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Cellular Functions of the Kinase-Coupled TRPM6/TRPM7 Channels” Von Mir Selbstständig Und Ohne Unerlaubte Hilfe Angefertigt Wurde

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Cellular Functions of the Kinase-Coupled TRPM6/TRPM7 Channels” Von Mir Selbstständig Und Ohne Unerlaubte Hilfe Angefertigt Wurde Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) an der Fakultät für Biologie der Ludwig-Maximilians-Universität München Cellular functions of the kinase- coupled TRPM6/TRPM7 channels durchgeführt am Walther-Straub-Institut für Pharmakologie und Toxikologie der Ludwig-Maximilians-Universität München vorgelegt von Silvia Ferioli aus Cento (FE), Italien München, November 2017 Die vorliegende Dissertation wurde von März 2012 bis November 2017 unter der Leitung von Prof. Dr. med. Thomas Gudermann und Dr. Vladimir Chubanov am Walther-Straub-Institut für Pharmakologie und Toxikologie der Ludwig-Maximilians- Universität München durchgeführt. Erster Gutachter: Prof. Dr. Barbara Conradt Zweiter Gutachter: Prof. Dr. med. Thomas Gudermann Datum der Abgabe: 23.11.2017 Datum der mündlichen Prüfung: 27.04.2018 Alla mia famiglia… Eidesstattliche Erklärung Eidesstattliche Erklärung Ich versichere hiermit an Eides statt, dass die vorgelegte Dissertation mit dem Titel ”Cellular functions of the kinase-coupled TRPM6/TRPM7 channels” von mir selbstständig und ohne unerlaubte Hilfe angefertigt wurde. München, den 23.11.2017 Silvia Ferioli Erklärung Hiermit erkläre ich, dass die Dissertation nicht ganz oder in wesentlichen Teilen einer anderen Prüfungskommission vorgelegt worden ist. dass ich mich anderweitig einer Doktorprüfung ohne Erfolg nicht unterzogen habe. München, den 23.11.2017 Silvia Ferioli i Abstract Abstract TRPM6 and TRPM7 are bifunctional proteins containing an ion channel segment covalently linked to a kinase domain. Both proteins function as divalent cation-selective channels highly permeable to Mg2+ and Ca2+, whose activity is regulated by intracellular levels of Mg2+ and Mg·ATP. TRPM7 has been found in all mammalian cells investigated so far, while expression of TRPM6 is restricted to epithelial cells of the kidney, intestine and placenta. TRPM6 and TRPM7 have been proposed to be required for cellular Mg2+ homeostasis. However, the exact role of TRPM7 in the regulation of Mg2+ metabolism remains poorly understood and discussed controversially. Loss-of- function mutations in the human TRPM6 gene give rise to an autosomal recessive disorder called hypomagnesemia with secondary hypocalcemia (HSH). It has been suggested that in epithelial cells TRPM6 functions primarily as a constituent of heteromeric TRPM6/M7 channel complexes, but this concept has not been thoroughly examined. Therefore, the main goal of this work was to define the cellular function of TRPM6 and TRPM7 in cells either expressing only TRPM7 or co-expressing both proteins, TRPM6 and TRPM7. In the first line of our experiments, we generated and functionally characterized three different cell lines deficient in TRPM7 such as mouse trophoblast stem (TS) cells, human haploid leukaemia (HAP1) cells and primary isolated mouse megakaryocytes (MKs). Using the patch-clamp approach, we showed that all three cell lines lacked endogenous TRPM7 divalent cation-selective currents. We also found that TRPM7 deficient TS and HAP1 cells developed Mg2+ deficiency and growth arrest, which could be rescued by increased levels of Mg2+ in the culture medium. TRPM7 deficient MKs were viable but exhibited reduced Mg2+ contents and impaired proplatelet formation. Similar to TS and HAP1 cells, the changes in MKs were reversed by Mg2+ supplementation. We concluded that the TRPM7 channel controls the cellular Mg2+ uptake necessary for the cell proliferation. To get insights into the cellular role of the native TRPM6 protein, we generated TRPM6 deficient TS cells. We observed that, in contrast to TRPM7 KO TS cells, TRPM6 deficient TS cells were able to proliferate in a medium not fortified by additional Mg2+. Furthermore, the currents in TRPM6 KO TS cells were reduced and more sensitive to cytosolic Mg·ATP compared to the WT TS cells. These findings are in line with the notion that endogenous TRPM6 functions as a subunit of heteromeric TRPM6/M7 channel complexes, where TRPM6 potentiate Mg2+ currents due to offset of the inhibitory effect of Mg·ATP. Our experiments with the endogenous TRPM6 and TRPM7 channels were further verified ii Abstract using recombinant TRPM6 and TRPM7 proteins overexpressed in HEK 293 cells. We observed that the recombinant TRPM6 and TRPM7 channels contribute differently to the functional characteristics of the heteromeric TRPM6/M7 channels mimicking the situation with TS cells lacking endogenous TRPM6. Most remarkably, we found that association of TRPM6 with TRPM7 results in large TRPM6/M7 currents insensitive to cytosolic levels of Mg·ATP. Taken together, we conclude that ubiquitously expressed TRPM7 is required for the cellular uptake of Mg2+ and that this function cannot be compensated by TRPM6. Association of TRPM6 in heteromeric TRPM6/M7 channel complexes allows to maintain a high rate of Mg2+ uptake in transporting epithelial cells. iii Abbreviations Abbreviations 2-APB 2-aminoethyl diphenylborinate Ab antibody APS ammonium peroxydisulphate ATP adenosine triphosphate BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid bp base pair BSA bovine serum albumin CC coiled-coil (domain) cDNA complementary DNA DCT distal convoluted tubule DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphates DPBS Dulbecco’s phosphate buffered saline DTT dithiothreitol DVF divalent cation-free solution ECL enhanced chemiluminescence EDTA ethylenediaminetetraacetic acid EGFP enhanced green fluorescent protein EGTA ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid FBS fetal bovine serum FGF4 fibroblast growth factor 4 Fwd forward GAPDH glyceraldehyde 3-phosphate dehydrogenase HAP1 cells human leukemia haploid cells HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid HEK 293 cells human embryonic kidney 293 cells HPR horseradish peroxidase HSH hypomagnesemia with secondary hypocalcemia IC50 half-maximal inhibitory concentration IgG immunoglobulin G IMDM Iscove's modified Dulbecco's medium IP immunoprecipitation IRES internal ribosome entry site ISH in situ hybridization I-V current-voltage relationship iv Abbreviations KO knockout MEM Eagle´s minimum essential medium MagNum magnesium nucleotide-regulated metal ion currents MIC Mg2+-inhibited cation currents MKs mekacaryocytes mRNA messenger RNA n.f. nominally free NMDG N-methyl-D-glucamine NEAA non-essential amino acids NeoR neomycin resistance cassette NTD neural tube defect PCR polymerase chain reaction Pf4 platelet factor 4 PIP2 phosphatidylinositol 4,5-bisphosphate PMEF primary mouse embryonic fibroblasts Rev revers RNA ribonucleic acid RPMI Roswell Park Memorial Institute medium Rs series resistance RT-PCR reverse transcription-polymerase chain reaction SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM standard error of the mean SP splice acceptor site sequence TAE tris-acetate-EDTA Taq DNA-polymerase from Thermus aquaticus TBS tris-buffered saline TBST tris-buffered saline with Tween 20 TGF-β1 transforming growth factor beta 1 TGM tris-glycine-methanol TGS tris-glycine-SDS Thpo thrombopoietin TMs transmembrane segments TRP transient receptor potential channels TRPM6 transient receptor potential melastatin 6 channel TRPM7 transient receptor potential melastatin 7 channel TS cells trophoblast stem cells UTR untranslated region WT wild type v Table of Contents Table of Contents Eidesstattliche Erklärung ............................................................................................ i Abstract ....................................................................................................................... ii Abbreviations ............................................................................................................. iv Table of Contents ...................................................................................................... vi 1. Introduction ............................................................................................................. 1 1.1. TRP channel superfamily ................................................................................... 1 1.1.1. Phylogenetic groups of TRP channels ......................................................... 1 1.1.1.1. Group 1 TRP subfamilies....................................................................... 3 1.1.1.2. Group 2 TRP subfamilies....................................................................... 5 1.2. Melastatin-related TRP (TRPM) channels .......................................................... 5 1.2.1. TRPM6 and TRPM7 kinase-coupled channels ............................................. 7 1.2.1.1. Expression patterns of TRPM6 and TRPM7 .......................................... 7 1.2.1.2. Domain organization of TRPM6 and TRPM7 ......................................... 9 1.2.1.3. Ion permeation profiles of TRPM6 and TRPM7 channels .................... 10 1.2.1.4. Regulation of TRPM6 and TRPM7 channels ....................................... 11 1.2.1.5. Modulation of TRPM6 and TRPM7 by small organic drug-like ligands . 12 1.2.1.6. The suggested physiological functions of TRPM7 ............................... 13 1.2.1.7. Physiological implications of the TRPM6 channel ................................ 14 1.3. Mg2+ homeostasis ............................................................................................. 16 1.3.1. Mg2+ channels and transporters ................................................................. 17 1.3.2. Roles of TRPM6 and TRPM7 in organismal
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