FSGS. Our study expands the mutational spectrum of TRPC6 associated with FSGS and furtherly supports the hypothesis of calcium dose-response dependency that a moderate increased calcium influx elicited a mild FSGS phenotype.

The high energetic cost of rapid force development in muscle.

Muscles consume metabolic energy for active movement, particularly when performing mechanical work or producing force. Less appreciated is the cost for activating muscle quickly, which adds considerably to the overall cost of cyclic force production. However, the cost magnitude relative to the cost of mechanical work, which features in many movements, is unknown. We therefore tested whether fast activation is costly compared with performing work or producing isometric force. We hypothesized that metabolic cost would increase with a proposed measure termed force rate (rate of increase in muscle force) in cyclic tasks, separate from mechanical work or average force level. We tested humans (N=9) producing cyclic knee extension torque against an isometric dynamometer (torque 22 N m, cyclic waveform frequencies 0.5-2.5 Hz), while also quantifying quadriceps muscle force and work against series elasticity (with ultrasonography), along with metabolic rate through respirometry. Net metabolic rate increased by more than four-fold (10.5 to 46.8 W) with waveform frequency. At high frequencies, the hypothesized force-rate cost accounted for nearly half (40%) of energy expenditure. This exceeded the cost for average force (17%) and was comparable to the cost for shortening work (43%). The force-rate cost is explained by additional active calcium transport necessary for producing forces at increasing waveform frequencies, owing to rate-limiting dynamics of force production. The force-rate cost could contribute substantially to the overall cost of movements that require cyclic muscle activation, such as locomotion.

Effects of inulin on calcium metabolism and bone health.

Inulin, a dietary fibre found in the roots of many plants, has positive effects on health. It is particularly noteworthy due to its positive impact on calcium metabolism. Inulin has significant functions, such as improving calcium absorption through passive diffusion, bolstering calcium absorption via ion exchange and expanding the absorption surface of the colon by stimulating cell growth. In addition, inulin boosts calcium absorption by increasing calcium solubility, stimulating levels of calcium-binding protein expression and increasing useful microorganisms. It increases calbindin levels and stimulates transcellular active calcium transport. An inulin intake of least 8-10g/day supports calcium absorption and total body bone mineral content/density in adolescents through its known mechanisms of action. It also significantly enhances calcium absorption and improves bone health in postmenopausal women and adult men. Sustained and sufficient inulin supplementation in adults has a positive effect on calcium metabolism and bone mineral density.

The influence of environmental calcium on the branchial morphology in a catadromous fish.

Eels are exposed to Ca2+ changes during migration between seawater and freshwater. The gill is the main organ of active calcium transport and has a large surface area to be particularly sensitive to environmental changes in the aquatic environment. In this research, we focused on the morphological changes of gill tissues when eels are faced with the environmental calcium challenges. Based on the results of hematoxylin and eosin (HE) staining and immunohistochemistry, compared with the control group (normal Ca2+ environment), the filament and lamella lengths and lamellar frequency (LF) appeared higher in high calcium environment and lower in deficient calcium environment, while the lamella width and filamental lamellar surface area (SAFL) decreased in high calcium environment and increased in deficient calcium environment. And there was no difference in the number filaments in first right gill arch in the three Ca2+ water environment. Transmission electron microscopy was employed to examine the ultrastructural changes in gills in different Ca2+ water environment. The nucleus and endoplasmic reticulum had a tendency to expand in calcium-deficient water, but had a tendency to shrink in high-calcium water comparing with the control group. This study provides the support that branchial surface areas are regulated in different Ca2+ waters through a list of calcium transporters including CACNB2.

Analysis of 1,25-Dihydroxyvitamin D3 Genomic Action Reveals Calcium-Regulating and Calcium-Independent Effects in Mouse Intestine and Human Enteroids.

Although vitamin D is critical for the function of the intestine, most studies have focused on the duodenum. We show that transgenic expression of the vitamin D receptor (VDR) only in the distal intestine of VDR null mice (KO/TG mice) results in the normalization of serum calcium and rescue of rickets. Although it had been suggested that calcium transport in the distal intestine involves a paracellular process, we found that the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated genes in the proximal intestine associated with active calcium transport (Trpv6, S100g, and Atp2b1) are also induced by 1,25(OH)2D3 in the distal intestine of KO/TG mice. In addition, Slc30a10, encoding a manganese efflux transporter, was one of the genes most induced by 1,25(OH)2D3 in both proximal and distal intestine. Both villus and crypt were found to express Vdr and VDR target genes. RNA sequence (RNA-seq) analysis of human enteroids indicated that the effects of 1,25(OH)2D3 observed in mice are conserved in humans. Using Slc30a10-/- mice, a loss of cortical bone and a marked decrease in S100g and Trpv6 in the intestine was observed. Our findings suggest an interrelationship between vitamin D and intestinal Mn efflux and indicate the importance of distal intestinal segments to vitamin D action.

Structure and regulation of the BsYetJ calcium channel in lipid nanodiscs

BsYetJ is a bacterial homolog of transmembrane BAX inhibitor-1 motif-containing 6 (TMBIM6) membrane protein that plays a key role in the control of calcium homeostasis. However, the BsYetJ (or TMBIM6) structure embedded in a lipid bilayer is uncharacterized, let alone the molecular mechanism of the calcium transport activity. Herein, we report structures of BsYetJ in lipid nanodiscs identified by double electron-electron resonance spectroscopy. Our results reveal that BsYetJ in lipid nanodiscs is structurally different from those crystallized in detergents. We show that BsYetJ conformation is pH-sensitive in apo state (lacking calcium), whereas in a calcium-containing solution it is stuck in an intermediate, inert to pH changes. Only when the transmembrane calcium gradient is established can the calcium-release activity of holo-BsYetJ occur and be mediated by pH-dependent conformational changes, suggesting a dual gating mechanism. Conformational substates involved in the process and a key residue D171 relevant to the gating of calcium are identified. Our study suggests that BsYetJ/TMBIM6 is a pH-dependent, voltage-gated calcium channel.

S2888 Tetany and Carpopedal Spasm as the Initial Presentation of Celiac Disease

INTRODUCTION: Celiac disease (CD) presents with a wide variety of symptoms, with gastrointestinal manifestations being the most common. We discuss a rare case of celiac disease diagnosed by severe hypocalcemia and carpopedal spasm. CASE DESCRIPTION/METHODS: A 25 year old male with no significant medical history presented with complaints of paresthesias in his hands and arms and a few weeks of diarrhea. Physical exam revealed carpopedal spasm. Initial lab work was significant for severe hypocalcemia (ionized calcium level of 0.66), hypomagnesemia and undetectable vitamin D 25-hydroxy level, along with elevated PTH (583.2). His electrolytes were aggressively replaced intravenously, and after stabilization, transitioned to oral supplementation. Vitamin D supplementation was also initiated. Patient was found to have elevated tissue transglutaminase IgA antibodies and gliadin peptide levels. EGD demonstrated scalloped mucosa with complete flattening of villi in the duodenum consistent with celiac disease, which was also confirmed on biopsy. Patient was initiated on calcium and vitamin D replacement and a gluten-free diet on discharge. DISCUSSION: CD usually presents with diarrhea and weight loss. However, >40% of patients present with atypical manifestations, many of which are secondary to malabsorption from a loss of absorptive area and the presence of immature surface epithelial cells whose have impaired absorptive and secretory functions. Hypocalcemia in CD occurs due to intestinal malabsorption of calcium, both from loss of villous surface area and depletion of calbindin from enterocytes leading to decreased active intestinal calcium transport mechanisms. Concurrent malabsorption of vitamin D may also play a role, although there have been reported cases with hypocalcemia despite a normal vitamin D. Hypocalcemia in CD is common, with studies demonstrating that average serum calcium is 0.02 mmol/l lower in asymptomatic CD patients that the general population. However, there are few reports, especially in the developed world, of severe hypocalcemia and neurological symptoms being the initial presentation of CD. Management includes intravenous or oral calcium replacement, with replacement of other electrolytes (magnesium, phosphorous and potassium) and vitamin D as well. These levels should be monitored at follow up visits, but it is expected that they will normalize after management of CD. It is important for a clinician to recognize atypical presentations of CD, especially in countries with high prevalence.Figure 1.: Endoscopic view of duodenal bulb showing scalloped mucosa and complete flattening of villi.Figure 2.: Microscopic view showing villous blunting and increased intraepithelial lymphocytes, H&E staining.

Vitamin D supplementation: cholecalciferol, calcifediol, and calcitriol.

The specific compound that is meant for use in the context of vitamin D supplementation is often ambiguous. The term "supplementation" has been used in the context of cholecalciferol, ergocalciferol, calcidiol, and calcitriol. In nature, by far the major form of vitamin D that nurtures the body is cholecalciferol. In contrast, ergocalciferol is primarily a synthetic and less stable product which is less potent per microgram dose than is cholecalciferol. Calcidol is the major circulating metabolite of cholecalciferol, while calcitriol is the hormone that upregulates the active transport of calcium from the gut, and which suppresses parathyroid hormone secretion. Nutrition policy papers and guidelines leave unstated the obvious fact that calcidiol and calcitriol are not nutrients, and that those metabolites are not pertinent to food fortification or dietary supplementation. Recent evidence shows that ergocalciferol is not stable with storage, and it is far more susceptible to breakdown with cooking and baking than is cholecalciferol. Therefore, it must be concluded that cholecalciferol is the only form of vitamin D that should be considered in the context of the nutritional functions of fortification and supplementation.

Glycocholic acid and butyrate synergistically increase vitamin D-induced calcium uptake in Caco-2 intestinal epithelial cell monolayers

BackgroundRoux-en-Y gastric bypass (RYGB) substantially decreases intestinal calcium absorption and may eventually lead to bone resorption. This is likely a consequence of bile diversion from the alimentary limb, as the presence of bile seems necessary for vitamin D-mediated calcium uptake. We recently suggested that the mediating mechanism may be a down-regulation of the vitamin D co-activator heat-shock protein (Hsp)90β. Recent evidence suggests that vitamin D may have effects on both active and passive calcium absorption. AimTo identify mechanisms in vitro that may be responsible for the decreased calcium absorption after RYGB. We hypothesized that bile, alone or in concert with nutritional compounds, could be of importance. Material & methodsCaco-2 cells were grown confluent on semi-permeable membranes in a double-chamber setup to mimic small intestinal mucosa. The effect of bile acids chenodeoxycholic, lithocholic, glycocholic and taurocholic acid, with and without the addition of the fatty-acid butyrate, were tested for their effects on Hsp90β expression and active and passive calcium-flux monitored using radioactive 45Ca. ResultsWe initially found that whole human bile, but only together with the fatty acid butyrate, potently induced Hsp90β expression. In line with this, a single bile acid, e.g. glycocholic acid (GCA), in combination with butyrate, increased Hsp90β expression (40 ± 13% vs. GCA, butyrate or vehicle alone; p < 0,001; n = 14–25). Further, this combination together with vitamin D increased the passive gradient-driven flux of calcium, compared to stimulation with vitamin D alone or in combination with either GCA or butyrate (880 ± 217% vs. vitamin D and GCA or butyrate, or vitamin D only; p = 0,01–0.006; n = 5–11). Surprisingly, this combination had no effect on active calcium transport in the absence of calcium gradient. ConclusionThe combination of GCA and butyrate increased gradient-driven calcium uptake up to 9-fold in Caco-2 intestinal epithelial cells, but had no effect on active calcium absorption. This effect was mediated via the vitamin D receptor co-activator Hsp90β.

Evidence of capacitation in the parasitoid wasp, Nasonia vitripennis, and its potential role in sex allocation.

The allocation of resources to the production of one sex or another has been observed in a large variety of animals. Its theoretical basis allows accurate predictions of offspring sex ratios in many species, but the mechanisms by which sex allocation is controlled are poorly understood. Using previously published data, we investigated whether alternative splicing, combined with differential gene expression, was involved with sex allocation in the parasitoid wasp, Nasonia vitripennis. We found that sex allocation is not controlled by alternative splicing but changes in gene and transcript‐specific expression, which were identified to be involved with oviposition, were shown to be similar to those involved in sperm motility and capacitation. Genes involved in cholesterol efflux, a key component of capacitation, along with calcium transport, neurotransmission, trypsin, and MAPKinase activity were regulated in ovipositing wasps. The results show evidence for regulation of sperm motility and of capacitation in an insect which, in the context of the physiology of the N. vitripennis spermatheca, could be important for sex allocation.

Metabolomic and transcriptomic profiling reveals the alteration of energy metabolism in oyster larvae during initial shell formation and under experimental ocean acidification

Marine bivalves secrete calcified shells to protect their soft bodies from predation and damages, which is of great importance for their survival, and for the safety of the coastal ecosystem. In recent years, larval shell formation of marine bivalves has been severely affected by ocean acidification (OA), and previous study indicated that OA might affect such process by disrupting endogenous energy metabolism. Developmental stages from trochophore to D-shape larvae are extremely important for initial shell formation in oyster since a calcified shell was formed to cover the chitin one. In the present study, metabolomic and transcriptomic approaches were employed to investigate the energy metabolism of oyster larvae during initial shell (prodissoconch I, PDI shell) formation and under experimental OA treatment. Totally 230 chemical compounds were identified from the present dataset, most of which were highly expressed in the “middle” stage (early D-shape larvae) which was critical for PDI shell formation since a calcified shell was formed to cover the chitin one. Several compounds such as glucose, glutarylcarnitine (C5), β-hydroxyisovaleroylcarnitine, 5-methylthioadenosine (MTA), myristoleate (14:1n5) and palmitoleate (16:1n7) were identified, which were involved in energy metabolic processes including amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. In addition, mRNA expressions of genes related to protein metabolism, glycolysis, lipid degradation, calcium transport and organic matrix formation activities were significantly down-regulated upon experimental OA. These results collectively suggested that formation of the initial shell in oyster larvae required endogenous energy coming from amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. These metabolic activities could be severely inhibited by experimental OA, which might alter the allocation of endogenous energy. Insufficient endogenous energy supply then suppressed the mobilization of calcium and resulted in a failure or delay in PDI shell formation.

High-Throughput FRET Screening in Living Cells Based on Lifetime Detection to Identify Small-Molecule Effectors of Serca

The development of an effective high-throughput screening (HTS) paradigm using FRET biosensors in living cells has enabled us to identify multiple classes of small-molecules that alter the activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). We aim to discover small-molecule activators that improve SERCA's function for the treatment of heart failure. We have validated an intramolecular SERCA FRET biosensor (red and green fluorescent proteins fused to distinct SERCA domains) by screening a small test library using novel plate readers (PRs) that detect the fluorescence lifetime (FLT) (Schaaf et al, 2017). Here we describe an FLT-PR that scans a 1536-well plate in ≤2.5 min. This instrument is equipped with multichannel FLT detection at two emission wavelengths, used to filter out fluorescent compounds that would be falsely identified as hits. False hits were further eliminated using FRET biosensors based on non-specific peptide linkers, and using spectral analysis. FRET biosensors were developed and tested based on two different isoforms of SERCA: the SERC2a cardiac-specific isoform and SERCA2b non-muscle isoform were used to discover compounds with specificity toward SERCA2a. Concentration-response studies were performed to determine potency of compounds. Hits were assessed by two separate functional assays to determine how they alter ATPase activity and calcium-uptake. The majority were found to be inhibitors, but some were activators, and some compounds affected the coupling of SERCA's ATPase and calcium transport activities Our future focus is to evaluate the structure-function relationships of the newly discovered SERCA activators, using medicinal chemistry to optimize drug-like properties. These new HTS methods are being applied to a wide range of drug targets.

Hetero-oligomerization of the Micropeptide Regulins that Modulate Calcium Transport Activity

Hetero-oligomerization of the Micropeptide Regulins that Modulate Calcium Transport Activity

Endolithic Algae Affect Modern Coral Carbonate Morphology and Chemistry

While burial diagenetic processes of tropical corals are well investigated, current knowledge about factors initiating early diagenesis remains fragmentary. In the present study we focus on recent Porites microatolls, growing in the intertidal zone. This growth form represents a model organism for elevated sea surface temperatures and provides important but rare archives for changes close to the seawater/atmosphere-interface with exceptional precision on sea level reconstruction. As other coral growth forms, microatolls are prone to the colonization by endolithic green algae. In this case the algae can facilitate earliest diagenetic alteration of the coral skeleton. Algae metabolic activity not only results in secondary coral porosity due to boring activities, but may also initiate reprecipitation of secondary aragonite within coral pore space, a process not exclusively restricted to microatoll settings. In the samples of this initial study, we quantified a mass transfer from primary to secondary aragonite of around 4% within endolithic green algae bands. Using δ18O, δ13C, Sr/Ca, U/Ca, Mg/Ca, and Li/Mg systematics suggest that the secondary aragonite precipitation followed abiotic precipitation principles. According to their individual distribution coefficients the different isotope and element ratios showed variable sensitivity to the presence of secondary aragonite in bulk samples, with implications for microatoll-based sea surface temperature (SST) reconstructions. The secondary precipitates formed on an organic template, presumably originating from endolithic green algae activity. Based on laboratory experiments with the green algae Ostreobium quekettii we propose a conceptual model that secondary aragonite formation is potentially accelerated by an active intracellular calcium transport through the algal thallus from the location of dissolution into coral pore spaces. The combined high-resolution imaging and geochemical approach applied in this study shows that endolithic algae can possibly act as a main driver for earliest diagenesis of coral aragonite starting already during a coral’s life span.