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Joint Meeting of the Federation of European Physiological Societies and the Austrian Physiological Society

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09/13/2017 - Hall C1 | 3:00pm - 5:00pm 
Symposium 1: Mitochondrial and cell membrane Ca2+ and Na+ signaling in health and disease

Kindly supported by The Physiological Society (UK)

Organizers: I. Sekler (Be’er Scheva, Israel); Alexej Verkhratsky (Manchester, United Kingdom)

Systematic identification of MCU modulators by orthogonal interspecies chemical screening
*Fabiana Perocchi1
1 LMU, Biochemistry, Munich, Germany
Abstract text :

The mitochondrial calcium uniporter complex is essential for calcium (Ca2 ) uptake in mitochondria of all mammalian tissues, whereby it regulates energy metabolism and cell death. An ever-growing number of human diseases linked to dysfunctions of mitochondrial Ca2 homeostasis qualify the uniporter as a target of broad pharmacological interest. However, at present we lack lead compounds for the selective regulation of its activity. Here, we introduce a high-throughput orthogonal, interspecies assay that identifies direct modulators of human MCU. Our strategy exploits a D-lactate- and mannitol/sucrose-based bioenergetic shunt that allows deconvolving false positive hits due to chemical impairment of mitochondrial membrane potential and upstream Ca2 signaling pathways. Out of more than 600 clinically approved drugs, we identify a direct, selective inhibitor of human MCU. Our approach is a highly effective tool for MCU-specific drug discovery, and, more generally, for therapeutic targeting of mitochondria.

Shaping cell motility and metabolism by coordinated Ca2+ and Na+ signals
*Mohamed Trebak1
1 Penn State University, Cellular and Molecular Physiology, Hershey, United States
Abstract text :



ORAI channel proteins (ORAI1/ORAI2/ORAI3) form highly Ca2 selective channels which control a wide variety of physiological and pathophysiological functions. ORAI proteins are gated and regulated by the endoplasmic reticulum Ca2 sensor proteins, STIM1 and STIM2 and contribute subunits to channels activated by either store depletion or by store-independent means.  Mitochondrial Ca2 homeostasis is crucial for cellular function and is controlled through Ca2 uptake by the mitochondrial Ca2 uniporter (MCU) and extrusion by the Na /Ca2 exchanger (NCLX).  Here, we discuss how MCU and NCLX regulate distinct ORAI-mediated Ca2 entry pathways across the plasma membrane and distinct downstream signaling pathways. We will also discuss the impact of altered STIM, ORAI and NCLX activity on mitochondrial function and cell migration in a model of invasive adenocarcinoma.

Dynamic aspects of calcium-dependent regulation in mammalian isoform/splice variants of the sodium-calcium exchanger
*Daniel Khananshvili1
1 Tel-Aviv University Medical School, Physiology and Pharmacology, Ramat-Aviv, Tel-Aviv, Israel
Abstract text :

Three gene products of the sodium-calcium exchanger (NCX1, NCX2 and NCX3) and their multiple splice-variants are expressed in a tissue-specific manner to control Ca2 -dependent events. NCXs are strongly regulated by Ca2 interaction with the regulatory CBD1 and CBD2 domains. NCX1 and NCX3 (but not NCX2) generate splice variants, where the splicing segment exclusively resides at CBD2. Recent findings reveal a unifying mechanism for decoding the allosteric signal upon Ca2 binding, describing the Ca2 -dependent tethering of CBDs, where Ca2 is "occluded" at the two-domain interface. A slow dissociation of occluded Ca2 results in NCX inactivation, where the exon-dependent interdomain interactions generate upto 100-fold differences in the Ca2 off-rates and inactivation rates of NCX. This represents the editing mechanism for dynamic regulation of tissue-specific (cardiac, skeletal, brain, kidney, etc) NCX variants. Ca2 binding to CBD1 rigidifies the backbone dynamics at the two-domain interface, where the strength, duration and remoteness of Ca2 -induced rigidification is exon dependent. This Ca2 -dependent rigidification is associated with dynamic shift of numerous conformational states in the absence of any global conformational changes ("population shift" mechanism). Thus, the exon-dependent conformational variances govern dynamic contributions of NCX variants to cell-specific affairs (excitation-contraction coupling, action potential duration, neurotransmitter secretion, etc). Collectively, NCX isoform/splice variants share a common mechanism for decoding the regulatory signal, where the splicing segment secondarily shapes dynamic features of NCX to match cell-specific oscillations in [Ca2 ]i

S01-4 (O)
Adrenergic stimulation leads to distinct intracellular Ca2+ and cAMP-dependent PKA responses in single rat astrocytes
*Anemari Horvat1, Robert Zorec1,2, Nina Vardjan1,2
1 University of Ljubljana, Medical Faculty, Institute of Pathophysiology, Laboratory of Neuroendocrinology-Molecular Cell Physiology, Ljubljana, Slovenia
2 Celica Biomedical, , Ljubljana, Slovenia
Abstract text :

During the arousal and startle response, locus coeruleus neurons, innervating practically all brain regions, release noradrenaline (NA), which reaches brain cells, including astrocytes. Astrocytes, a subset of glial cells, express both α- and β-adrenergic receptors (ARs) and thus represent an important target for NA. Although devoid of electrical excitability, astrocytes respond to NA by activation of α- and β-ARs with increased cytosolic levels of secondary messengers Ca2 and cAMP, i.e. cytoplasmic excitability. AR-activation controls many processes in astrocytes, including cell morphology and metabolism. It is known from biochemical studies that Ca2 and cAMP signals in astrocytes can interact. However, it is presently unclear whether the temporal properties of the second messengers are time associated upon AR-activation. We used confocal microscopy to study AR agonist-induced intracellular changes in Ca2 and cAMP in single cultured rat astrocytes by real-time monitoring of the Ca2 indicator Fluo4-AM, and the fluorescence resonance energy transfer-based nanosensor A-kinase activity reporter 2, which reports the activity of cAMP via its downstream effector protein kinase A (PKA). We have observed that temporal profiles of the respective secondary messenger systems are distinct in astrocytes. While the activation of α1-ARs triggers Ca2 oscillations within 10 s, the activation of β-ARs leads to a ~10-fold slower persistent increase in cAMP-dependent PKA activity devoid of oscillations. Moreover, the results revealed that β-AR activation in cultured astrocytes potentiates the α1-AR induced Ca2 response and vice versa, indicating that the pathways control and tune the activity of each other at the single-cell level.

S01-5 (O)
Function and distribution of the mitochondria in pancreatic ductal epithelial cells
Emese Tóth1, *József Maléth1, Réka Erdős1, Zsolt Rázga2, László Tretter3, Gergő Horváth3, Zoltán Jr. Rakonczay4, Péter Hegyi5,6
1 University of Szeged, First Department of Medicine, Szeged, Hungary
2 University of Szeged, Department of Pathology, Szeged, Hungary
3 Semmelweis University, Department of Medical Biochemistry, Budapest, Hungary
4 University of Szeged, Department of Pathophysiology, Szeged, Hungary
5 University of Szeged, MTA-SZTE Momentum Translational Gastroenterology Research Group, Szeged, Hungary
6 University of Pécs, Institute for Translational Medicine/1st Department of Medicine, Pécs, Hungary
Abstract text :

Mitochondrial dysfunction is a hallmark of several disease pathogenesis including acute pancreatitis (AP). Our previous results suggest that mitochondrial damage is crucial in bile acid induced inhibition of pancreatic ductal HCO3- secretion, however the details of mitochondrial function and dysfunction in pancreatic ductal epithalial cells (PDEC) is not known yet.

Guinea pig and Cyclophilin D WT and knock out (KO) mouse pancreatic ducts were used. Mitochondrial distribution was studied by electron microscopy (EM), membrane potential (Δψm) was measured by confocal microscopy and pancreatic ductal HCO3- secretion by microfluorometry.

EM measurements revealed that the mitochondrial density is significantly higher on the apical side of the guinea pig PDEC compared to the middle or the basal segment in HEPES solution. The apical mitochondrial density increased further in CO2/HCO3- buffered solution, or during the administration of 5μM forskolin. This redistribution was also confirmed by the Δψm measurements as we detected increased TMRM fluorescence on the apical side of the PDEC during stimulation. The genetic KO of cyclophilin D significantly reduced the loss of Δψm and protected pancreatic ductal HCO3- secretion during the administration of 500μM chenodeoxycholic acid.

Our results suggest that mitochondrial function has a central role in the function of PDEC presumably by providing ATP for fluid and ion secretion. The opening of MPTP seems to be crucial in the bile acid induced toxicity offering a potential therapeutic target in AP.