Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein: Is PAM involved in the capacitative calcium entry?

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca²⁺ signalling and maintenance of Ca²⁺ homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca²⁺-ATPase, Na⁺, K⁺-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca²⁺ ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca²⁺ entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca²⁺ entry, and their formation and rebuilding have an important regulatory role in cellular Ca²⁺ homeostasis.     

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.     

The single pore residue Asp in PKD2L1 determines Ca permeation of the PKD1L3/PKD2L1 complex

The polycystic kidney disease 1-like 3 (PKD1L3)–polycystic kidney disease 2-like 1 (PKD2L1) complex functions as a Ca²⁺-permeable, non-selective cation channel that is activated by acid and its subsequent removal; this is called an off-response. In this study, we identified a single aspartic residue in PKD2L1 that is responsible for the Ca²⁺ permeation of the PKD1L3/PKD2L1 complex. Calcium imaging analysis using point mutants of negatively charged amino acids present in the putative pore regions of PKD1L3 and PKD2L1 revealed that neutralization of the aspartic residue in PKD2L1 (D523N), which is conserved among PKD2 family members, abolished Ca²⁺ permeation, despite robust cell surface expression. In contrast, neutralization of the other negatively charged residues of PKD1L3 (D2049N and E2072Q) and PKD2L1 (D525N and D530N) as well as substitution of Asp⁵²³ with a glutamate residue (D523E) had little effect on Ca²⁺ permeation properties. These results demonstrate that Asp⁵²³ in PKD2L1 is a key determinant of Ca²⁺ permeation into the PKD1L3/PKD2L1 complex and that PKD2L1 contributes to forming the pore of the PKD1L3/PKD2L1 channel.     

Crystallographic and spectroscopic characterization of a molecular hinge: Conformational changes in bothropstoxin I,a dimeric Lys49-phospholipase A2 homologue

Bothropstoxin I (BthTX-I) from the venom of Bothrops jararacussuis a myotoxic phospholipase A2 (PLA2) homologue which, although catalytically inactive due to an Asp49→Lys substitution, disrupts the integrity of lipid membranes by a Ca²⁺-independent mechanism. The crystal structures of two dimeric forms of BthTX-I which diffract X-rays to resolutions of 3.1 and 2.1 Å have been determined. The monomers in both structures are related by an almost perfect twofold axis of rotation and the dimer interfaces are defined by contacts between the N-terminal α-helical regions and the tips of the β-wings of partner monomers. Significant differences in the relative orientation of the monomers in the two crystal forms results in “open” and “closed” dimer conformations. Spectroscopic investigations of BthTX-I in solution have correlated these conformational differences with changes in the intrinsic fluorescence emission of the single tryptophan residues located at the dimer interface. The possible relevance of this structural transition in the Ca²⁺-independent membrane damaging activity is discussed. Proteins 30:442–454, 1998. © 1998 Wiley-Liss, Inc.     

Antimicrotubular drugs binding to vinca domain of tubulin

The effect of oxidative stress on the Ca²⁺-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO₄/EDTA (0.2 mol Fe²⁺ per mg of protein) at 37°C for 1 h in the presence or absence of antioxidants. FeSO₄/EDTA decreased the maximum velocity of Ca²⁺-ATPase reaction without a change of affinity for Ca²⁺ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca²⁺-ATPase and decrease in tryptophan fluorescence, while the loss of –SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca²⁺-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca²⁺-ATPase activity.     

Calcium Binding to Plasma Membranes from Normal and SV40 Transformed Hamster Lymphocytes

Abstract

The calcium binding properties of isolated plasma membranes from normal and SV40 transformed hamster lymphocytes were compared over the Ca²⁺ concentration range of 10^?5M to 5 × 10^?3M and at physiological ionic strength. At all Ca²⁺ concentrations, normal membranes bound more Ca²⁺ than tumor membranes; at blood Ca²⁺ levels (1–2 mM) plasma membranes of normal cells bind twice as much as membranes from tumor cells. Normal plasma membranes demonstrated positive cooperative Ca²⁺ binding whereas tumor membranes displayed non-interacting Ca²⁺-binding sites. Ca²⁺ binding to both membranes was insensitive to Mg²⁺ (0.1 to 2.5 mM). A pH shift from 7 to 6 resulted in a 70% decrease of normal membrane-bound Ca²⁺ compared to a 40% decrease observed with tumor membranes. Extracellular surface Ca²⁺ binding to intact cells was also studied after a 72-hour equilibration of cells with ⁴⁵Ca²⁺ and with ethylene-glycol-bis-(β-amino-ethyl ether) N, N′-tetraacetate chelation as marker for surface Ca²⁺. Tumor cell surface Ca²⁺ binding was only 10% of that observed with quiescent lymphocytes. Normal lymphocytes stimulated to divide with phytohemagglutinin also showed a decreased level of surface Ca²⁺ (50%). However, plasma membranes isolated from non-dividing and phytohemagglutinin-stimulated lymphocytes exhibited equivalent Ca²⁺ binding.     

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca²⁺ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca²⁺ binding to the SARS-CoV-2-FP, the role of Ca²⁺ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca²⁺-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca²⁺-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca²⁺ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca²⁺ ions. These findings provide novel mechanistic insights regarding the role of Ca²⁺ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.     

(Ca2+ + Mg2+)-ATPase of density-separated human red cells. Effects of calcium and a soluble cytoplasmic activator (Calmodulin)

The effect of calcium and a soluble cytoplasmic activator on (Ca²⁺ + Mg²⁺)-ATPase of density-separated human red cells was investigated. At all calcium concentrations tested, dense (old) lysed cells and their isolated membranes displayed lower activities as compared to the light (young) cells and their membranes. Isolated membranes from all density red cell fractions showed two distinct (Ca²⁺ + Mg²⁺)-ATPase activities; one at low calcium and another at moderate calcium concentrations. At high calcium concentration, (Ca²⁺ + Mg²⁺)-ATPase activity of isolated membranes was low in all cell fractions. In contrast to the isolated membranes, lysed cells from all density fractions had a maximum (Ca²⁺ + Mg²⁺)-ATPase activity only at a low concentration of calcium, while moderate and high calcium concentrations produced low activity. Upon isolation of membranes, a substantial loss of (Ca²⁺ + Mg²⁺)-ATPase activity took place from all density cell fractions. Upon membrane isolation, the relative loss of (Ca²⁺ + Mg²⁺)-ATPase activity at low Ca²⁺ concentration was greater in older cells. The extent of stimulation of (Ca²⁺ + Mg²⁺)-ATPase by the activator at low calcium concentration was 3–4-fold greater in older cell membranes than in the young ones.These data suggest that the lower (Ca²⁺ + Mg²⁺)-ATPase activity in old cells could be accounted for by a selective loss of (Ca²⁺ + Mg²⁺)-ATPase activity at low Ca²⁺ concentration presumably due to reduced affinity of old cell membranes to activator protein.     

IN VITRO Ca2+ DEPENDENT PROTEOLYSIS IN BRAIN TISSUES: A POSSIBLE SOURCE OF ERROR IN TRANSMITTER METABOLISM STUDIES

—The effects of Ca²⁺ ions on the metabolism of [³H]serotonin and [³H]-labelled catecholamines have been examined in hippocampal slices or synaptosomes. The formation of [³H]-5 hydroxyindoles ([³H]serotonin + [³H]-5 hydroxyindoleacetic acid) from [³H]tryptophan and that of [³H]-labelled catecholamines from [³H]tyrosine were increased when Ca²⁺ was omitted from the incubating medium. However, the total synthesis of 5-HT from tryptophan and that of catecholamines from tyrosine did not seem to be significantly changed. Altered formation of tritiated amines were due to changes in the specific activities of respective precursor amino acids. This reflected altered sizes of the free amino acid pools caused by Ca²⁺-dependent in vitro proteolysis. This must be taken into consideration when studying in vitro Ca²⁺ dependency of neutrotransmitter metabolism.     

Modeling the structural and dynamical changes of the epithelial calcium channel TRPV5 caused by the A563T variation based on the structure of TRPV6

TRPV5, transient receptor potential cation channel vanilloid subfamily member 5, is an epithelial Ca²⁺ channel that plays a key role in the active Ca²⁺ reabsorption process in the kidney. A single nucleotide polymorphism (SNP) rs4252499 in the TRPV5 gene results in an A563T variation in the sixth transmembrane (TM) domain of TRPV5. Our previous study indicated that this variation increases the Ca²⁺ transport function of TRPV5. To understand the molecular mechanism, a model of TRPV5 was established based on the newly deposited structure of TRPV6 that has 83.1% amino acid identity with TRPV5 in the modeled region. Computational simulations were performed to study the structural and dynamical differences between the TRPV5 variants with A563 and T563. Consistent with the TRPV1-based simulation, the results indicate that the A563T variation increases the contacts between residues 563 and V540, which is one residue away from the key residue D542 in the Ca²⁺-selective filter. The variation enhanced the stability of the secondary structure of the pore region, decreased the fluctuation of residues around residue 563, and reduced correlated and anti-correlated motion between monomers. Furthermore, the variation increases the pore radius at the selective filter. These findings were confirmed using simulations based on the recently determined structure of rabbit TRPV5. The simulation results provide an explanation for the observation of enhanced Ca²⁺ influx in TRPV5 caused by the A563T variation. The A563T variation is an interesting example of how a residue distant from the Ca²⁺-selective filter influences the Ca²⁺ transport function of the TRPV5 channel.

Communicated by Ramaswamy H. Sarma     

Strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures

We evaluate the effects of strontium ranelate on the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures, a system that gave us the advantage of obtaining mineral samples produced exclusively during treatment. Cells were treated with strontium ranelate at concentrations of 0.05 and 0.5 mM Sr²⁺. Mineral substances were isolated and analyzed by using a combination of methods: Fourier transform infrared spectroscopy, solid-state ¹H nuclear magnetic resonance, X-ray diffraction, micro-Raman spectroscopy and energy dispersive X-ray spectroscopy. The minerals produced in all cell cultures were typical bone-like apatites. No changes occurred in the local structural order or crystal size of the minerals. However, we noticed several relevant changes in the mineral produced under 0.5 mM Sr²⁺: (1) increase in type-B CO₃ ^2? substitutions, which often lead to the creation of vacancies in Ca²⁺ and OH^? sites; (2) incorporation of Sr²⁺ by substituting slightly less than 10 % of Ca²⁺ in the apatite crystal lattice, resulting in an increase in both lattice parameters a and c; (3) change in the PO₄ ^3? environments, possibly because of the expansion of the lattice; (4) the Ca/P ratio of this mineral was reduced, but its (Ca+Sr)/P ratio was the same as that of the control, indicating that its overall cation/P ratio was preserved. Thus, strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures.     

The effect of taurine on the ion transport system in mitochondria

The effect of taurine on the ATP-dependent mitochondrial swelling that characterizes the activity of mitochondrial ATP-dependent K⁺ channel and the formation of Ca²⁺-dependent pores, different in sensitivity to cyclosporin A, has been studied in rat liver mitochondria. It has been shown that taurine in micromolar concentrations (0.5–125 μM) stimulates the energy-dependent swelling of mitochondria. Taurine in physiological concentrations (0.5–20 mM) has no effect on the ATP-dependent swelling and the formation of cyclosporin A-insensitive Pal/Ca²⁺-activated pore in mitochondria. Taurine in these concentrations increased the rate of cyclosporin A-sensitive swelling of mitochondria induced by Ca²⁺ and P_i and reduced the Ca²⁺ capacity of mitochondria. The different effects of physiological taurine concentrations on the ATP-dependent transport of K⁺ and Ca²⁺ ions in mitochondrial membranes as compared with cell membranes are discussed.     

Down‐regulation of Orai3 arrests cell‐cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells

Breast cancer (BC) is the leading cancer in the world in terms of incidence and mortality in women. However, the mechanism by which BC develops remains largely unknown. The increase in cytosolic free Ca²⁺ can result in different physiological changes including cell growth and death. Orai isoforms are highly Ca²⁺ selective channels. In the present study, we analyzed Orai3 expression in normal and cancerous breast tissue samples, and its role in MCF‐7 BC and normal MCF‐10A mammary epithelial cell lines. We found that the expression of Orai3 mRNAs was higher in BC tissues and MCF‐7 cells than in normal tissues and MCF‐10A cells. Down‐regulation of Orai3 by siRNA inhibited MCF‐7 cell proliferation and arrested cell cycle at G1 phase. This phenomenon is associated with a reduction in CDKs 4/2 (cyclin‐dependent kinases) and cyclins E and D1 expression and an accumulation of p21^Waf1/Cip1 (a cyclin‐dependent kinase inhibitor) and p53 (a tumor‐suppressing protein). Orai3 was also involved in MCF‐7 cell survival. Furthermore, Orai3 mediated Ca²⁺ entry and contributed to intracellular calcium concentration ([Ca²⁺]_i). In MCF‐10A cells, silencing Orai3 failed to modify [Ca²⁺]_i, cell proliferation, cell‐cycle progression, cyclins (D1, E), CDKs (4, 2), and p21^Waf1/Cip1 expression. Our results provide strong evidence for a significant effect of Orai3 on BC cell growth in vitro and show that this effect is associated with the induction of cell cycle and apoptosis resistance. Our study highlights a possible role of Orai3 as therapeutic target in BC therapy. J. Cell. Physiol. 226: 542–551, 2011. © 2010 Wiley‐Liss, Inc.     

Mitochondrial Ca uptake is inhibited by a concerted action of p38 MAPK and protein kinase D

Angiotensin II elicits cytosolic Ca²⁺ signal that is transferred into the mitochondria. Previously we found in H295R cells that this signal transfer is enhanced by both the inhibition of p38 MAPK and a novel isoform of PKC [G. Szanda, P. Koncz, A. Rajki, A. Spät, Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca²⁺ uptake, Cell Calcium 43 (2008) 250–259]. Now we report that simultaneous activation of these protein kinases (by TNFα and PMA + an inhibitor of the conventional PKC isoforms, respectively) attenuates the transfer of cytosolic Ca²⁺ signal, elicited by depolarisation or store-operated Ca²⁺ influx, into the mitochondria. The Ca²⁺ uptake enhancing effect of the p38 MAPK inhibitor SB202190 is due to the inhibition of p38 MAPK and not to a direct mitochondrial action. Protein kinases reduce mitochondrial [Ca²⁺] by inhibiting the uptake mechanism. The threshold of mitochondrial Ca²⁺ uptake may depend on the activity of p38 MAPK. The silencing of protein kinase D (PKD) also results in enhanced transfer of Ca²⁺ signal from the cytosol into the mitochondria. Our data indicate that Ca²⁺ mobilising agonists, through the simultaneous activation of p38 MAPK, a novel PKC isoform and PKD, exert a negative feed-forward action on mitochondrial Ca²⁺ uptake, thus reducing the risk of Ca²⁺ overload.     

Proliferation-associated increase in sensitivity of mammary epithelial cells to inositol-1,4,5-trisphosphate

Injection of D -myo-inositol-1,4,5-trisphosphate (IP3) was found to induce a transient increase of intracellular Ca²⁺ concentration in cancerous mammary cells (MMT060562) and in normal mammary cells treated with epidermal growth factor. Responses to injection of either D -myo-inositol-1,4-bisphosphate (IP2) or D -myo-inositol-1,3,4,5-tetrakisphosphate (IP4) were small or absent. Furthermore, normal mammary cells cultivated with low-protein serum replacement alone or in the presence of differentiation-inducing hormones (insulin + cortisol + prolactin) were less sensitive to IP3. Thapsigargin induced a transient increase of Ca²⁺ due to the release of Ca²⁺ from an intracellular pool. There was no difference in the peak heights of the thapsigargin-induced Ca²⁺ increase when mammary cells were cultivated in the presence or absence of epidermal growth factor or insulin + cortisol + prolactin. These findings suggest that the releasable intracellular Ca²⁺ pool remained unchanged whereas sensitivity to IP3 increases during the proliferation stage. Mechanical stimulus of a mammary cell induces an increase of intracellular Ca²⁺ in the stimulated cell. A certain stimulating factor is released from the mechanically stimulated cell into the extracellular space, and it induces an increase of Ca²⁺ in surrounding cells.¹⁸ In contrast, the IP3-induced Ca²⁺ increase in both cancerous and epidermal growth factor-treated normal mammary cells did not spread to adjacent cells. Therefore, increase of Ca²⁺ is not sufficient to account for the release of stimulating substances from mammary cells in the mechanically-induced spreading response.     

Fluorescence study on transmembrane Ca2+ gradient-mediated conformation changes of sarcoplasmic reticulum Ca2+-ATPase

The conformational states of Ca²⁺-ATPase in sarcoplasmic reticulum (SR) vesicles with or without a thousand-fold transmembrane Ca²⁺ gradient have been studied by fluorescence spectroscopy and fluorescence quenching. In consequence of the establishment of the transmembrane Ca²⁺ gradient, the steady-state fluorescence results revealed a reproducible 8% decrease in the intrinsic fluorescence while time-resolved fluorescence measurements showed that 13 tryptophan residues in SR · Ca²⁺-ATPase could be divided into three groups. The fluorescence lifetime of one of these groups increased from 5.5 ns to 5.95 ns in the presence of a Ca²⁺ gradient. Using KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus, growing in Yunnan, China), the fluorescence quenching further indicated that the dynamic change of this tryptophan group, located at the protein-lipid interface, is a characteristic of transmembrane Ca²⁺ gradient-mediated conformational changes in SR · Ca²⁺-ATPase.Abbreviations SR sarcoplasmic reticulum - HB hypocrellin B - Trp tryptophan - DMSO dimethysulfoxide - Hepes N-2-hydroxyethyl piperazine-N-ethanesulfonic acad - SR(50005) SR vesicles with 1000-fold transmembrane Ca²⁺ gradient - SR(5050) SR vesicles without Ca²⁺ gradient - K_sv(app) apparent Stern-Volmer constant - K_svi Stern-Volmer constant of component i for dynamic quenching     

Ca2+-dependent Structural Changes in the B-cell Receptor CD23 Increase Its Affinity for Human Immunoglobulin E

Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca²⁺-free and Ca²⁺-bound forms, as well as the crystal structure of the Ca²⁺-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca²⁺ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca²⁺ binds at the principal and evolutionarily conserved binding site in CD23. Ca²⁺ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca²⁺-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca²⁺ brings an extra degree of modulation to CD23 function.     

大电导钾离子通道(BK)结构与功能的研究进展

大电导钙离子激活钾通道(BK)是细胞膜上唯一接受细胞内Ca2+和膜电位双重调控的离子通道.最新发表的关于BK通道电镜结构及其胞质功能域的晶体结构的文章,第一次展示了BK通道各亚基的组装,并证实通道各功能域在通道门控机制中存在紧密的相互作用.近年来,针对BK通道的功能调节及其门控动力学模拟的研究取得较多进展,有助于更好地理解BK通道发挥生理功能的门控机制,并揭示BK通道相关疾病的病理生理学基础.     

Calcium antagonists and calmodulin inhibitors block cytokinin-induced bud formation in Funaria

The plant hormone cytokinin stimulates nuclear migration followed by an asymmetric cell division in target cells of the protonema of the moss Funaria hygrometrica, leading to bud formation. The role of calcium in this developmental event was investigated by examining the effects of various calcium antagonists on the cytokinin-induced division. Calcium-free medium (buffered with EGTA), the extracellular Ca²⁺ antagonist La³⁺ (lanthanum), and the Ca²⁺ channel inhibitors D 600 and verapamil all block bud formation. These inhibitions are partially reversed by washing the cells or by raising the extracellular [Ca²⁺]. The Ca²⁺ ionophore A23187 partially reversed the effects of D 600 and verapamil. Bud formation is also inhibited by the intracellular Ca²⁺ antagonist TMB-8 (8-diethylamino)ocytl 3,4,5-trimethoxybenzoate HCl), and this inhibition is partially reversed by washing or raising the extracellular [Ca²⁺]. The cross walls of both the filaments and bud initial cells formed during TMB-8 exposure exhibit a distorted morphology. High concentrations of TMB-8 block nuclear migration. The calmodulin inhibitor trifluoperazine stops cytokinin-induced budding more effectively than the related compound chlorpromazine. Low concentrations of these two compounds do not affect nuclear migration; however, the target cell does not enter mitosis. These results support the hypothesis that a rise in intracellular calcium mediates cytokinin-induced bud formation in Funaria. It is concluded that the proposed cytokinin-induced rise in intracellular calcium may be effected in part by the activation of calmodulin. The essential source of Ca²⁺ appears to be extracellular, because blocking Ca²⁺ uptake with Ca²⁺ transport inhibitors can block both nuclear migration and subsequent division.     

Cytotoxicity of equinatoxin II from the sea anemoneActinia equina involves ion channel formation and an increase in intracellular calcium activity

Summary Equinatoxin Il is a 20-kDa basic protein isolated from the sea anemoneActinia equina. The aim of our work was to investigate the primary molecular basis for the cytotoxic effects of equinatoxin II in two model systems: single bovine lactotrophs and planar lipid bilayers. Previous work has shown that equinatoxin II produces rapid changes in cell morphology, which are dependent on external calcium. It has also been reported that addition of equinatoxin II increases membrane electrical conductance, which suggests that the cytotoxic action of equinatoxin II involves an increase in the permeability of membranes to Ca²⁺. Extensive changes in cytosolic Ca²⁺ activity are thought to invoke irreversible changes in cell physiology and morphology. In this paper, we show that morphological changes brought about by equinatoxin II in bovine lactotrophs are associated with a rapid rise in cytosolic Ca²⁺ activity, monitored with a fura-2 video imaging apparatus. Moreover, incorporation of equinatoxin II into planar lipid bilayers produces Ca²⁺ permeable ion channels. This suggests that the mode of equinatoxin II cytotoxicity involves the formation of cation (Ca²⁺) permeable channels in cell membranes.