Ca Homeostasis Research Articles

Aluminum Chloride Induces Osteoblasts Apoptosis via Disrupting Calcium Homeostasis and Activating Ca(2+)/CaMKII Signal Pathway.

Aluminum promotes osteoblast (OB) apoptosis. Apoptosis is induced by the disordered calcium homeostasis. Therefore, to investigate the relationship between Al-induced OB apoptosis and calcium homeostasis, calvarium OBs from neonatal rats (3-4days) were cultured and exposed to 0.048-mg/mL Al(3+) or 0.048-mg/mL Al(3+) combined with 5μM BAPTA-AM (OBs were pretreated with 5μM BAPTA-AM for 1h, then added 0.048mg/mL Al(3+)), respectively. Then OB apoptosis rate, intracellular calcium ions concentration ([Ca(2+)]i), mRNA expression level of calmodulin (CaM), and protein expression levels of CaM and p-CaMKII in OBs were examined. The result showed that AlCl3 increased OB apoptosis rate, and [Ca(2+)]i and p-CaMKII expression levels and decreased CaM expression levels, whereas BAPTA-AM relieved the effects. These results proved that AlCl3 induced OB apoptosis by disrupting the intracellular Ca(2+) homeostasis and activating the Ca(2+)/CaMKII signal pathway. Our findings can provide new insights for revealing the apoptosis mechanism of OBs exposed to AlCl3.

Increased TRPC5 glutathionylation contributes to striatal neuron loss in Huntington's disease.

Aberrant glutathione or Ca(2+) homeostasis due to oxidative stress is associated with the pathogenesis of neurodegenerative disorders. The Ca(2+)-permeable transient receptor potential cation (TRPC) channel is predominantly expressed in the brain, which is sensitive to oxidative stress. However, the role of the TRPC channel in neurodegeneration is not known. Here, we report a mechanism of TRPC5 activation by oxidants and the effect of glutathionylated TRPC5 on striatal neurons in Huntington's disease. Intracellular oxidized glutathione leads to TRPC5 activation via TRPC5 S-glutathionylation at Cys176/Cys178 residues. The oxidized glutathione-activated TRPC5-like current results in a sustained increase in cytosolic Ca(2+), activated calmodulin-dependent protein kinase and the calpain-caspase pathway, ultimately inducing striatal neuronal cell death. We observed an abnormal glutathione pool indicative of an oxidized state in the striatum of Huntington's disease transgenic (YAC128) mice. Increased levels of endogenous TRPC5 S-glutathionylation were observed in the striatum in both transgenic mice and patients with Huntington's disease. Both knockdown and inhibition of TRPC5 significantly attenuated oxidation-induced striatal neuronal cell death. Moreover, a TRPC5 blocker improved rearing behaviour in Huntington's disease transgenic mice and motor behavioural symptoms in littermate control mice by increasing striatal neuron survival. Notably, low levels of TRPC1 increased the formation of TRPC5 homotetramer, a highly Ca(2+)-permeable channel, and stimulated Ca(2+)-dependent apoptosis in Huntington's disease cells (STHdh(Q111/111)). Taken together, these novel findings indicate that increased TRPC5 S-glutathionylation by oxidative stress and decreased TRPC1 expression contribute to neuronal damage in the striatum and may underlie neurodegeneration in Huntington's disease.

Molecular aspects of intestinal calcium absorption.

Intestinal Ca(2+) absorption is a crucial physiological process for maintaining bone mineralization and Ca(2+) homeostasis. It occurs through the transcellular and paracellular pathways. The first route comprises 3 steps: the entrance of Ca(2+) across the brush border membranes (BBM) of enterocytes through epithelial Ca(2+) channels TRPV6, TRPV5, and Cav1.3; Ca(2+) movement from the BBM to the basolateral membranes by binding proteins with high Ca(2+) affinity (such as CB9k); and Ca(2+) extrusion into the blood. Plasma membrane Ca(2+) ATPase (PMCA1b) and sodium calcium exchanger (NCX1) are mainly involved in the exit of Ca(2+) from enterocytes. A novel molecule, the 4.1R protein, seems to be a partner of PMCA1b, since both molecules co-localize and interact. The paracellular pathway consists of Ca(2+) transport through transmembrane proteins of tight junction structures, such as claudins 2, 12, and 15. There is evidence of crosstalk between the transcellular and paracellular pathways in intestinal Ca(2+) transport. When intestinal oxidative stress is triggered, there is a decrease in the expression of several molecules of both pathways that inhibit intestinal Ca(2+) absorption. Normalization of redox status in the intestine with drugs such as quercetin, ursodeoxycholic acid, or melatonin return intestinal Ca(2+) transport to control values. Calcitriol [1,25(OH)₂D₃] is the major controlling hormone of intestinal Ca(2+) transport. It increases the gene and protein expression of most of the molecules involved in both pathways. PTH, thyroid hormones, estrogens, prolactin, growth hormone, and glucocorticoids apparently also regulate Ca(2+) transport by direct action, indirect mechanism mediated by the increase of renal 1,25(OH)₂D₃ production, or both. Different physiological conditions, such as growth, pregnancy, lactation, and aging, adjust intestinal Ca(2+) absorption according to Ca(2+) demands. Better knowledge of the molecular details of intestinal Ca(2+) absorption could lead to the development of nutritional and medical strategies for optimizing the efficiency of intestinal Ca(2+) absorption and preventing osteoporosis and other pathologies related to Ca(2+) metabolism.

The toxic effects of Bisphenol A on the mouse spermatocyte GC-2 cell line: the role of the Ca2+-calmodulin-Ca2+/calmodulin-dependent protein kinase II axis.

Bisphenol A (BPA), an endocrine-disrupting chemical (EDC), is known to induce male reproductive toxicity in rodents. However, its toxic effects on the germ cells are still poorly understood. It has been proposed that Ca(2+) homeostasis and Ca(2+) sensors, including calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII), play critical roles in spermatogenesis. Therefore, in the present study, we aimed to investigate whether a perturbation in Ca(2+)-CaM-CaMKII signaling was involved in the BPA-induced injury to mouse spermatocyte GC-2spd (ts) (GC-2) cells. Our results showed that BPA (range from 0.2 to 20 μM) induced obvious GC-2 cell injury, including decreased cell viability, the release of mitochondrial cytochrome c and the activation of caspase-3. However, these processes could be partially abrogated by pretreatment with a Ca(2+) chelator (BAPTA/AM), a CaM antagonist (W7) or a CaMKII inhibitor (KN93). These results, taken together, indicate that BPA exposure contributes to male germ cell injury, which may be partially mediated through a perturbation in Ca(2+)/CaM/CaMKII signaling and the mitochondrial apoptotic process.

Monitoring dynamic alterations in calcium homeostasis by T1-mapping manganese-enhanced MRI (MEMRI) in the early stage of small intestinal ischemia-reperfusion injury.

Manganese-enhanced MRI studies have proven to be useful in monitoring physiological activities associated with calcium ions (Ca(2+)) due to the paramagnetic property of the manganese ion (Mn(2+)), which makes it an excellent probe of Ca(2+) . In this study, we developed a method in which a Mn(2+)-enhanced T1 -map MRI could enable the monitoring of Ca(2+) influx during the early stages of intestinal ischemia-reperfusion (I/R) injury. The Mn(2+) infusion protocol was optimized by obtaining dose-dependent and time-course wash-out curves using a Mn(2+)-enhanced T1-map MRI of rabbit abdomens following an intravenous infusion of 50 mmol/l MnCl2 (5-10 nmol/g body weight (BW)). In the rabbit model of intestinal I/R injury, T1 values were derived from the T1 maps in the intestinal wall region and revealed a relationship between the dose of the infused MnCl2 and the intestinal wall relaxation time. Significant Mn(2+) clearance was also observed over time in control animals after the infusion of Mn(2+) at a dose of 10 nmol/g BW. This technique was also shown to be sensitive enough to monitor variations in calcium ion homeostasis in vivo after small intestinal I/R injury. The T1 values of the intestinal I/R group were significantly lower (P < 0.05) than that of the control group at 5, 10, and 15 min after Mn(2+) infusion. Our data suggest that MnCl2 has the potential to be an MRI contrast agent that can be effectively used to monitor changes in intracellular Ca(2+) homeostasis during the early stages of intestinal I/R injury.

A background Ca2+ entry pathway mediated by TRPC1/TRPC4 is critical for development of pathological cardiac remodelling.

Pathological cardiac hypertrophy is a major predictor for the development of cardiac diseases. It is associated with chronic neurohumoral stimulation and with altered cardiac Ca(2+) signalling in cardiomyocytes. TRPC proteins form agonist-induced cation channels, but their functional role for Ca(2+) homeostasis in cardiomyocytes during fast cytosolic Ca(2+) cycling and neurohumoral stimulation leading to hypertrophy is unknown. In a systematic analysis of multiple knockout mice using fluorescence imaging of electrically paced adult ventricular cardiomyocytes and Mn(2+)-quench microfluorimetry, we identified a background Ca(2+) entry (BGCE) pathway that critically depends on TRPC1/C4 proteins but not others such as TRPC3/C6. Reduction of BGCE in TRPC1/C4-deficient cardiomyocytes lowers diastolic and systolic Ca(2+) concentrations both, under basal conditions and under neurohumoral stimulation without affecting cardiac contractility measured in isolated hearts and in vivo. Neurohumoral-induced cardiac hypertrophy as well as the expression of foetal genes (ANP, BNP) and genes regulated by Ca(2+)-dependent signalling (RCAN1-4, myomaxin) was reduced in TRPC1/C4 knockout (DKO), but not in TRPC1- or TRPC4-single knockout mice. Pressure overload-induced hypertrophy and interstitial fibrosis were both ameliorated in TRPC1/C4-DKO mice, whereas they did not show alterations in other cardiovascular parameters contributing to systemic neurohumoral-induced hypertrophy such as renin secretion and blood pressure. The constitutively active TRPC1/C4-dependent BGCE fine-tunes Ca(2+) cycling in beating adult cardiomyocytes. TRPC1/C4-gene inactivation protects against development of maladaptive cardiac remodelling without altering cardiac or extracardiac functions contributing to this pathogenesis.

A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations

The particular importance of Ca(2+) signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca(2+) ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca(2+). A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca(2+) ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca(2+) transients generated by cell stimulation and impairs its Ca(2+) extrusion function under conditions of low resting cytosolic Ca(2+) as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca(2+)-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca(2+) homeostasis and the previous finding that PMCAs act as digenic modulators in Ca(2+)-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype.

Regulation of RYR2 by sarcoplasmic reticulum Ca(2+).

Ca(2+) is arguably the most important ion involved in the contraction of the heart. The cardiac ryanodine receptor (RyR2), the major Ca(2+) release channel located in the sarcoplasmic reticulum (SR) membrane, is responsible for releasing the bulk of Ca(2+) required for contraction. Moreover, RyR2 is also crucial for maintaining SR Ca(2+) homeostasis by releasing Ca(2+) from the SR when it becomes overloaded with Ca(2+) . During normal contraction, RyR2 is activated by cytosolic Ca(2+) , whereas during store overload conditions, the opening of RyR2 is governed by SR Ca(2+) . Although the process of the cytosolic control of RyR2 is well established, the molecular mechanism by which SR luminal Ca(2+) regulates RyR2 has only recently been elucidated and remains controversial. In addition to the activation of RyR2, SR luminal Ca(2+) also determines when the RyR2 channel closes. RyR2-mediated Ca(2+) release from the SR does not continue until the SR is completely depleted. Rather, it ceases when SR luminal Ca(2+) falls below a certain level. Given the importance of SR Ca(2+) , it is not surprising that the SR luminal Ca(2+) level is tightly controlled by SR Ca(2+) -buffering proteins. Consequently, the opening and closing of RyR2 is heavily influenced by the presence of such proteins, particularly those associated with RyR2, such as calsequestrin and the histidine-rich Ca(2+) -binding protein. These proteins appear to indirectly alter RyR2 activity by modifying the microdomain SR Ca(2+) level surrounding RyR2.

Pharmacological characterization of the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) as a potent NCX3 inhibitor that worsens anoxic injury in cortical neurons, organotypic hippocampal cultures, and ischemic brain.

The Na(+)/Ca(2+) exchanger (NCX), a 10-transmembrane domain protein mainly involved in the regulation of intracellular Ca(2+) homeostasis, plays a crucial role in cerebral ischemia. In the present paper, we characterized the effect of the newly synthesized compound 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) on the activity of the three NCX isoforms and on the evolution of cerebral ischemia. BED inhibited NCX isoform 3 (NCX3) activity (IC50 = 1.9 nM) recorded with the help of single-cell microflorimetry, (45)Ca(2+) radiotracer fluxes, and patch-clamp in whole-cell configuration. Furthermore, this drug displayed negligible effect on NCX2, the other isoform expressed within the CNS, and it failed to modulate the ubiquitously expressed NCX1 isoform. Concerning the molecular site of action, the use of chimera strategy and deletion mutagenesis showed that α1 and α2 repeats of NCX3 represented relevant molecular determinants for BED inhibitory action, whereas the intracellular regulatory f-loop was not involved. At 10 nM, BED worsened the damage induced by oxygen/glucose deprivation (OGD) followed by reoxygenation in cortical neurons through a dysregulation of [Ca(2+)]i. Furthermore, at the same concentration, BED significantly enhanced cell death in CA3 subregion of hippocampal organotypic slices exposed to OGD and aggravated infarct injury after transient middle cerebral artery occlusion in mice. These results showed that the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride is one of the most potent inhibitor of NCX3 so far identified, representing an useful tool to dissect the role played by NCX3 in the control of Ca(2+) homeostasis under physiological and pathological conditions.

Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII.

AimsEnhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca2+-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca2+-leak in atrial cardiomyocytes (CMs).Methods and resultsIn murine atrial CMs, SR-Ca2+-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca2+/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late INa (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca2+-leak. The SR-Ca2+-leak induction by ATX-II was not detected when either the Na+/Ca2+ exchanger was inhibited (KBR) or in CaMKIIδc-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late INa-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late INa did not alter Ca2+-transient amplitude or SR-Ca2+-load. However, upon late INa activation and simultaneous CaMKII inhibition, Ca2+-transient amplitude and SR-Ca2+-load were increased, whereas PKA inhibition reduced Ca2+-transient amplitude and load and additionally slowed Ca2+ elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late INa, CaMKII, or PKA reduced the SR-Ca2+-leak.ConclusionLate INa exerts distinct effects on Ca2+ homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late INa represents a potential approach to attenuate CaMKII activation and decreases SR-Ca2+-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late INa inhibition.

Adaptation by the collecting duct to an exogenous acid load is blunted by deletion of the proton-sensing receptor GPR4.

We previously reported that the deletion of the pH sensor GPR4 causes a non-gap metabolic acidosis and defective net acid excretion (NAE) in the GPR4 knockout mouse (GPR4-/-) (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, and Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). Since the major regulatory site of NAE in the kidney is the collecting duct (CD), we examined acid-base transport proteins in intercalated cells (ICs) of the CD and found comparable mRNA expression of kidney anion exchanger 1 (kAE1), pendrin, and the a4 subunit of H(+)-ATPase in GPR4-/- vs. +/+. However, NH4Cl loading elicited adaptive doubling of AE1 mRNA in GPR4+/+, but a 50% less pronounced response in GPR4-/-. In GPR4+/+, NH4Cl loading evoked a cellular response characterized by an increase in AE1-labeled and a decrease in pendrin-labeled ICs similar to what was reported in rabbits and rats. This response did not occur in GPR4-/-. Microperfusion experiments demonstrated that the activity of the basolateral Cl(-)/HCO3(-) exchanger, kAE1, in CDs isolated from GPR4-/- failed to increase with NH4Cl loading, in contrast to the increase observed in GPR4+/+. Therefore, the deficiency of GPR4 blunted, but did not eliminate the adaptive response to an acid load, suggesting a compensatory response from other pH/CO2/bicarbonate sensors. Indeed, the expression of the calcium-sensing receptor (CaSR) was nearly doubled in GPR4-/- kidneys, in the absence of apparent disturbances of Ca(2+) homeostasis. In summary, the expression and activity of the key transport proteins in GPR4-/- mice are consistent with spontaneous metabolic acidosis, but the adaptive response to a superimposed exogenous acid load is blunted and might be partially compensated for by CaSR.

The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport

The sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) is responsible for intracellular Ca(2+) homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca(2+)-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca(2+) transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. In this study we demonstrate that deletion of SLN N-terminal residues (2)ERSTQ leads to loss of the uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Furthermore, molecular dynamics simulations of SLN and SERCA interaction showed a rearrangement of SERCA residues that is altered when the SLN N terminus is deleted. Interestingly, transfer of the PLB cytosolic domain to the SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. Further introduction of the PLB TM region into this chimera resulted in conversion to full PLB-like function. We also found that swapping PLB N and C termini with those from SLN caused the resulting chimera to acquire SLN-like function. Swapping the C terminus alone was not sufficient for this conversion. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however, the resulting chimeras acquire functions different from the parent molecules. Importantly, our studies highlight that the N termini of SLN and PLB influence their respective unique functions.

Testosterone modulates cardiac contraction and calcium homeostasis: cellular and molecular mechanisms.

The incidence of cardiovascular disease rises dramatically with age in both men and women. Because a woman’s risk of cardiovascular disease rises markedly after the onset of menopause, there has been growing interest in the effect of estrogen on the heart and its role in the pathophysiology of these diseases. Much less attention has been paid to the impact of testosterone on the heart, even though the levels of testosterone also decline with age and low-testosterone levels are linked to the development of cardiovascular diseases. The knowledge that receptors for all major sex steroid hormones, including testosterone, are present on individual cardiomyocytes suggests that these hormones may influence the heart at the cellular level. Indeed, it is well established that there are male-female differences in intracellular Ca2+ release and contraction in isolated ventricular myocytes. Growing evidence suggests that these differences arise from effects of sex steroid hormones on processes involved in intracellular Ca2+ homeostasis. This review considers how myocardial contractile function is modified by testosterone, with a focus on the impact of testosterone on processes that regulate Ca2+ handling at the level of the ventricular myocyte. The idea that testosterone regulates Ca2+ handling in the heart is important, as Ca2+ dysregulation plays a key role in the pathogenesis of a variety of different cardiovascular diseases. A better understanding of sex hormone regulation of myocardial Ca2+ homeostasis may reveal new targets for the treatment of cardiovascular diseases in all older adults.

Tracing the Evolutionary History of Inositol, 1, 4, 5-Trisphosphate Receptor: Insights from Analyses of Capsaspora owczarzaki Ca2+ Release Channel Orthologs.

Cellular Ca(2+) homeostasis is tightly regulated and is pivotal to life. Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) are the major ion channels that regulate Ca(2+) release from intracellular stores. Although these channels have been extensively investigated in multicellular organisms, an appreciation of their evolution and the biology of orthologs in unicellular organisms is largely lacking. Extensive phylogenetic analyses reveal that the IP3R gene superfamily is ancient and diverged into two subfamilies, IP3R-A and IP3R-B/RyR, at the dawn of Opisthokonta. IP3R-B/RyR further diversified into IP3R-B and RyR at the stem of Filozoa. Subsequent evolution and speciation of Holozoa is associated with duplication of IP3R-A and RyR genes, and loss of IP3R-B in the vertebrate lineages. To gain insight into the properties of IP3R important for the challenges of multicellularity, the IP3R-A and IP3R-B family orthologs were cloned from Capsaspora owczarzaki, a close unicellular relative to Metazoa (designated as CO.IP3R-A and CO.IP3R-B). Both proteins were targeted to the endoplasmic reticulum. However, CO.IP3R-A, but strikingly not CO.IP3R-B, bound IP3, exhibited robust Ca(2+) release activity and associated with mammalian IP3Rs. These data indicate strongly that CO.IP3R-A as an exemplar of ancestral IP3R-A orthologs forms bona fide IP3-gated channels. Notably, however, CO.IP3R-A appears not to be regulated by Ca(2+), ATP or Protein kinase A-phosphorylation. Collectively, our findings explore the origin, conservation, and diversification of IP3R gene families and provide insight into the functionality of ancestral IP3Rs and the added specialization of these proteins in Metazoa.

The Role of Na+/Ca2+ Exchanger Subtypes in Neuronal Ischemic Injury

The Na(+)/Ca(2+) exchanger (NCX) plays an important role in the maintenance of Na(+) and Ca(2+) homeostasis in most cells including neurons under physiological and pathological conditions. It exists in three subtypes (NCX1-3) with different tissue distributions but all of them are present in the brain. NCX transports Na(+) and Ca(2+) in either Ca(2+)-efflux (forward) or Ca(2+)-influx (reverse) mode, depending on membrane potential and transmembrane ion gradients. During neuronal ischemia, Na(+) and Ca(2+) ionic disturbances favor NCX to work in reverse mode, giving rise to increased intracellular Ca(2+) levels, while it may regain its forward mode activity on reperfusion. The exact significance of NCX in neuronal ischemic and reperfusion states remains unclear. The differential role of NCX subtypes in ischemic neuronal injury has been extensively investigated using various pharmacological tools as well as genetic models. This review discusses the mode of action of NCX in ischemic and reperfusion states, the differential roles played by NCX subtypes in these states as well as the role of NCX in pre- and postconditioning. NCX subtypes carry variable roles in ischemic injury. Furthermore, the mode of action of each subtype varies in ischemia and reperfusion states. Thus, therapeutic targeting of NCX in stroke should be based on appropriate timing of the administration of NCX subtype-specific strategies.

The solution structure of human calcium-bound S100A4 mutated at four cysteine loci.

The S100 proteins constitute the largest family within the EF-hand protein superfamily, shown to bind and control various proteins involved in several cellular functions such as proliferation, differentiation, apoptosis, Ca homeostasis, and energy metabolism (Donato et al. 2013; Hermann et al. 2012; Leclerc and Heizmann 2011). They regulate a wide range of important cellular processes via protein–protein interactions (Schafer and Heizmann 1996). Calcium interactions with the EF-hand motifs result in a conformation change of target protein binding by exposing hydrophobic regions in S100 proteins (Smith and Shaw 1998). The calcium binding EF-hand motif starts the action with structural changes in the S100 proteins, allowing them to interact through target selectivity (Yap et al. 1999; Zimmer and Weber 2010). The protein S100A4 was first derived from both tumor and stroma. It is a homodimeric protein in solution and has been shown to function as a metastasis-promoting protein (Ambartsumian et al. 2005; Ismail et al. 2010). Its presence has now been well documented in many cancers including breast, colorectal, gastric, pancreatic, and bladder cancers. It plays a role in tumor formation and angiogenesis (Ambartsumian et al. 2001; Barraclough et al. 2009; Ford and Zain 1995). The hinge region and C-terminal EF-hand of the S100A4 protein are unique compared to other S100 proteins; however most S100 proteins are involved in target protein binding. The binding of calcium ions produces conformational changes in proteins resulting in exposure of the hydrophobic pocket of residues in helices 3 and 5, the hinge region, and the C-terminal EF-hand (Malashkevich et al. 2008; Mishra et al. 2012; Semov et al. 2005). Previous results have provided insights into the dynamic mechanism of the C-terminal in S100A4 as a mediator of S100A4-driven metastasis, and they highlight its role in tuning the Ca-binding affinity of S100A4. These results also suggest that locking the C-terminus to the core domain may be an alternative strategy for inhibiting its metastasispromoting activities (Duelli et al. 2014). In this study, we mutated four cysteine residues (Cys3, Cys76, Cys81, and Cys86) on S100A4 to serine. The Cys3 residue is located very near the N-terminus, and the remaining three are in helix 4 (H4) (Pathuri et al. 2008). We labeled this protein ‘mutant S100A4’ and determined the three-dimensional structure of the calcium-bound of this protein using NMR. With this structure solved, we could explain why full length S100A4 has weaker calcium-binding affinity than its truncated form, where the last 13 amino acids in the C-terminal are deleted.

Experimental Colitis Is Associated with Transcriptional Inhibition of Na+/Ca2+ Exchanger Isoform 1 (NCX1) Expression by Interferon γ in the Renal Distal Convoluted Tubules

NCX1 is a Na(+)/Ca(2+) exchanger, which is believed to provide a key route for basolateral Ca(2+) efflux in the renal epithelia, thus contributing to renal Ca(2+) reabsorption. Altered mineral homeostasis, including intestinal and renal Ca(2+) transport may represent a significant component of the pathophysiology of the bone mineral density loss associated with Inflammatory Bowel Diseases (IBD). The objective of our research was to investigate the effects of TNBS and DSS colitis and related inflammatory mediators on renal Ncx1 expression. Colitis was associated with decreased renal Ncx1 expression, as examined by real-time RT-PCR, Western blotting, and immunofluorescence. In mIMCD3 cells, IFNγ significantly reduced Ncx1 mRNA and protein expression. Similar effects were observed in cells transiently transfected with a reporter construct bearing the promoter region of the kidney-specific Ncx1 gene. This inhibitory effect of IFNγ is mediated by STAT1 recruitment to the proximal promoter region of Ncx1. Further in vivo study with Stat1(-/-) mice confirmed that STAT1 is indeed required for the IFNγ mediated Ncx1 gene regulation. These results strongly support the hypothesis that impaired renal Ca(2+) handling occurs in experimental colitis. Negative regulation of NCX1- mediated renal Ca(2+) absorption by IFNγ may significantly contribute to the altered Ca(2+) homeostasis in IBD patients and to IBD-associated loss of bone mineral density.

Calcium dysregulation via L-type voltage-dependent calcium channels and ryanodine receptors underlies memory deficits and synaptic dysfunction during chronic neuroinflammation

BackgroundChronic neuroinflammation and calcium (Ca+2) dysregulation are both components of Alzheimer’s disease. Prolonged neuroinflammation produces elevation of pro-inflammatory cytokines and reactive oxygen species which can alter neuronal Ca+2 homeostasis via L-type voltage-dependent Ca+2 channels (L-VDCCs) and ryanodine receptors (RyRs). Chronic neuroinflammation also leads to deficits in spatial memory, which may be related to Ca+2 dysregulation.MethodsThe studies herein use an in vivo model of chronic neuroinflammation: rats were infused intraventricularly with a continuous small dose of lipopolysaccharide (LPS) or artificial cerebrospinal fluid (aCSF) for 28 days. The rats were treated with the L-VDCC antagonist nimodipine or the RyR antagonist dantrolene.ResultsLPS-infused rats had significant memory deficits in the Morris water maze, and this deficit was ameliorated by treatment with nimodipine. Synaptosomes from LPS-infused rats had increased Ca+2 uptake, which was reduced by a blockade of L-VDCCs either in vivo or ex vivo.ConclusionsTaken together, these data indicate that Ca+2 dysregulation during chronic neuroinflammation is partially dependent on increases in L-VDCC function. However, blockade of the RyRs also slightly improved spatial memory of the LPS-infused rats, demonstrating that other Ca+2 channels are dysregulated during chronic neuroinflammation. Ca+2-dependent immediate early gene expression was reduced in LPS-infused rats treated with dantrolene or nimodipine, indicating normalized synaptic function that may underlie improvements in spatial memory. Pro-inflammatory markers are also reduced in LPS-infused rats treated with either drug. Overall, these data suggest that Ca+2 dysregulation via L-VDCCs and RyRs play a crucial role in memory deficits resulting from chronic neuroinflammation.

Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites

The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca(2+) homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt-mediated ER-PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Syt-dependent contacts were by far the predominant contacts, ER-PM distance (19-22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca(2+) shortened the ER-PM distance at E-Syt1-dependent contacts sites. E-Syt-mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca(2+) channel Orai1 as well as store operated Ca(2+) entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt-dependent ER-PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.

Novel insights into mechanisms for Pak1-mediated regulation of cardiac Ca(2+) homeostasis.

A series of recent studies report novel roles for Pak1, a key member of the highly conserved family of serine-threonine protein kinases regulated by Ras-related small G-proteins, Cdc42/Rac1, in cardiac physiology and cardioprotection. Previous studies had identified Pak1 in the regulation of hypertrophic remodeling that could potentially lead to heart failure. This article provides a review of more recent findings on the roles of Pak1 in cardiac Ca2+ homeostasis. These findings identified crucial roles for Pak1 in cardiomyocyte Ca2+ handling and demonstrated that it functions through unique mechanisms involving regulation of the post-transcriptional activity of key Ca2+-handling proteins, including the expression of Ca2+-ATPase SERCA2a, along with the speculative possibility of an involvement in the maintenance of transverse (T)-tubular structure. They highlight important regulatory functions of Pak1 in Ca2+ homeostasis in cardiac cells, and identify novel potential therapeutic strategies directed at manipulation of Pak1 signaling for the management of cardiac disease, particularly heart failure.