A Ca2+‐binding protein with numerous roles and uses: parvalbumin in molecular biology and physiology

Parvalbumins (PVs) are acidic, intracellular Ca²⁺‐binding proteins of low molecular weight. They are associated with several Ca²⁺‐mediated cellular activities and physiological processes. It has been suggested that PV might function as a “Ca²⁺ shuttle” transporting Ca²⁺ from troponin‐C (TnC) to the sarcoplasmic reticulum (SR) Ca²⁺ pump during muscle relaxation. Thus, PV may contribute to the performance of rapid, phasic movements by accelerating the contraction–relaxation cycle of fast‐twitch muscle fibers. Interestingly, PVs promote the generation of power stroke in fish by speeding up the rate of relaxation and thus provide impetus to attain maximal sustainable speeds. However, immunological monitoring of diverse tissues demonstrated that PVs are also present in non‐muscle cells. The axoplasmic transport and various intracellular secretory mechanisms including the endocrine secretions seem to be controlled by the Ca²⁺ regulation machinery. Any defect in the Ca²⁺ handling apparatus may cause several clinical problems; for instance, PV deficiency alters the neuronal activity, a key mechanism leading to epileptic seizures. Moreover, atypical relaxation of the heart results in diastolic dysfunction, which is a major cause of heart failure predominantly among the aged people. PV may offer a unique potential to correct defective relaxation in energetically compromised failing hearts through PV gene transfer. Consequently, PV gene transfer may present a new therapeutic approach to correct cellular disturbances in Ca²⁺ signaling pathways of diseased organs. Hence, PVs appear to be amazingly useful candidate proteins regulating a variety of cellular functions through action on Ca²⁺ flux management.     

Characterization of a 1,25(OH)2-vitamin D3-responsive capacitative Ca2+ entry pathway in rat osteoblast-like cells

We investigated the existence of a capacitative Ca2+ entry (CCE) pathway in ROS 17/2.8 osteoblast-like cells and its responsiveness to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3]. Depletion of inner Ca2+ stores with thapsigargin or 1,25(OH)2D3 in the absence of extracellular Ca2+ transiently elevated cytosolic Ca2+ ([Ca2+]i); after recovery of basal values, Ca2+ re-addition to the medium markedly increased Ca2+ entry, reflecting pre-activation of a CCE pathway. Recovery of the Ca2+ overshoot that followed the induced CCE was mainly mediated by the plasma membrane Ca2+-ATPase. Addition of 1,25(OH)2D3 to the declining phase of the thapsigargin-induced CCE did not modify further [Ca2+]i, indicating that steroid activation of CCE was dependent on store depletion. Pre-treatment with 1 microM Gd3+ inhibited 30% both thapsigargin- and 1,25(OH)2D3-stimulated CCE, whereas 2.5 microM Gd3+ was required for maximal inhibition ( approximately 85%). The activated CCE was permeable to both Mn2+ and Sr2+. Mn2+ entry sensitivity to Gd3+ was the same as that of the CCE. However, 1-microM Gd3+ completely prevented capacitative Sr2+ influx, whereas subsequent Ca2+ re-addition was reduced only 30%. These results suggest that in ROS 17/2.8 cells CCE induced by thapsigargin or 1,25(OH)2D3 is contributed by at least two cation entry pathways: a Ca2+/Mn2+ permeable route insensitive to very low micromolar (1 microM) Gd3+ accounting for most of the CCE and a minor Ca2+/Sr2+/Mn2+ permeable route highly sensitive to 1 microM Gd3+. The Ca2+-mobilizing agonist ATP also stimulated CCE resembling the Ca2+/Sr2+/Mn2+ permeable entry activated by 1,25(OH)2D3. The data demonstrates for the first time, the presence of a hormone-responsive CCE pathway in an osteoblast cell model, raising the possibility that it could be an alternative Ca2+ influx route through which osteotropic agents influence osteoblast Ca2+ homeostasis. Copyright Wiley-Liss, Inc.     

Ca~(2+)转运通路对金雀异黄酮舒张大鼠脑血管作用的影响

目的:研究Ca2+转运通路对金雀异黄酮舒张大鼠脑血管作用的影响。方法:75只大鼠被随机分为3组,分别经由二甲亚砜、金雀异黄酮和酪氨酸磷酸化抑制剂A47处理基底动脉及Willis环血管。每组大鼠进一步划分成5个亚组,每个亚组用不同浓度的细胞外Ca2+处理,分为:0、0.6、1.2、1.8和3.6 m M Ca2+组。5-羟色胺诱导血管收缩。测定大鼠基底动脉管壁厚度与官腔周长的比值;荧光成像分析法测定血管平滑肌细胞细胞内Ca2+浓度;免疫印迹分析检测肌球蛋白轻链激酶(MLCK),蛋白质磷酸酶催化亚基1(PP1),肌凝蛋白磷酸酶目标亚基1(MYPT1)的表达来测定血管平滑肌细胞Ca2+敏感性。结果:金雀异黄酮和酪氨酸磷酸化抑制剂A47显著降低大鼠基底动脉管壁厚度与官腔周长的比值(P0.01),Ca2+内流(P0.01,P0.05)及MLCK的表达(P0.01);增加PP1和MYPT1的表达(P0.01)。细胞外Ca2+与金雀异黄酮及酪氨酸磷酸化抑制剂A47有协同效应。硝苯地平和毒胡萝卜素可废除该效应。结论:低细胞外Ca2+水平增强了金雀异黄酮和酪氨酸磷酸化抑制剂A47的血管舒张作用。L型电压门控Ca2+通道(L-VGCC)和肌浆网Ca2+库(SR)参与交互效应。     

MicroRNA‐129‐1‐3p protects cardiomyocytes from pirarubicin‐induced apoptosis by down‐regulating the GRIN2D‐mediated Ca2+ signalling pathway

Pirarubicin (THP), an anthracycline anticancer drug, is a first‐line therapy for various solid tumours and haematologic malignancies. However, THP can cause dose‐dependent cumulative cardiac damage, which limits its therapeutic window. The mechanisms underlying THP cardiotoxicity are not fully understood. We previously showed that MiR‐129‐1‐3p, a potential biomarker of cardiovascular disease, was down‐regulated in a rat model of THP‐induced cardiac injury. In this study, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses to determine the pathways affected by miR‐129‐1‐3p expression. The results linked miR‐129‐1‐3p to the Ca²⁺ signalling pathway. TargetScan database screening identified a tentative miR‐129‐1‐3p‐binding site at the 3′‐UTR of GRIN2D, a subunit of the N‐methyl‐D‐aspartate receptor calcium channel. A luciferase reporter assay confirmed that miR‐129‐1‐3p directly regulates GRIN2D. In H9C2 (rat) and HL‐1 (mouse) cardiomyocytes, THP caused oxidative stress, calcium overload and apoptotic cell death. These THP‐induced changes were ameliorated by miR‐129‐1‐3p overexpression, but exacerbated by miR‐129‐1‐3p knock‐down. In addition, miR‐129‐1‐3p overexpression in cardiomyocytes prevented THP‐induced changes in the expression of proteins that are either key components of Ca²⁺ signalling or important regulators of intracellular calcium trafficking/balance in cardiomyocytes including GRIN2D, CALM1, CaMKⅡδ, RyR2‐pS2814, SERCA2a and NCX1. Together, these bioinformatics and cell‐based experiments indicate that miR‐129‐1‐3p protects against THP‐induced cardiomyocyte apoptosis by down‐regulating the GRIN2D‐mediated Ca²⁺ pathway. Our results reveal a novel mechanism underlying the pathogenesis of THP‐induced cardiotoxicity. The miR‐129‐1‐3p/Ca²⁺ signalling pathway could serve as a target for the development of new cardioprotective agents to control THP‐induced cardiotoxicity.     

Temporal switching and cell‐to‐cell variability in Ca2+ release activity in mammalian cells

Genetically identical cells in a uniform external environment can exhibit different phenotypes, which are often masked by conventional measurements that average over cell populations. Although most studies on this topic have used microorganisms, differentiated mammalian cells have rarely been explored. Here, we report that only approximately 40% of clonal human embryonic kidney 293 cells respond with an intracellular Ca²⁺ increase when ryanodine receptor Ca²⁺ release channels in the endoplasmic reticulum are maximally activated by caffeine. On the other hand, the expression levels of ryanodine receptor showed a unimodal distribution. We showed that the difference in the caffeine sensitivity depends on a critical balance between Ca²⁺ release and Ca²⁺ uptake activities, which is amplified by the regenerative nature of the Ca²⁺ release mechanism. Furthermore, individual cells switched between the caffeine‐sensitive and caffeine‐insensitive states with an average transition time of approximately 65 h, suggestive of temporal fluctuation in endogenous protein expression levels associated with caffeine response. These results suggest the significance of regenerative mechanisms that amplify protein expression noise and induce cell‐to‐cell phenotypic variation in mammalian cells.     

Spermidine oxidase‐derived H2O2 regulates pollen plasma membrane hyperpolarization‐activated Ca2+‐permeable channels and pollen tube growth

Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca²⁺ concentration that accompanies pollen tube growth. Using the whole‐cell patch clamp and outside‐out single‐channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization‐activated Ca²⁺ current: the addition of Spd cannot induce the channel open probability increase in excised outside‐out patches, indicating that the effect of Spd in the induction of Ca²⁺ currents is exerted via a second messenger. This messenger is hydrogen peroxide (H₂O₂), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization‐activated Ca²⁺‐permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca²⁺‐permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal‐encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca²⁺‐permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca²⁺ influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd‐derived H₂O₂ signals Ca²⁺ influx, thereby regulating pollen tube growth.     

Dynamic regulation of guard cell anion channels by cytosolic free Ca2+ concentration and protein phosphorylation

In guard cells, activation of anion channels (I_anion) is an early event leading to stomatal closure. Activation of I_anion has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). However, the dynamics of the action of [Ca²⁺]_i on I_anion has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca²⁺]_i dynamics of I_anion in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca²⁺]_i using Fura‐2 fluorescence imaging. We found that I_anion rises with [Ca²⁺]_i only at concentrations substantially above the mean resting value of 125 ± 13 nm , yielding an apparent K_d of 720 ± 65 nm and a Hill coefficient consistent with the binding of three to four Ca²⁺ ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of I_anion activity, but without a dependence of the current on [Ca²⁺]_i. The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca²⁺]_i sensitivity of I_anion, displacing the apparent K_d for [Ca²⁺]_i to 573 ± 38 nm . These findings support previous evidence for different modes of regulation for I_anion, only one of which depends on [Ca²⁺]_i, and they underscore an independence of [Ca²⁺]_i from protein (de‐)phosphorylation in controlling I_anion. Most importantly, our results demonstrate a significant displacement of I_anion sensitivity to higher [Ca²⁺]_i compared with that of the guard cell K⁺ channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.     

Design of Ca2+‐independent Staphylococcus aureus sortase A mutants

The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca²⁺, because binding of Ca²⁺ to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca²⁺‐independent SaSrtA, we substituted two Glu residues in the Ca²⁺‐binding pocket (Glu¹⁰⁵ and Glu¹⁰⁸). Although single mutations decreased SaSrtA activity, mutations of both Glu¹⁰⁵ and Glu¹⁰⁸ resulted in Ca²⁺‐independent activity. Kinetic analysis suggested that the double mutations affect the substrate binding site, without affecting substrate specificity. This approach will allow us to develop SaSrtA variants suitable for various applications, including in vivo site‐specific protein modification and labeling. Biotechnol. Bioeng. 2012; 109: 2955–2961. © 2012 Wiley Periodicals, Inc.     

Predicting Ca2+‐binding sites using refined carbon clusters

Identifying Ca²⁺‐binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca²⁺‐binding proteins. Currently, these sites are identified in structures either through X‐ray crystallography or NMR analysis. However, Ca²⁺‐binding sites are not always visible in X‐ray structures due to flexibility in the binding region or low occupancy in a Ca²⁺‐binding site. Similarly, both Ca²⁺ and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca²⁺‐binding sites in both X‐ray and NMR structures, we report a new graph theory algorithm (MUG^C) to predict Ca²⁺‐binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side‐chain oxygen ligand co‐ordinates, MUG^C is able to achieve 94% sensitivity with 76% selectivity on a dataset of X‐ray structures composed of 43 Ca²⁺‐binding proteins. Additionally, prediction of Ca²⁺‐binding sites in NMR structures was obtained by MUG^C using a different set of parameters, which were determined by the analysis of both Ca²⁺‐constrained and unconstrained Ca²⁺‐loaded structures derived from NMR data. MUG^C identified 20 of 21 Ca²⁺‐binding sites in NMR structures inferred without the use of Ca²⁺ constraints. MUG^C predictions are also highly selective for Ca²⁺‐binding sites as analyses of binding sites for Mg²⁺, Zn²⁺, and Pb²⁺ were not identified as Ca²⁺‐binding sites. These results indicate that the geometric arrangement of the second‐shell carbon cluster is sufficient not only for accurate identification of Ca²⁺‐binding sites in NMR and X‐ray structures but also for selective differentiation between Ca²⁺ and other relevant divalent cations. © Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Neurite-specific Ca2+ dynamics underlying sound processing in an auditory interneurone

Concepts on neuronal signal processing and integration at a cellular and subcellular level are driven by recording techniques and model systems available. The cricket CNS with the omega-1-neurone (ON1) provides a model system for auditory pattern recognition and directional processing. Exploiting ON1's planar structure we simultaneously imaged free intracellular Ca(2+) at both input and output neurites and recorded the membrane potential in vivo during acoustic stimulation. In response to a single sound pulse the rate of Ca(2+) rise followed the onset spike rate of ON1, while the final Ca(2+) level depended on the mean spike rate. Ca(2+) rapidly increased in both dendritic and axonal arborizations and only gradually in the axon and the cell body. Ca(2+) levels were particularly high at the spike-generating zone. Through the activation of a Ca(2+)-sensitive K(+) current this may exhibit a specific control over the cell's electrical response properties. In all cellular compartments presentation of species-specific calling song caused distinct oscillations of the Ca(2+) level in the chirp rhythm, but not the faster syllable rhythm. The Ca(2+)-mediated hyperpolarization of ON1 suppressed background spike activity between chirps, acting as a noise filter. During directional auditory processing, the functional interaction of Ca(2+)-mediated inhibition and contralateral synaptic inhibition was demonstrated. Upon stimulation with different sound frequencies, the dendrites, but not the axonal arborizations, demonstrated a tonotopic response profile. This mirrored the dominance of the species-specific carrier frequency and resulted in spatial filtering of high frequency auditory inputs.     

Inhibition of Orai1‐mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5‐fluorouracil

Increasing evidence supports that activation of store‐operated Ca²⁺ entry (SOCE) is implicated in the chemoresistance of cancer cells subjected to chemotherapy. However, the molecular mechanisms underlying chemoresistance are not well understood. In this study, we aim to investigate whether 5‐FU induces hepatocarcinoma cell death through regulating Ca²⁺‐dependent autophagy. [Ca²⁺]_i was measured using fura2/AM dye. Protein expression was determined by Western blotting and immunohistochemistry. We found that 5‐fluorouracil (5‐FU) induced autophagic cell death in HepG2 hepatocarcinoma cells by inhibiting PI3K/AKT/mTOR pathway. Orai1 expression was obviously elevated in hepatocarcinoma tissues. 5‐FU treatment decreased SOCE and Orai1 expressions, but had no effects on Stim1 and TRPC1 expressions. Knockdown of Orai1 or pharmacological inhibition of SOCE enhanced 5‐FU‐induced inhibition of PI3K/AKT/mTOR pathway and potentiated 5‐FU‐activated autophagic cell death. On the contrary, ectopic overexpression of Orai1 antagonizes 5‐FU‐induced autophagy and cell death. Our findings provide convincing evidence to show that Orai1 expression is increased in hepatocarcinoma tissues. 5‐FU can induce autophagic cell death in HepG2 hepatocarcinoma cells through inhibition of SOCE via decreasing Orai1 expression. These findings suggest that Orai1 expression is a predictor of 5‐FU sensitivity for hepatocarcinoma treatment and blockade of Orai1‐mediated Ca²⁺ entry may be a promising strategy to sensitize hepatocarcinoma cells to 5‐FU treatment.     

Solution structures of polcalcin Phl p 7 in three ligation states: Apo‐, hemi‐Mg2+‐bound,and fully Ca2+‐bound

Polcalcins are small EF‐hand proteins believed to assist in regulating pollen‐tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain‐swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg²⁺, or 100 μM Ca²⁺. Phl p 7 is monomeric in all three ligation states. In the apo‐form, both EF‐hand motifs reside in the closed conformation, with roughly antiparallel N‐ and C‐terminal helical segments. In 5.0 mM Mg²⁺, the divalent ion is bound by EF‐hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca²⁺‐bound Phl p 7 resembles that previously reported for Bet v 4—likewise exposing apolar surface to the solvent. Occluded in the apo‐ and Mg²⁺‐bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF‐hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at –x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca²⁺. When Mg²⁺ resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer‐sphere ligand. ¹⁵N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Ca2+‐regulated and diurnal rhythm‐regulated Na+/Ca2+ exchanger AtNCL affects flowering time and auxin signalling in Arabidopsis

Calcium (Ca²⁺) is vital for plant growth, development, hormone response and adaptation to environmental stresses, yet the mechanisms regulating plant cytosolic Ca²⁺ homeostasis are not fully understood. Here, we characterize an Arabidopsis Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger AtNCL that regulates Ca²⁺ and multiple physiological processes. AtNCL was localized to the tonoplast in yeast and plant cells. AtNCL appeared to mediate sodium (Na⁺) vacuolar sequestration and meanwhile Ca²⁺ release. The EF‐hand domains within AtNCL regulated Ca²⁺ binding and transport of Ca²⁺ and Na⁺. Plants with diminished AtNCL expression were more tolerant to high CaCl₂ but more sensitive to both NaCl and auxin; heightened expression of AtNCL rendered plants more sensitive to CaCl₂ but tolerant to NaCl. AtNCL expression appeared to be regulated by the diurnal rhythm and suppressed by auxin. DR5::GUS expression and root responses to auxin were altered in AtNCL mutants. The auxin‐induced suppression of AtNCL was attenuated in SLR/IAA14 and ARF6/8 mutants. The mutants with altered AtNCL expression also altered flowering time and FT and CO expression; FT may mediate AtNCL‐regulated flowering time change. Therefore, AtNCL is a vacuolar Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger that regulates auxin responses and flowering time.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

Ca2+‐dependent activation of guard cell anion channels,triggered by hyperpolarization,is promoted by prolonged depolarization

Rapid stomatal closure is driven by the activation of S‐type anion channels in the plasma membrane of guard cells. This response has been linked to Ca²⁺ signalling, but the impact of transient Ca²⁺ signals on S‐type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca²⁺ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S‐type anion channels were monitored using intracellular triple‐barrelled micro‐electrodes. In cells kept at a holding potential of ?100 mV, voltage steps to ?180 mV triggered elevation of the cytosolic free Ca²⁺ concentration. The increase in the cytosolic Ca²⁺ level was accompanied by activation of S‐type anion channels. Guard cell anion channels were activated by Ca²⁺ with a half maximum concentration of 515 nm (SE = 235) and a mean saturation value of ?349 pA (SE = 107) at ?100 mV. Ca²⁺ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to ?100 mV, a transient increase in the cytosolic Ca²⁺ level was observed, activating S‐type channels without measurable delay. These data show that cytosolic Ca²⁺ elevation can activate S‐type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca²⁺ transport proteins, resulting in an overshoot of the cytosolic Ca²⁺ level after returning the membrane potential to ?100 mV.     

Alteration of Ca2+‐ATPase activity in the homogenate,plasma membrane and microsomes of the salivary glands of streptozotocin‐induced diabetic rats

Diabetes has been implicated in the dryness of the mouth, loss of taste sensation, sialosis, and other disorders of the oral cavity, by impairment of the salivary glands. The aim of the present study was to examine the plasma membrane, microsomal, and homogenate Ca²⁺‐ATPase activity in the rat submandibular and parotid salivary glands of streptozotocin‐induced diabetes. We have also examined the influence of the acidosis state on this parameter. Diabetes was induced by an intraperitoneal injection of streptozotocin and acidosis was induced by daily injection of NH₄Cl. At 15 and 30 days after diabetes induction, the animals were euthanized and the submandibular and parotid salivary glands were removed and analyzed. Ca²⁺‐ATPase (total, independent, and dependent) was determined in the homogenate, microsomal, and plasma membranes of the salivary glands of diabetic and control rats. Calcium concentration was also determined in the glands and showed to be higher in the diabetic animals. Ca²⁺‐ATPase activity was found to be reduced in all cell fractions studied in the diabetic animals compared with control. Similar results were obtained for the submandibular salivary glands of acidotic animals; however in the parotid salivary glands it was found an increase in the enzyme activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Hierarchical domain‐motion analysis of conformational changes in sarcoplasmic reticulum Ca2+‐ATPase

Sarco(endo)plasmic reticulum Ca²⁺‐ATPase transports two Ca²⁺ per ATP‐hydrolyzed across biological membranes against a large concentration gradient by undergoing large conformational changes. Structural studies with X‐ray crystallography revealed functional roles of coupled motions between the cytoplasmic domains and the transmembrane helices in individual reaction steps. Here, we employed “Motion Tree (MT),” a tree diagram that describes a conformational change between two structures, and applied it to representative Ca²⁺‐ATPase structures. MT provides information of coupled rigid‐body motions of the ATPase in individual reaction steps. Fourteen rigid structural units, “common rigid domains (CRDs)” are identified from seven MTs throughout the whole enzymatic reaction cycle. CRDs likely act as not only the structural units, but also the functional units. Some of the functional importance has been newly revealed by the analysis. Stability of each CRD is examined on the morphing trajectories that cover seven conformational transitions. We confirmed that the large conformational changes are realized by the motions only in the flexible regions that connect CRDs. The Ca²⁺‐ATPase efficiently utilizes its intrinsic flexibility and rigidity to response different switches like ligand binding/dissociation or ATP hydrolysis. The analysis detects functional motions without extensive biological knowledge of experts, suggesting its general applicability to domain movements in other membrane proteins to deepen the understanding of protein structure and function. Proteins 2015; 83:746–756. © 2015 Wiley Periodicals, Inc.     

Ca2+‐dependent down‐regulation of human histamine H1 receptors in Chinese hamster ovary cells

G_q/11 protein‐coupled human histamine H₁ receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin‐dependent endocytosis followed by proteasome/lysosome‐mediated down‐regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca²⁺ concentrations induced by a receptor‐bypassed stimulation with ionomycin, a Ca²⁺ ionophore, on the endocytosis and down‐regulation of H₁ receptors in Chinese hamster ovary cells. All cellular and cell‐surface H₁ receptors were detected by the binding of [³H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H₁ receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine‐ and pirdonium‐sensitive binding sites of [³H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca²⁺ and partially by a ubiquitin‐activating enzyme inhibitor (UBEI‐41), but were not affected by inhibitors of calmodulin (W‐7 or calmidazolium) and protein kinase C (chelerythrine or GF109203X). These ionomycin‐induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E‐64, leupeptin, chloroquine, or NH₄Cl), proteasomes (lactacystin or MG‐132), and a Ca²⁺‐dependent non‐lysosomal cysteine protease (calpain) (MDL28170). Since H₁ receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H₁ receptors, even after the ionomycin treatment, H₁ receptors appeared to exist in a form to which [³H]mepyramine was unable to bind. These results suggest that H₁ receptors are apparently down‐regulated by a sustained increase in intracellular Ca²⁺ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.