The TRPM2 ion channel in nucleotide-gated calcium signaling Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Ingo Lange aus Erlangen Als Dissertation genehmigt von den Naturwissen- schaftlichen Fakultäten der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 14. Juli 2008 Vorsitzender der Prüfungskommission: Prof. Dr. Eberhart Bänsch Erstberichterstatter: Prof. Dr. Lars Nitschke Zweitberichterstatter: Prof. Dr. Andrea Fleig 2 3 TABLE OF CONTENT TABLE OF CONTENT....................................................................................................................................... 4 INTRODUCTION TO CALCIUM SIGNALING ........................................................................................... 6 CALCIUM AS A SECOND MESSENGER ................................................................................................................. 6 INFORMATION IS PROCESSED THROUGH CALCIUM–BINDING MOTIFS............................................................... 7 CALCIUM SIGNALING ACROSS THE PLASMA MEMBRANE .................................................................................. 7 CLASSICAL CALCIUM RELEASE CHANNELS ....................................................................................................... 8 INFORMATION THROUGH CALCIUM CAN BE MOBILIZED AND PROCESSED FROM DIFFERENT SOURCES ........... 8 CALCIUM SIGNALING IN THE COMPLEX NETWORK OF CELL SPECIFIC PHYSIOLOGY......................................... 9 INTRODUCTION TO TRP ION CHANNELS ......................................................................................................... 11 TRPM2 IN THE NETWORK OF CALCIUM SIGNALING ....................................................................................... 11 TRP-CHANNELS AND CALCIUM RELEASE........................................................................................................ 13 AIM OF THE THESIS .......................................................................................................................................... 14 MATERIAL........................................................................................................................................................ 15 AGONISTS AND ANTAGONISTS AND PHARMACOLOGICAL INHIBITORS ........................................................... 15 CELL CULTURE AND MEDIA ............................................................................................................................. 15 BUFFERS AND SOLUTIONS................................................................................................................................ 16 ENZYMES ......................................................................................................................................................... 16 CELL SEPARATION REAGENTS AND TOOLS...................................................................................................... 17 ANTIBODIES, BEADS AND STAINING REAGENTS.............................................................................................. 17 TRANSFECTION ................................................................................................................................................ 18 CALCIUM DYES AND CHELATORS .................................................................................................................... 18 ANIMALS.......................................................................................................................................................... 18 EQUIPMENT ...................................................................................................................................................... 18 METHODS.......................................................................................................................................................... 20 CELL CULTURE AND ISOLATION ...................................................................................................................... 20 HEK-293 cells ............................................................................................................................................ 20 INS-1 cells .................................................................................................................................................. 20 Isolation of pancreatic beta cells............................................................................................................... 20 Isolation of human blood-derived neutrophils.......................................................................................... 21 Isolation of human T-lymphocytes............................................................................................................. 21 Isolation of blood-derived monocytes ....................................................................................................... 21 Isolation of murine spleen-derived neutrophils ........................................................................................ 22 Neutrophil isolation from bone marrow (mouse) ..................................................................................... 22 Culture of bone marrow-derived dendritic cells....................................................................................... 22 INS-1 SIRNA EXPERIMENTS ........................................................................................................................... 23 TRPM2 AND ER FLUORESCENCE LABELING IN INS-1 CELLS........................................................................ 23 TRPM2 IMMUNOFLUORESCENCE IN MOUSE NEUTROPHILS AND DENDRITIC CELLS ...................................... 24 ELECTROPHYSIOLOGY AND FLUORESCENCE MEASUREMENTS ....................................................................... 24 Voltage clamp protocols ............................................................................................................................ 24 Fluorescence measurements...................................................................................................................... 25 Fura-2 Ca2+ measurements and perforated patch .................................................................................... 26 Single channel measurements.................................................................................................................... 26 SOLUTIONS....................................................................................................................................................... 27 RESULTS............................................................................................................................................................ 28 OVERVIEW ....................................................................................................................................................... 28 ADP-RIBOSE IS A MULTIMODAL AGONIST FOR PURINERGIC RECEPTORS AND TRPM2 CHANNELS IN THE PLASMA MEMBRANE AND INTRACELLULAR STORES OF BETA CELLS.............................................................. 29 Extracellular ADPR triggers calcium release through P2Y receptors in HEK293 cells........................ 29 Effects of extracellular NAD+ and cADPR in HEK293 cells ................................................................... 30 4 Effect of intracellular ADPR in HEK293 wild-type and TRPM2-expressing cells ................................. 31 INS-1 cells as a model for endogenous TRPM2 ....................................................................................... 33 Extracellular ADPR triggers calcium release through P2Y and adenosine receptors in INS-1 cells.... 33 Effects of extracellular NAD+ and cADPR in INS-1 cells ........................................................................ 34 Effects of intracellular ADPR and pharmacological characterization of stores in INS-1 cells ............. 37 TRPM2 in primary mouse beta cells ......................................................................................................... 40 Extracellular ADPR acts on P2Y receptors.............................................................................................. 40 Intracellular ADPR mediates calcium release through TRPM2.............................................................. 41 cADPR causes calcium release in beta cells............................................................................................. 41 TRPM2 function is limited to calcium release in mouse dendritic cells.................................................. 43 SYNERGISTIC REGULATION OF ENDOGENOUS TRPM2 CHANNELS BY ADENINE DINUCLEOTIDES IN PRIMARY HUMAN NEUTROPHILS...................................................................................................................... 45 Regulation of TRPM2 by intracellular Ca2+ ............................................................................................. 45 Regulation of TRPM2 by cADPR and H2O2.............................................................................................. 50 Regulation of TRPM2 by NAADP ............................................................................................................. 52 TRPM2 IN MOUSE NEUTROPHILS .................................................................................................................... 54 Regulation of TRPM2 by ADPR in wild-type, TRPM2 and CD38 deficient mouse neutrophils............