Aus dem Institut für Veterinär-Physiologie des Fachbereichs Veterinärmedizin der Freien Universität Berlin SLC41A1, SLC41A3 and CNNM2: Magnesium responsive genes with potential involvement in human ailments Inaugural-Dissertation zur Erlangung des akademischen Doktorgrades philosophiae doctor (Ph.D.) in ’Biomedical Science’ an der Freien Universität Berlin vorgelegt von Lucia Mastrototaro Biotechnologin aus Lecce (Italien) Berlin 2016 Journal-Nr. : 3915 Gefördert durch ein Elsa-Neumann-Stipendium des Landes Berlin Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. Jürgen Zentek Erster Gutachter: Univ.-Prof. Dr. Jörg Rudolf Aschenbach Zweiter Gutachter: Univ.-Prof. Dr. Heidrun Gehlen Dritter Gutachter: Prof. Dr. Jürgen Vormann Deskriptoren (nach CAB-Thesaurus): magnesium, SLC41A1, SLC41A3, CNNM2, neurodegeneration, Parkinson’s disease, insulin, intracellular magnesium homeostasis, magnesium mitochondrial efflux system, protein expression, protein localization, transport studies Tag der Promotion: 05.10.2016 To my family Table of contents TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... II LIST OF ABBREVIATIONS ........................................................................................................III 1. GENERAL INTRODUCTION ................................................................................................ 1 2. LITERATURE REVIEW ...................................................................................................... ..3 2.1. Magnesium and its importance in the human body ....................................................... ..3 2.2. Magnesium transport into the cell and between intracellular stores ............................. ..4 2.3. Hormonal regulation of intracellular magnesium .......................................................... ...83 2.4. SLC41 family ............................................................................................................... ..9 2.4.1. SLC41A1 as Na+/Mg2+ exchanger ........................................................................ 10 2.4.2. SLC41A3 .............................................................................................................. 14 2.5. CNNM2 ....................................................................................................................... 16 3. AIMS AND OBJECTIVES OF THE THESIS ....................................................................... 20 4. RESULTS .......................................................................................................................... 21 4.1. Publication I .................................................................................................................... 21 4.2. Publication II ................................................................................................................... 33 4.3. Publication III .................................................................................................................. 45 4.4. Publication IV .................................................................................................................. 54 4.5. Publication V ................................................................................................................... 73 5. DISCUSSION .................................................................................................................... 94 SUMMARY .............................................................................................................................. 101 ZUSAMMENFASSUNG ........................................................................................................... 103 REFERENCES ........................................................................................................................ 105 PUBLICATIONS ...................................................................................................................... 116 DANKSAGUNG ....................................................................................................................... 120 Selbständigkeitserklärung ....................................................................................................... 123 I List of figures LIST OF FIGURES Figure 1. Various proposed Mg2+ transporters and their predicted transport mechanisms in vertebrate cells (56) ................................................................................................................... ..5 Figure 2. Structural overview of the prokaryotic MgtE transporters. ............................................9 Figure 3. Computer model of hSLC41A1 (56kDa). .................................................................... 11 Figure 4. Computer model of hSLC41A1 (56kDa). .................................................................... 12 Figure 5. Computer-predicted model (TMpred) of SLC41A3 topology (106) ............................. 16 Figure 6. Localization of the mutations in the predicted secondary structure of CNNM2 ........... 17 Figure 7. Schematic model of the structure of CNNM2 after endoplasmic processing .............. 18 II List of abbreviations LIST OF ABBREVIATIONS aa Amino acid ACCA1 Acetyl-/propionyl-coenzyme A carboxylase alpha chain 1 ACD Ancient conserved domain ACDP2 Ancient conserved domain protein 2 ADP Adenosine diphosphate AD Alzheimer’s disease ADHD Attention deficit hyperactivity disorder ALS Amyotrophic lateral sclerosis AMBRA1 Autophagy/beclin-1 regulator AR Androgen receptor AREs Androgen receptor elements ATP Adenosine triphosphate cAMP Cyclic adenosine monophosphate CBS Cystathionine-beta-synthase CNNM2 Cyclin M2 COX Cytochrome C oxidase DM2 Diabetes mellitus 2 DCT Distal convoluted tubule EBP Emopamil binding protein ER Endoplasmic reticulum FFS Fast-filter spectrofluorometry GA Golgi apparatus HD Huntington’s disease HEK293 Human embryonic kidney 293 I1/2 Isophorm 1/2 IMH Intracellular Mg homeostasis MagT1 Magnesium transporter 1 MDCT Mouse distal convoluted tubule cells Mg Magnesium MgtE Magnesium transporter E MPC1 Mitochondrial pyruvate carrier 1 MRG Magnesium responsive gene III List of abbreviations NMDG N-Methyl-D-glucamine NME Na+/Mg2+ exchanger PARK7/DJ1 Parkinson disease 7/ deglycase protein PCR Polymerase chain reaction PD Parkinson’s disease PKA Protein kinase A PKC Protein kinase C PINK1 PTEN Induced Putative Kinase 1 RBC Red blood cells SLC Solute carrier SLC41A1 Solute carrier family 41 member A1 SLC41A3 Solute carrier family 41 member A3 SNP Single nucleotide polymorphism SPTBN1 Spectrin β chain 1 SU-YTH Split-ubiquitin yeast two hybrid TAL Thick ascending limb TM Transmembrane TRPM6/7 Transient receptor potential melastatin ion channel 6/7 TUSC3 Tumor suppressor candidate 3 IV General introduction 1. GENERAL INTRODUCTION Magnesium (Mg) plays several crucial roles in eukaryotic cells and in the whole organism. In mitochondria it is a key factor of the adenosine triphosphate (ATP)-synthesizing machinery and its “deficit” or defects in its transport impairs cellular functions, e. g. proliferation rate, which can be reversed by magnesium supplementation. Therefore it is not surprising that Mg deficiency and/or a change in the intracellular Mg homeostasis (IMH) might lead to a multitude of serious ailments, such as neurodegenerative disorders (Parkinson’s disease (PD) and Alzheimer’s disease (AD)), stroke, diabetes and cardiovascular diseases (1). The intracellular free Mg2+ concentration is in the order of 0.5 mM and it is maintained below the concentration predicted from the transmembrane electrochemical potential. This control is achieved through a balance of Mg2+ uptake, intracellular Mg2+ storage and Mg2+ efflux; clearly, specific magnesium transporters exist for each step and impairment in their function can lead to altered intracellular magnesium levels or magnesium homeostasis. In the last decades, roughly a dozen of candidate genes have been proposed to encode for proteins regulating Mg2+ transport in eukaryotic cells, but only few of them have been well characterized as Mg2+ transporters. The first mammalian Mg2+ transporter to be identified at the molecular level was Mrs2 of the inner mitochondrial membrane (2). Other well characterized channels and transporters are TRPM6/7, MagT1, and SLC41A1 (3). Importantly, SLC41A1 has been identified and characterized as the main Mg2+ efflux mechanism in the plasma membrane (4, 5) by members of the supervising team at the Institute of Veterinary Physiology, Berlin, and as Na+/Mg2+ exchanger overexpressed in pre-eclamptic women (6). Several other genes have been described as magnesium responsive genes (MRG) and meanwhile molecularly characterized, but their role in the cellular physiology has long remained uncovered. For example, CNNM2 and SLC41A3 are both listed among the MRG, indeed their mRNA levels rise in mouse distal convoluted tubule cells (MDCT) grown in nominally Mg2+-free medium (7, 8), but data about their cellular localization and their role in magnesium transport are missing. Thus this thesis has focused on the further investigation of Mg2+ efflux mediated by SLC41A1 and its impact on human aliments such as PD and diabetes and on the functional and molecular characterization of SLC41A3 and CNNM2 in order to have a complete frame of the magnesium transport and mobilization in the cells and eventually to better understand the importance of these processes