T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Identification of a Calmodulin-Regulated Ca2+-ATPase in the Endoplasmic Reticulum1

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical ���ER-type��� Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.

Impairment of calcium ATPases by high glucose and potential pharmacological protection

The review deals with impairment of Ca2+-ATPases by high glucose or its derivatives in vitro, as well as in human diabetes and experimental animal models. Acute increases in glucose level strongly correlate with oxidative stress. Dysfunction of Ca2+-ATPases in diabetic and in some cases even in nondiabetic conditions may result in nitration of and in irreversible modification of cysteine-674. Nonenyzmatic protein glycation might lead to alteration of Ca2+-ATPase structure and function contributing to Ca2+ imbalance and thus may be involved in development of chronic complications of diabetes. The susceptibility to glycation is probably due to the relatively high percentage of lysine and arginine residues at the ATP binding and phosphorylation domains. Reversible glycation may develop into irreversible modifications (advanced glycation end products, AGEs). Sites of SERCA AGEs are depicted in this review. Finally, several mechanisms of prevention of Ca2+-pump glycation, and their advantages and disadvantages are discussed. �� 2013 Informa UK, Ltd.

Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.

Control of ocular blood flow occurs predominantly at the level of the retinal and choroidal arterioles. The present article provides an overview of the Ca2 + handling mechanisms and plasmalemmal ion channels involved in the regulation of retinal and choroidal arteriolar smooth muscle tone. Increases in global intracellular free Ca2 + ([Ca2 +]i) involve multiple mechanisms, including agonist-dependent release of Ca2 + from intracellular stores through activation of the inositol trisphosphate (IP3) pathway. Ca2 + enters by voltage-dependent L-type Ca2 + channels and novel dihydropyridine-sensitive store-operated nonselective cation channels. Ca2 + extrusion is mediated by plasmalemmal Ca2 +-ATPases and through Na+/Ca2+ exchange. Local Ca2 + transients (Ca2 + sparks) play an important excitatory role, acting as the building blocks for more global Ca2 + signals that can initiate vasoconstriction. K+ and Cl- channels may also affect cell function by modulating membrane potential. The precise contribution of each of these mechanisms to the regulation of retinal and choroidal perfusion in vivo warrants future investigation.

Subcellular localization and kinetic characterization of a gill (Na +, K+)-ATPase from the giant freshwater prawn macrobrachium rosenbergii

The stimulation by Mg2+, Na+, K+, NH 4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na +, K+)-ATPase ��-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single ��-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 �� 2.3 U mg-1, K 0.5 = 0.10 �� 0.01 mmol L-1. Stimulation by Na+ (V M = 110.0 �� 3.3 U mg-1, K 0.5 = 1.30 �� 0.03 mmol L -1), Mg2+ (V M = 115.0 �� 4.6 U mg -1, K 0.5 = 0.96 �� 0.03 mmol L-1), NH4 + (V M = 141.0 �� 5.6 U mg -1, K 0.5 = 1.90 �� 0.04 mmol L-1), and K+ (V M = 120.0 �� 2.4 U mg-1, K M = 2.74 �� 0.08 mmol L-1) followed single saturation curves and, except for K+, exhibited site-site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 �� 1.3 mol L-1. Complementary inhibition studies suggest the presence of F0F1-, Na+-, or K +-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (���25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. �� 2013 Springer Science+Business Media New York.

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -beta and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The Delta pmcA and Delta pmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the Delta calA and Delta crzA mutant strains. However, only the A. fumigatus Delta pmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Effetti dell'acido urico sulle cellule glomerulari mesangiali: meccanismi intracellulari di trasduzione del segnale e possibili implicazioni nella progressione del danno renale e nella sindrome infiammatoria in corso di nefropatie croniche

Uric acid is a major inducer of inflammation in renal interstitium and may play a role in the progression of renal damage in hyperuricemic subjects with primary nephropathies, renal vascular disease, and essential hypertension. At the same time, UA also acts as a water-soluble scavenger of reactive oxygen species. We evaluated the cellular effects of UA on cultured HMC as a potential interstitial target for abnormally elevated levels in acute and chronic renal disease. Intracellular free Ca2+ ([Ca2+]i) was monitored by microfluorometry of fura 2-loaded cells, while oxidation of intracellularly trapped non-fluorescent 2��,7��-dichlorofluorescein diacetate (DCFHDA, 20 uM) was employed to assess the generation of reactive oxygen species during 12-hr incubations with various concentrations of UA or monosodium urate. Fluorescent metabolites of DCFH-DA in the culture media of HMC were detected at 485/530 nm excitation/emission wavelengths, respectively. UA dose-dependently lowered resting [Ca2+]i (from 102��9 nM to 95��3, 57��2, 48��6 nM at 1-100 uM UA, respectively, p <0.05), leaving responses to vasoconstrictors such as angiotensin II unaffected. The effect was not due to Ca2+/H+ exchange upon acidification of the bathing media, as acetate, glutamate, lactate and other organic acids rather increased [Ca2+]i (to max. levels of 497��42 nM with 0.1 mM acetate). The decrease of [Ca2+]i was abolished by raising extracellular Ca2+ and not due to effects on Ca2+ channels or activation of Ca2+-ATPases, since unaffected by thapsigargin. The process rather appeared sensitive to removal of extracellular Na+ in combination with blockers of Na+/Ca2+ exchange, such as 2���,4���-dichlorobenzamil, pointing to a countertransport mechanism. UA dose-dependently prompted the extracellular release of oxidised DCFH (control 37��2 relative fluorescence units (RFU)/ml, 0.1uM 47��2, 1 uM 48��2, 10 uM 51��4, 0.1 mM 53��4; positive control, 10 uM sodium nitroprusside 92��5 RFU/ml, p<0.01). In summary, UA interferes with Ca2+ transport in cultured HMC, triggering oxidative stress which may initiate a sequence of events leading to interstitial injury and possibly amplifying renal vascular damage and/or the progression of chronic disease.

Consequences of depleted SERCA2-gated calcium stores in the skin

Sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) pumps belong to the family of Ca2+-ATPases responsible for the maintenance of calcium in the endoplasmic reticulum. In epidermal keratinocytes, SERCA2-controlled calcium stores are involved in cell cycle exit and onset of terminal differentiation. Hence, their dysfunction was thought to provoke impaired keratinocyte cohesion and hampered terminal differentiation. Here, we assessed cultured keratinocytes and skin biopsies from a canine family with an inherited skin blistering disorder. Cells from lesional and phenotypically normal areas of one of these dogs revealed affected calcium homeostasis due to depleted SERCA2-gated stores. In phenotypically normal patient cells, this defect compromised upregulation of p21(WAF1) and delayed the exit from the cell cycle. Despite this abnormality it failed to impede the terminal differentiation process in the long term but instead coincided with enhanced apoptosis and appearance of chronic wounds, suggestive of secondary mutations. Collectively, these findings provide the first survey on phenotypic consequences of depleted SERCA-gated stores for epidermal homeostasis that explain how depleted SERCA2 calcium stores provoke focal lesions rather than generalized dermatoses, a phenotype highly reminiscent of the human genodermatosis Darier disease.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

<p>Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.</p>

ECA1 complements yeast mutants defective in Ca2+ pumps and encodes an endoplasmic reticulum-type Ca2+-ATPase in Arabidopsis���thaliana

To understand the structure, role, and regulation of individual Ca2+ pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca2+ pumps (EC 3.6.1.38). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca2+ pumps. Yeast mutants defective in a Golgi Ca2+ pump (pmr1) or both Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn2+. Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca2+-pumping ATPases. In the presence of [��-32P]ATP, ECA1p formed a Ca2+-dependent [32P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+ pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca2+ pump in Arabidopsis. The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn2+, and the formation of a Mn2+-dependent phosphoprotein suggested that ECA1p may also regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compartments of plants.

Human atrial beta1L-adrenoceptor but not beta3-adrenoceptor activation increases force and Ca2+ current at physiological temperature

BACKGROUND AND PURPOSE It has been proposed that BRL37344, SR58611 and CGP12177 activate b3-adrenoceptors in human atrium to increase contractility and L-type Ca2+ current (ICa-L). b3-adrenoceptor agonists are potentially beneficial for the treatment of a variety of diseases but concomitant cardiostimulation would be potentially harmful. It has also been proposed that (-)-CGP12177 activates the low affinity binding site of the b1-adrenoceptor in human atrium. We therefore used BRL37344, SR58611 and (-)-CGP12177 with selective b-adrenoceptor subtype antagonists to clarify cardiostimulant b-adrenoceptor subtypes in human atrium. EXPERIMENTAL APPROACH Human right atrium was obtained from patients without heart failure undergoing coronary artery bypass or valve surgery. Cardiomyocytes were prepared to test BRL37344, SR58611 and CGP12177 effects on ICa-L. Contractile effects were determined on right atrial trabeculae. KEY RESULTS BRL37344 increased force which was antagonized by blockade of b1- and b2-adrenoceptors but not by blockade of b3-adrenoceptors with b3-adrenoceptor-selective L-748,337 (1 mM). The b3-adrenoceptor agonist SR58611 (1 nM���10 mM) did not affect atrial force. BRL37344 and SR58611 did not increase ICa-L at 37��C, but did at 24��C which was prevented by L-748,337. (-)-CGP12177 increased force and ICa-L at both 24��C and 37��C which was prevented by (-)-bupranolol (1���10 mM), but not L-748,337. CONCLUSIONS AND IMPLICATIONS We conclude that the inotropic responses to BRL37344 are mediated through b1- and b2-adrenoceptors. The inotropic and ICa-L responses to (-)-CGP12177 are mediated through the low affinity site b1L-adrenoceptor of the b1-adrenoceptor. b3-adrenoceptor-mediated increases in ICa-L are restricted to low temperatures. Human atrial b3-adrenoceptors do not change contractility and ICa-L at physiological temperature.

A raman and infrared spectroscopic study of Ca2+ dominant members of the mixite group from the Czech Republic

Raman microscopy of two mixite minerals BiCu6(AsO4)3(OH)6.3H2O from J��chymov and from Smrkovec (both Czech Republic) has been used to study their molecular structure, which is interpreted and the presence of (AsO4)3-, (AsO3OH)2-, (PO4)3- and (PO3OH)2- units, molecular water and hydroxyl ions were inferred. O-H���O hydrogen bond lengths were calculated from the Raman and infrared spectra using Libowitzky���s empirical relation. Small differences in the Raman spectra between both samples were observed and attributed to compositional and hydrogen bonding network differences.

A Raman spectroscopic study of the ���cave��� mineral ardealite Ca2(HPO4)(SO4)���4H2O

The mineral ardealite Ca2(HPO4)(SO4)���4H2O is a ���cave��� mineral and is formed through the reaction of calcite with bat guano. The mineral shows disorder and the composition varies depending on the origin of the mineral. Raman spectroscopy complimented with infrared spectroscopy has been used to characterise the mineral ardealite. The Raman spectrum is very different from that of gypsum. Bands are assigned to SO42- and HPO42- stretching and bending modes.