BBA - Molecular Cell Research (v.1853, #9)

Preface by Jacques Haiech; Claus W. Heizmann; Joachim Krebs (1919-1920).

Sense and specificity in neuronal calcium signalling by Robert D. Burgoyne; Lee P. Haynes (1921-1932).
Changes in the intracellular free calcium concentration ([Ca2 +]i) in neurons regulate many and varied aspects of neuronal function over time scales from microseconds to days. The mystery is how a single signalling ion can lead to such diverse and specific changes in cell function. This is partly due to aspects of the Ca2 + signal itself, including its magnitude, duration, localisation and persistent or oscillatory nature. The transduction of the Ca2 + signal requires Ca2 + binding to various Ca2 + sensor proteins. The different properties of these sensors are important for differential signal processing and determine the physiological specificity of Ca2 + signalling pathways. A major factor underlying the specific roles of particular Ca2 + sensor proteins is the nature of their interaction with target proteins and how this mediates unique patterns of regulation. We review here recent progress from structural analyses and from functional analyses in model organisms that have begun to reveal the rules that underlie Ca2 + sensor protein specificity for target interaction. We discuss three case studies exemplifying different aspects of Ca2 + sensor/target interaction. This article is part of a special issue titled the 13th European Symposium on Calcium.
Keywords: Caenorhabditis elegans; Ca2 +-binding proteins; Ca2 + channel; Ca2 + sensors; NCS-1; Neuronal signalling;

Action potentials are usually considered as the smallest unit of neuronal information conveyed by presynaptic neurons to their postsynaptic target. Thus, neuronal signaling in brain circuits is all-or-none or digital. However, recent studies indicate that subthreshold analog variation in presynaptic membrane potential modulates spike-evoked transmission. The informational content of each presynaptic action potential is therefore greater than initially expected. This property constitutes a form of fast activity-dependent modulation of functional coupling. Therefore, it could have important consequences on information processing in neural networks in parallel with more classical forms of presynaptic short-term facilitation based on repetitive stimulation, modulation of presynaptic calcium or modifications of the release machinery. We discuss here how analog voltage shift in the presynaptic neuron may regulate spike-evoked release of neurotransmitter through the modulation of voltage-gated calcium and potassium channels in the axon and presynaptic terminal. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Calcium channel; Cav2.1; Potassium channel; Kv1; Synaptic transmission; Axon;

SOCE in neurons: Signaling or just refilling? by Lukasz Majewski; Jacek Kuznicki (1940-1952).
In this review we describe the present knowledge about store operated Ca2 + entry (SOCE) in neurons and the proteins involved in this process: STIM, as well as Orai and TRP channels. We address the issue of whether SOCE is used only to refill Ca2 + in the ER or whether Ca2 + that enters the neuronal cell during SOCE also performs signaling functions. We collected the data indicating that SOCE and its components participate in the important processes in neurons. This has implications for identifying new drug targets for the treatment of brain diseases. Evidence indicates that in neurodegenerative diseases Ca2 + homeostasis and SOCE components become dysregulated. Thus, different targets and strategies might be identified for the potential treatment of these diseases. This article is part of a Special Issue entitled: 13th European symposium on calcium.
Keywords: Store operated calcium entry (SOCE); Neuron; STIM; Orai; TRPC; Voltage-operated calcium channel;

Distinct roles of multiple isoforms of CaMKII in signaling to the nucleus by Huan Ma; Boxing Li; Richard W. Tsien (1953-1957).
Long-lasting synaptic changes following information acquisition are critical steps for memory. In this process, long-term potentiation (LTP) is widely considered as one of the major cellular mechanisms modifying synaptic strength. It can be classified into early phase LTP (E-LTP) and late phase LTP (L-LTP) based on its duration. Using genetically modified mice, investigators have recognized the critical role of CaMKII in E-LTP and memory. However, its function in L-LTP, which is strongly dependent on gene transcription and protein synthesis, is still unclear. In this review, we discuss how different isoforms of CaMKII are coordinated to regulate gene expression in an activity-dependent manner, and thus contribute to L-LTP and memory. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Long-term potentiation (LTP); Excitation–transcription coupling; CaM translocation; γCaMKII; βCaMKII; αCaMKII;

Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain the metastatic switch in a number of solid cancers, including breast cancer (BC) and renal cellular carcinoma (RCC). Preventing EPC mobilization causes tumor shrinkage. Novel anti-angiogenic treatments have been introduced in therapy to inhibit VEGFR-2 signaling; unfortunately, these drugs blocked tumor angiogenesis in pre-clinical murine models, but resulted far less effective in human patients. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis in cancer patients could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca2 + entry (SOCE) regulates the growth of human EPCs, and it is mediated by the interaction between the endoplasmic reticulum Ca2 +-sensor, Stim1, and the plasmalemmal Ca2 + channels, Orai1 and TRPC1. EPCs do not belong to the neoplastic clone: thus, unlike tumor endothelium and neoplastic cells, they should not remodel their Ca2 + toolkit in response to tumor microenvironment. However, our recent work demonstrated that EPCs isolated from naïve RCC patients (RCC-EPCs) undergo a dramatic remodeling of their Ca2 + toolkit by displaying a remarkable drop in the endoplasmic reticulum Ca2 + content, by down-regulating the expression of inositol-1,4,5-receptors (InsP3Rs), and by up-regulating Stim1, Orai1 and TRPC1. Moreover, EPCs are dramatically less sensitive to VEGF stimulation both in terms of Ca2 + signaling and of gene expression when isolated from tumor patients. Conversely, the pharmacological abolition of SOCE suppresses proliferation in these cells. These results question the suitability of VEGFR-2 as a therapeutically relevant target for anti-angiogenic treatments and hint at Orai1 and TRPC1 as more promising alternatives. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Tumor vascularization; Endothelial progenitor cel; Vascular endothelial growth facto; Stim1; Orai1; TRPC1;

Calcium homeostasis in cancer: A focus on senescence by Valerio Farfariello; Oksana Iamshanova; Emmanuelle Germain; Ingrid Fliniaux; Natalia Prevarskaya (1974-1979).
Senescence is one of the primary responses to the activation of oncoproteins or down-regulation of tumor suppressors in normal cells and is therefore considered as being anti-tumorigenic but the mechanisms controlling this process are still much unknown. Calcium (Ca2+) plays a major role in many cellular processes and calcium channels control many of the “hallmarks of cancer” but their involvement in tumor initiation is poorly understood and remains unclear.Therefore, in this article we review some striking senescence-associated characteristics and their potential regulation by Ca2+. The main aim is to produce plausible hypothesis on how calcium homeostasis may participate in cancer-related senescence. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Senescence; Cancer; Calcium homeostasis; Calcium channels;

Structural insights into endoplasmic reticulum stored calcium regulation by inositol 1,4,5-trisphosphate and ryanodine receptors by Min-Duk Seo; Masahiro Enomoto; Noboru Ishiyama; Peter B. Stathopulos; Mitsuhiko Ikura (1980-1991).
The two major calcium (Ca2 +) release channels on the sarco/endoplasmic reticulum (SR/ER) are inositol 1,4,5-trisphosphate and ryanodine receptors (IP3Rs and RyRs). They play versatile roles in essential cell signaling processes, and abnormalities of these channels are associated with a variety of diseases. Structural information on IP3Rs and RyRs determined using multiple techniques including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (EM), has significantly advanced our understanding of the mechanisms by which these Ca2 + release channels function under normal and pathophysiological circumstances. In this review, structural advances on the understanding of the mechanisms of IP3R and RyR function and dysfunction are summarized. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Calcium signaling; Calcium release channel; Inositol 1,4,5-trisphosphate receptor (IP3R); Ryanodine receptor (RyR); Protein structure; X-ray crystallography; Nuclear magnetic resonance (NMR); Electron microscopy (EM);

The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca2 +-release channel by Tamara Vervloessem; David I. Yule; Geert Bultynck; Jan B. Parys (1992-2005).
The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) type 2 (IP3R2) is an intracellular Ca2 +-release channel located on the endoplasmic reticulum (ER). IP3R2 is characterized by a high sensitivity to both IP3 and ATP and is biphasically regulated by Ca2 +. Furthermore, IP3R2 is modulated by various protein kinases. In addition to its regulation by protein kinase A, IP3R2 forms a complex with adenylate cyclase 6 and is directly regulated by cAMP. Finally, in the ER, IP3R2 is less mobile than the other IP3R isoforms, while its functional properties appear dominant in heterotetramers. These properties make the IP3R2 a Ca2 + channel with exquisite properties for setting up intracellular Ca2 + signals with unique characteristics. IP3R2 plays a crucial role in the function of secretory cell types (e.g. pancreatic acinar cells, hepatocytes, salivary gland, eccrine sweat gland). In cardiac myocytes, the role of IP3R2 appears more complex, because, together with IP3R1, it is needed for normal cardiogenesis, while its aberrant activity is implicated in cardiac hypertrophy and arrhythmias. Most importantly, its high sensitivity to IP3 makes IP3R2 a target for anti-apoptotic proteins (e.g. Bcl-2) in B-cell cancers. Disrupting IP3R/Bcl-2 interaction therefore leads in those cells to increased Ca2 + release and apoptosis. Intriguingly, IP3R2 is not only implicated in apoptosis but also in the induction of senescence, another tumour-suppressive mechanism. These results were the first to unravel the physiological and pathophysiological role of IP3R2 and we anticipate that further progress will soon be made in understanding the function of IP3R2 in various tissues and organs.
Keywords: Apoptosis; Cancer; IP3; Heart; Secretion; Senescence;

Structure and function of the mitochondrial calcium uniporter complex by Diego De Stefani; Maria Patron; Rosario Rizzuto (2006-2011).
The mitochondrial calcium uniporter (MCU) is the critical protein of the inner mitochondrial membrane mediating the electrophoretic Ca2 + uptake into the matrix. It plays a fundamental role in the shaping of global calcium signaling and in the control of aerobic metabolism as well as apoptosis. Two features of mitochondrial calcium signaling have been known for a long time: i) mitochondrial Ca2 + uptake widely varies among cells and tissues, and ii) channel opening strongly relies on the extramitochondrial Ca2 + concentration, with low activity at resting [Ca2 +] and high capacity as soon as calcium signaling is activated. Such complexity requires a specialized molecular machinery, with several primary components can be variably gathered together in order to match energy demands and protect from toxic stimuli. In line with this, MCU is now recognized to be part of a macromolecular complex known as the MCU complex. Our understanding of the structure and function of the MCU complex is now growing promptly, revealing an unexpected complexity that highlights the pleiotropic role of mitochondrial Ca2 + signals. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Mitochondria; Calcium; MCU; MCUb; MICU1; MICU2;

Calcium signaling at ER membrane contact sites by Thomas Burgoyne; Sandip Patel; Emily R. Eden (2012-2017).
Communication between organelles is a necessary consequence of intracellular compartmentalization. Membrane contact sites (MCSs) are regions where the membranes of two organelles come into close apposition allowing exchange of small molecules and ions including Ca2 +. The ER, the cell's major Ca2 + store, forms an extensive and dynamic network of contacts with multiple organelles. Here we review established and emerging roles of ER contacts as platforms for Ca2 + exchange and further consider a potential role for Ca2 + in the regulation of MCS formation. We additionally discuss the challenges associated with the study of MCS biology and highlight advances in microscopy-based solutions. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Calcium; Membrane contact sites (MCSs); ER; Ca2 + exchange;

In this review the four different genes of the mammalian plasma membrane calcium ATPase (PMCA) and their spliced isoforms are discussed with respect to their tissue distribution, their differences during development and their importance for regulating Ca2 + homeostasis under different conditions. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: PMCA; Plasma membrane calcium ATPase; Alternative splicing; Spliced isoform;

Calcium signaling in taste cells by Kathryn F. Medler (2025-2032).
The sense of taste is a common ability shared by all organisms and is used to detect nutrients as well as potentially harmful compounds. Thus taste is critical to survival. Despite its importance, surprisingly little is known about the mechanisms generating and regulating responses to taste stimuli. All taste responses depend on calcium signals to generate appropriate responses which are relayed to the brain. Some taste cells have conventional synapses and rely on calcium influx through voltage-gated calcium channels. Other taste cells lack these synapses and depend on calcium release to formulate an output signal through a hemichannel. Beyond establishing these characteristics, few studies have focused on understanding how these calcium signals are formed. We identified multiple calcium clearance mechanisms that regulate calcium levels in taste cells as well as a calcium influx that contributes to maintaining appropriate calcium homeostasis in these cells. Multiple factors regulate the evoked taste signals with varying roles in different cell populations. Clearly, calcium signaling is a dynamic process in taste cells and is more complex than has previously been appreciated. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.Display Omitted
Keywords: Taste; Calcium influx; Calcium release; Ryanodine receptors; Mitochondria; Sodium–calcium exchangers;

Annexin-A5 promotes membrane resealing in human trophoblasts by Romain Carmeille; Séverine A. Degrelle; Laurent Plawinski; Flora Bouvet; Céline Gounou; Danièle Evain-Brion; Alain R. Brisson; Anthony Bouter (2033-2044).
Annexin-A5 (AnxA5) is the smallest member of the annexins, a group of soluble proteins that bind to membranes containing negatively-charged phospholipids, principally phosphatidylserine, in a Ca2 +-dependent manner. AnxA5 presents unique properties of binding and self-assembling on membrane surfaces, forming highly ordered two-dimensional (2D) arrays. We showed previously that AnxA5 plays a central role in the machinery of cell membrane repair of murine perivascular cells, promoting the resealing of membrane damages via the formation of 2D protein arrays at membrane disrupted sites and preventing the extension of membrane ruptures. As the placenta is one of the richest source of AnxA5 in humans, we investigated whether AnxA5 was involved in membrane repair in this organ. We addressed this question at the level of human trophoblasts, either mononucleated cytotrophoblasts or multinucleated syncytiotrophoblasts, in choriocarcinoma cells and primary trophoblasts. Using established procedure of laser irradiation and fluorescence microscopy, we observed that both human cytotrophoblasts and syncytiotrophoblasts repair efficiently a μm2-size disruption. Compared to wild-type cells, AnxA5-deficient trophoblasts exhibit severe defect of membrane repair. Through specifically binding to the disrupted site as early as a few seconds after membrane wounding, AnxA5 promotes membrane resealing of injured human trophoblasts. In addition, we observed that a large membrane area containing the disrupted site was released in the extracellular milieu. We propose mechanisms ensuring membrane resealing and subsequent lesion removal in human trophoblasts. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.Display Omitted
Keywords: Membrane repair; Annexin-A5; Placenta; Trophoblast; Membrane resealing; Lesion removal;

Ca2 +-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins by Heidi Wolfmeier; Roman Schoenauer; Alexander P. Atanassoff; Daniel R. Neill; Aras Kadioglu; Annette Draeger; Eduard B. Babiychuk (2045-2054).
Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood.We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca2 + entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2 + sequestration that prevents excessive Ca2 + elevation during the execution phase of plasmalemmal repair is of no less importance.The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca2 + signals in cells that were able to survive after PLY attack. Intracellular Ca2 + dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2 + signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects.Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Plasma membrane; Calcium; Annexin; Non-immune defense;

Structural effects of Mg2 + on the regulatory states of three neuronal calcium sensors operating in vertebrate phototransduction by Valerio Marino; Stefan Sulmann; Karl-Wilhelm Koch; Daniele Dell'Orco (2055-2065).
The effects of physiological concentration of magnesium on the switch states of the neuronal calcium sensor proteins recoverin, GCAP1 and GCAP2 were investigated. Isothermal titration calorimetry was applied for binding studies. Circular dichroism spectroscopy was used to characterize protein thermal stability, secondary and tertiary structure in conditions of high and low [Ca2 +], mimicking respectively the dark-adapted and light-exposed photoreceptor states during the phototransduction cascade. Further, molecular dynamics (MD) simulations were run to investigate the dynamical structural properties of GCAP1 in its activator, inhibitor and putative transitory states.Our results confirmed that Mg2 + is unable to trigger the typical Ca2 +-induced conformational change of recoverin (myristoyl switch) while it decreases its thermal stability. Interestingly, Mg2 + seems to affect the conformation of GCAP2 both at high and low [Ca2 +], however the variations are more substantial for myristoylated GCAP2 in the absence of Ca2 +. GCAP1 is responsive to Mg2 + only in its low [Ca2 +] state and Mg2 +-GCAP1 tertiary structure slightly differs from both apo and Ca2 +-bound states. Finally, MD simulations suggest that the GCAP1 state harboring one Mg2 + ion bound to EF2 acquires structural characteristics that are thought to be relevant for the activation of the guanylate cyclase. Moreover, all the putative Mg2 +-bound states of myristoylated GCAP1 are structurally less flexible than Ca2 +-bound states. GCAP1 acquires a more compact tertiary structure that is less accessible to the solvent, thereby inducing a different conformation to the myristoyl moiety, which might be crucial for the activation of the guanylate cyclase. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Phototransduction; Neuronal calcium sensor; Conformational switch; Guanylate cyclase-activating protein; Magnesium; Molecular dynamics;

Selective dendritic susceptibility to bioenergetic, excitotoxic and redox perturbations in cortical neurons by Philip Hasel; Sean Mckay; Jing Qiu; Giles E. Hardingham (2066-2076).
Neurodegenerative and neurological disorders are often characterised by pathological changes to dendrites, in advance of neuronal death. Oxidative stress, energy deficits and excitotoxicity are implicated in many such disorders, suggesting a potential vulnerability of dendrites to these situations. Here we have studied dendritic vs. somatic responses of primary cortical neurons to these types of challenges in real-time.Using a genetically encoded indicator of intracellular redox potential (Grx1-roGFP2) we found that, compared to the soma, dendritic regions exhibited more dramatic fluctuations in redox potential in response to sub-lethal ROS exposure, and existed in a basally more oxidised state. We also studied the responses of dendritic and somatic regions to excitotoxic NMDA receptor activity. Both dendritic and somatic regions experienced similar increases in cytoplasmic Ca2+. Interestingly, while mitochondrial Ca2+ uptake and initial mitochondrial depolarisation were similar in both regions, secondary delayed mitochondrial depolarisation was far weaker in dendrites, potentially as a result of less NADH depletion. Despite this, ATP levels were found to fall faster in dendritic regions. Finally we studied the responses of dendritic and somatic regions to energetically demanding action potential burst activity. Burst activity triggered PDH dephosphorylation, increases in oxygen consumption and cellular NADH:NAD ratio. Compared to somatic regions, dendritic regions exhibited a smaller degree of mitochondrial Ca2+ uptake, lower fold-induction of NADH and larger reduction in ATP levels. Collectively, these data reveal that dendritic regions of primary neurons are vulnerable to greater energetic and redox fluctuations than the cell body, which may contribute to disease-associated dendritic damage. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Antioxidant defences; Oxidative stress; Excitotoxicity; Mitochondria; Calcium signalling; Bioenergetics;

Kcnip1 a Ca2 +-dependent transcriptional repressor regulates the size of the neural plate in Xenopus by Isabelle Néant; Britt Mellström; Paz Gonzalez; Jose R. Naranjo; Marc Moreau; Catherine Leclerc (2077-2085).
In amphibian embryos, our previous work has demonstrated that calcium transients occurring in the dorsal ectoderm at the onset of gastrulation are necessary and sufficient to engage the ectodermal cells into a neural fate by inducing neural specific genes. Some of these genes are direct targets of calcium. Here we search for a direct transcriptional mechanism by which calcium signals are acting. The only known mechanism responsible for a direct action of calcium on gene transcription involves an EF-hand Ca2 + binding protein which belongs to a group of four proteins (Kcnip1 to 4). Kcnip protein can act in a Ca2 +-dependent manner as a transcriptional repressor by binding to a specific DNA sequence, the Downstream Regulatory Element (DRE) site. In Xenopus, among the four kcnips, we show that only kcnip1 is timely and spatially present in the presumptive neural territories and is able to bind DRE sites in a Ca2 +-dependent manner. The loss of function of kcnip1 results in the expansion of the neural plate through an increased proliferation of neural progenitors. Later on, this leads to an impairment in the development of anterior neural structures. We propose that, in the embryo, at the onset of neurogenesis Kcnip1 is the Ca2 +-dependent transcriptional repressor that controls the size of the neural plate.This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: kcnip; KChIP; DREAM; Ca2 + signaling; Early neurogenesis; Xenopus laevis; Embryo; Neural plate;

Ezrin interacts with the scaffold protein IQGAP1 and affects its cortical localization by Rathangadhara Chakrapani Nammalwar; Annika Heil; Volker Gerke (2086-2094).
The cortical cytoskeleton constitutes an important subcellular structure that determines cell shape and regulates cell migration as well as membrane traffic to and from the plasma membrane. Many components of the cortical cytoskeleton have been identified including structural and scaffolding proteins, membrane–cytoskeleton linker proteins and signaling intermediates. We describe here an association of the membrane-F-actin linker protein ezrin with the scaffolding protein IQGAP1 that serves as a hub for concentrating different signaling complexes. Both, ezrin and IQGAP1 bind in a Ca2 +-dependent manner to the EF hand protein S100P and complexes consisting of Ca2 +-bound S100P, IQGAP1 and ezrin can be isolated by immunoprecipitation. Ezrin and IQGAP1 also interact in the absence of Ca2 +, thus independent of S100P. Direct ezrin–IQGAP1 interaction can be shown with the purified proteins. It is mediated via the N-terminal FERM domain of ezrin and the IQ domain of IQGAP1, respectively. Ezrin and IQGAP1 colocalize in the submembraneous cytoskeleton and in cellular protrusions of human epithelial cells and knockdown of ezrin reduces the cortical localization of IQGAP1. Thus, ezrin appears to participate in recruiting IQGAP1 to the cell cortex thereby establishing a close connection between membrane-F-actin contacts and actin regulators that can be assembled by IQGAP1. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Actin; Calcium-binding protein; Cell cortex; EF hand; Membrane;

Determinants of the membrane orientation of a calcium signaling enzyme CD38 by Yong Juan Zhao; Wen Jie Zhu; Xian Wang Wang; Li-He Zhang; Hon Cheung Lee (2095-2103).
CD38 catalyzes the synthesis of two structurally distinct messengers for Ca2 +-mobilization, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), from cytosolic substrates, NAD and NADP, respectively. CD38 is generally thought of as a type II membrane protein with its catalytic site facing outside. We recently showed that CD38 exists, instead, in two opposite membrane orientations. The determinant for the membrane topology is unknown. Here, specific antibodies against type III CD38 were designed and produced. We show that mutating the positively charged residues in the N-terminal tail of CD38 converted its orientation to type III, with the catalytic domain facing the cytosol and it was fully active in producing intracellular cADPR. Changing the serine residues to aspartate, which is functionally equivalent to phosphorylation, had a similar effect. The mutated CD38 was expressed intracellularly and was un-glycosylated. The membrane topology could also be modulated by changing the highly conserved di-cysteine. The results indicate that the net charge of the N-terminal segment is important in determining the membrane topology of CD38 and that the type III orientation can be a functional form of CD38 for Ca2 +-signaling. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.Display Omitted
Keywords: CD38; Cyclic ADP-ribose; Membrane topology; ADP-ribosyl cyclase; Calcium signaling;

The transmembrane Bax inhibitor motif (TMBIM) containing protein family: Tissue expression, intracellular localization and effects on the ER CA2+-filling state by Dmitrij A. Lisak; Teresa Schacht; Vitalij Enders; Jörn Habicht; Santeri Kiviluoto; Julia Schneider; Nadine Henke; Geert Bultynck; Axel Methner (2104-2114).
Bax inhibitor-1 (BI-1) is an evolutionarily conserved pH-dependent Ca2+ leak channel in the endoplasmic reticulum and the founding member of a family of six highly hydrophobic mammalian proteins named transmembrane BAX inhibitor motif containing (TMBIM) 1-6 with BI-1 being TMBIM6. Here we compared the structure, subcellular localization, tissue expression and the effect on the cellular Ca2+ homeostasis of all family members side by side. We found that all TMBIM proteins possess the di-aspartyl pH sensor responsible for pH sensing identified in TMBIM6 and its bacterial homologue BsYetJ. TMBIM1-3 and TMBIM4-6 represent two phylogenetically distinct groups that are localized in the Golgi apparatus (TMBIM1-3), endoplasmic reticulum (TMBIM4-6) or mitochondria (TMBIM5) but share a common structure of at least seven transmembrane domains with the last domain being semi-hydrophobic. TMBIM1 is mainly expressed in muscle, TMBIM2 and 3 in the nervous system, TMBIM4 and 5 are ubiquitously expressed and TMBIM6 in skeletal muscle, kidney, liver and spleen. All TMBIM proteins reduce the Ca2+ content of the endoplasmic reticulum, and all but TMBIM5 also reduce the cytosolic resting Ca2+ concentration. These results suggest that the TMBIM family has comparable functions in the maintenance of intracellular Ca2+ homeostasis in a wide variety of tissues. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. Guest Editors: Jacques Haiech, Claus Heizmann and Joachim Krebs.
Keywords: RECS1; FAIM2; GRINA; GAAP; GHITM; MICS1;

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane protein that is expressed in the central nervous system. MOG has a single N-glycosylation site within its extracellular domain. MOG has been linked with pathogenesis of multiple sclerosis; anti-MOG antibodies have been detected in the sera of multiple sclerosis patients. N-glycosylation is an important post-translational modification of protein that might impact their folding, localization and function. However, the role of sugar in the biology of MOG is not well understood. In this study, we created a mutant MOG lacking N-linked glycan and tested its properties. We concluded that the lack of sugar did not impact on MOG abundance in the absence of endoplasmic reticulum molecular chaperone calnexin. We also show that the absence of N-glycan did not interfere with MOG's subcellular localization and it did not result in activation of endoplasmic reticulum stress. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Myelin oligodendrocyte glycoprotein (MOG); Endoplasmic reticulum; Molecular chaperones; Calnexin; Calreticulin; ERp57;

TRPC1 regulates fMLP-stimulated migration and chemotaxis of neutrophil granulocytes by O. Lindemann; C. Strodthoff; M. Horstmann; N. Nielsen; F. Jung; S. Schimmelpfennig; M. Heitzmann; A. Schwab (2122-2130).
Neutrophils form the first line of defense of the innate immune system and are rapidly recruited by chemotactic signals to sites of inflammation. Understanding the mechanisms of neutrophil chemotaxis is therefore of great interest for the potential development of new immunoregulatory therapies. It has been shown that members of the transient receptor potential (TRP) family of cation channels are involved in both cell migration and chemotaxis. In this study, we demonstrate that TRPC1 channels play an important role in fMLP mediated chemotaxis and migration of murine neutrophils. The knock-out of TRPC1 channels leads to an impaired migration, transmigration and chemotaxis of the neutrophils. In contrast, Ca2 + influx but not store release after activation of the TRPC1−/− neutrophils with fMLP is strongly enhanced. We show that the enhanced Ca2+ influx in the TRPC1−/− neutrophils is associated with a steepened front to rear gradient of the intracellular Ca2+ concentration with higher levels at the cell rear. Taken together, this paper highlights a distinct role of TRPC1 in neutrophil migration and chemotaxis. We propose that TRPC1 controls the activity of further Ca2+ influx channels and thus regulates the maintenance of intracellular Ca2+ gradients which are critical for cell migration. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Chemotaxis; Neutrophil; TRP-channel; Migration; Ca2+; FPR;

Atrial myocytes in a number of species lack transverse tubules. As a consequence the intracellular calcium signals occurring during each heartbeat exhibit complex spatio-temporal dynamics. These calcium patterns arise from saltatory calcium waves that propagate via successive rounds of diffusion and calcium-induced calcium release. The many parameters that impinge on calcium-induced calcium release and calcium signal propagation make it difficult to know a priori whether calcium waves will successfully travel, or be extinguished. In this study, we describe in detail a mathematical model of calcium signalling that allows the effect of such parameters to be independently assessed. A key aspect of the model is to follow the triggering and evolution of calcium signals within a realistic three-dimensional cellular volume of an atrial myocyte, but with low computational costs. This is achieved by solving the linear transport equation for calcium analytically between calcium release events and by expressing the onset of calcium liberation as a threshold process. The model makes non-intuitive predictions about calcium signal propagation. For example, our modelling illustrates that the boundary of a cell produces a wave-guiding effect that enables calcium ions to propagate further and for longer, and can subtly alter the pattern of calcium wave movement. The high spatial resolution of the modelling framework allows the study of any arrangement of calcium release sites. We demonstrate that even small variations in randomly positioned release sites cause highly heterogeneous cellular responses. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Calcium wave; Fire–diffuse–fire; Computational cell biology; Atrial myocyte; Green's function;

Extracellular S100A4 induces smooth muscle cell phenotypic transition mediated by RAGE by Chiraz Chaabane; Claus W. Heizmann; Marie-Luce Bochaton-Piallat (2144-2157).
We identified S100A4 as a marker of rhomboid (R) smooth muscle cells (SMCs) in vitro (the synthetic phenotype, typical of intimal SMCs) in the porcine coronary artery and of intimal SMCs in vivo in both pigs and humans. S100A4 is an intracellular Ca2 + signaling protein and can be secreted; it has extracellular functions via the receptor for advanced glycation end products (RAGE). Our objective was to explore the role of S100A4 in SMC phenotypic change, a phenomenon characteristic of atherosclerotic plaque formation. Transfection of a human S100A4-containing plasmid in spindle-shaped (S) SMCs (devoid of S100A4) led to approximately 10% of S100A4-overexpressing SMCs, S100A4 release, and a transition towards a R-phenotype of the whole SMC population. Furthermore treatment of S-SMCs with S100A4-rich conditioned medium collected from S100A4-transfected S-SMCs induced a transition towards a R-phenotype, which was associated with decreased SMC differentiation markers and increased proliferation and migration by activating the urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). It yielded NF-κB activation in a RAGE-dependent manner. Blockade of extracellular S100A4 in R-SMCs with S100A4 neutralizing antibody induced a transition from R- to S-phenotype, decreased proliferative activity and upregulation of SMC differentiation markers. By contrast, silencing of S100A4 mRNA in R-SMCs did not change the level of extracellular S100A4 or SMC morphology in spite of decreased proliferative activity. Our results show that extracellular S100A4 plays a pivotal role in SMC phenotypic changes. It could be a new target to prevent SMC accumulation during atherosclerosis and restenosis. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: EF-hand; S100A4; RAGE; PDGF-BB; Atherosclerosis; Restenosis;

The calcium-sensing receptor: A promising target for prevention of colorectal cancer by Abhishek Aggarwal; Maximilian Prinz-Wohlgenannt; Samawansha Tennakoon; Julia Höbaus; Cedric Boudot; Romuald Mentaverri; Edward M. Brown; Sabina Baumgartner-Parzer; Enikö Kállay (2158-2167).
The inverse correlation between dietary calcium intake and the risk of colorectal cancer (CRC) is well known, but poorly understood. Expression of the calcium-sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is downregulated in CRC leading us to hypothesize that the CaSR has tumor suppressive roles in the colon. The aim of this study was to understand whether restoration of CaSR expression could reduce the malignant phenotype in CRC.In human colorectal tumors, expression of the CaSR negatively correlated with proliferation markers whereas loss of CaSR correlated with poor tumor differentiation and reduced apoptotic potential. In vivo, dearth of CaSR significantly increased expression of proliferation markers and decreased levels of differentiation and apoptotic markers in the colons of CaSR/PTH double knock-out mice confirming the tumor suppressive functions of CaSR.In vitro CRC cells stably overexpressing wild-type CaSR showed significant reduction in proliferation, as well as increased differentiation and apoptotic potential. The positive allosteric modulator of CaSR, NPS R-568 further enhanced these effects, whereas treatment with the negative allosteric modulator, NPS 2143 inhibited these functions. Interestingly, the dominant-negative mutant (R185Q) was able to abrogate these effects.Our results demonstrate a critical tumor suppressive role of CaSR in the colon. Restoration of CaSR expression and function is linked to regulation of the balance between proliferation, differentiation, and apoptosis and provides a rationale for novel strategies in CRC therapy.
Keywords: Calcium-sensing receptor; Tumor suppressor; Colorectal cancer; Colon; Calcium; Calcimimetic; Calcilytic;

Calcium is an organizer of cell polarity in plants by Ellie Himschoot; Tom Beeckman; Jiří Friml; Steffen Vanneste (2168-2172).
Cell polarity is a fundamental property of pro- and eukaryotic cells. It is necessary for coordination of cell division, cell morphogenesis and signaling processes. How polarity is generated and maintained is a complex issue governed by interconnected feed-back regulations between small GTPase signaling and membrane tension-based signaling that controls membrane trafficking, and cytoskeleton organization and dynamics. Here, we will review the potential role for calcium as a crucial signal that connects and coordinates the respective processes during polarization processes in plants. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Calcium; Polarity; Trafficking; GTPase; Tension; Arabidopsis;

A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca2 + microdomain in the caveolar compartment by Ilari Pulli; Tomas Blom; Christoffer Löf; Melissa Magnusson; Alessandro Rimessi; Paolo Pinton; Kid Törnquist (2173-2182).
Caveolae are plasma membrane invaginations enriched in sterols and sphingolipids. Sphingosine kinase 1 (SK1) is an oncogenic protein that converts sphingosine to sphingosine 1-phosphate (S1P), which is a messenger molecule involved in calcium signaling. Caveolae contain calcium responsive proteins, but the effects of SK1 or S1P on caveolar calcium signaling have not been investigated. We generated a Caveolin-1–Aequorin fusion protein (Cav1–Aeq) that can be employed for monitoring the local calcium concentration at the caveolae ([Ca2+]cav). In HeLa cells, Cav1–Aeq reported different [Ca2+] as compared to the plasma membrane [Ca2+] in general (reported by SNAP25–Aeq) or as compared to the cytosolic [Ca2+] (reported by cyt-Aeq). The Ca2+ signals detected by Cav1–Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-β-cyclodextrin, suggesting that the caveolae are specific targets for Ca2+ signaling. HeLa cells overexpressing SK1 showed increased [Ca2+]cav during histamine-induced Ca2+ mobilization in the absence of extracellular Ca2+ as well as during receptor-operated Ca2+ entry (ROCE). The SK1-induced increase in [Ca2+]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca2+]cav during ROCE. S1P treatment stimulated the [Ca2+]cav upon ROCE. The Ca2+ responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca2+ signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Keywords: Caveolin-1; Aequorin; Sphingosine kinase 1; Sphingosine-1-phosphate; Calcium; Plasma membrane;