Localization and functional analysis of the calcium permeable melastatin-like channel TRPM3 [Elektronische Ressource] / vorgelegt von Chen, Xiaodi

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Aus dem Institut fur Pharmakologie und ToxikologieGesch aftsfuhrender Direktor: Prof. Dr. Frank Czubaykodes Fachbereichs Medizin der Philipps{Universitut MarburgLocalization and Functional Analysis of theCalcium Permeable Melastatin{like ChannelTRPM3Inaugural{Dissertation zur Erlangungdes Doktorgrades der Humanbiologiedem Fachbereich Medizin der Philipps{Universit at Marburgvorgelegt vonCHEN, XIAODIaus Shanghai, ChinaMarburg/Lahn 2009Angenommen vom Fachbereich Medizin der Philipps{Universit at Marburgam: 10.12.2009Gedruckt mit Genehmigung des Fachbereichs.Dekan: Prof. Dr. Matthias RothmundReferent: Prof. Dr. Tim PlantKorreferent: Prof. Dr. Dr. Jurgen DautTo my wife and daughterAcknowledgementI would like to acknowledge all those very helpful people who have assisted me in my work.First and foremost, I would like to sincerely thank my mentor, Dr. Thomas Hofmann,for his guidance, understanding, insightful criticisms, patience, and most importantly, hisfriendship during my doctoral thesis at Philipps{University Marburg. He encouraged meto develop independent thinking and research skills, and helped me in the nal stage ofwriting the thesis.I wish to express my sincere gratitude to Prof. Dr. Tim Plant for his kindness, supportand suggestions throughout all stages of the thesis. I would also like to thank Dr. VladimirChubanov for his assistance and guidance in various molecular biologic approaches.My thanks go to all of the members of the Dr.

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Aus dem Institut fur Pharmakologie und Toxikologie
Gesch aftsfuhrender Direktor: Prof. Dr. Frank Czubayko
des Fachbereichs Medizin der Philipps{Universitut Marburg
Localization and Functional Analysis of the
Calcium Permeable Melastatin{like Channel
TRPM3
Inaugural{Dissertation zur Erlangung
des Doktorgrades der Humanbiologie
dem Fachbereich Medizin der Philipps{Universit at Marburg
vorgelegt von
CHEN, XIAODI
aus Shanghai, China
Marburg/Lahn 2009Angenommen vom Fachbereich Medizin der Philipps{Universit at Marburg
am: 10.12.2009
Gedruckt mit Genehmigung des Fachbereichs.
Dekan: Prof. Dr. Matthias Rothmund
Referent: Prof. Dr. Tim Plant
Korreferent: Prof. Dr. Dr. Jurgen DautTo my wife and daughterAcknowledgement
I would like to acknowledge all those very helpful people who have assisted me in my work.
First and foremost, I would like to sincerely thank my mentor, Dr. Thomas Hofmann,
for his guidance, understanding, insightful criticisms, patience, and most importantly, his
friendship during my doctoral thesis at Philipps{University Marburg. He encouraged me
to develop independent thinking and research skills, and helped me in the nal stage of
writing the thesis.
I wish to express my sincere gratitude to Prof. Dr. Tim Plant for his kindness, support
and suggestions throughout all stages of the thesis. I would also like to thank Dr. Vladimir
Chubanov for his assistance and guidance in various molecular biologic approaches.
My thanks go to all of the members of the Dr. Thomas Hofmann research group, especially,
Fatma Aktuna, Anna Dietz, for helping me in the experiments and giving some needed
humor and entertainment in a somewhat stressful laboratory environment. Thanks to Prof.
Dr. Alexander Dietrich, Prof. Dr. Achim Aigner, and Prof. Dr. Frank Czubayko, whose
advice, guidance and wisdom aided the writing of this thesis. My thanks also go to Daniel
Schulze, Meike Fahlbusch, Tim Mayer, Sabrina H obel, Ahmed Ibrahim, Winfried Lorenz,
Susanne Ziegler, with whom I worked e ciently and solved many of the same problems.
I am deeply grateful to the Department of Pharmacology and Toxicology at Philipps{
University Marburg, especially to those members who gave me their valuable input and
advice.
Philipps University
Marburg
December 11, 2009Table of Contents
Acknowledgement iv
List of Figures xi
List of Tables xiii
List of Abbreviations xiv
1 Introduction 1
2+1.1 Ca signaling and phospholipase C{mediated signaling pathways . . . . . 1
1.2 TRP channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1 The molecular structure of mammalian TRP channels . . . . . . . . 3
1.2.1.1 Transmembrane domains, N{ and C{termini of TRP channels 3
1.2.1.2 Multimerization of mammalian TRP channel subunits . . . 5
1.2.2 Tissue expression and biological functions of mammalian TRP channels 5
1.2.3 TRPM channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3 TRPM3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.1 Structural features of TRPM3 . . . . . . . . . . . . . . . . . . . . . 9
1.3.1.1 Genomic regions and transcript variants . . . . . . . . . . . 9
1.3.1.2 Protein structure . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3.2 Tissue distribution of TRPM3 . . . . . . . . . . . . . . . . . . . . . 10
1.3.3 Channel properties of . . . . . . . . . . . . . . . . . . . . . 11
1.3.4 Inhibition, blockage and activation of TRPM3 . . . . . . . . . . . . 12
1.3.5 Biological role and relevance of TRPM3 . . . . . . . . . . . . . . . . 13
1.4 Objectives of this thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
ivTABLE OF CONTENTS
2 Materials and Methods 14
2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.1.1 Animals, bacteria, cell lines and yeast cells . . . . . . . . . . . . . . 14
2.1.2 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.1.3 Cell culture materials . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.1.4 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.1.5 Consumables and laboratory equipment . . . . . . . . . . . . . . . . 17
2.1.6 Enzymes and peptides . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.1.7 Molecular biology reagent systems . . . . . . . . . . . . . . . . . . . 18
2.1.8 Nucleic acids and plasmids . . . . . . . . . . . . . . . . . . . . . . . 19
2.1.9 Bioinformatic tools and online databases . . . . . . . . . . . . . . . . 19
2.1.10 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.2.1 DNA analysis and preparation . . . . . . . . . . . . . . . . . . . . . 21
2.2.1.1 General DNA methods . . . . . . . . . . . . . . . . . . . . 21
2.2.1.2 Polymerase chain reaction (PCR) . . . . . . . . . . . . . . 21
2.2.1.3 Preparation of chemocompetent DH5 E.coli cells . . . . . 22
2.2.1.4 DNA transfection . . . . . . . . . . . . . . . . . . . . . . . 23
2.2.1.5 Rapid ampli cation of cDNA ends (RACE) . . . . . . . . . 23
2.2.1.6 Site{directed mutagenesis . . . . . . . . . . . . . . . . . . . 25
®2.2.1.7 TOPO {cloning . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2.2 RNA analysis and preparation . . . . . . . . . . . . . . . . . . . . . 26
2.2.2.1 Isolation of total RNA from eukaryotic cells and tissues . . 26
+2.2.2.2 Selection of Poly(A) RNA from total RNA . . . . . . . . 26
2.2.2.3 Electrophoresis of RNA with agarose{formaldehyde gels . . 27
2.2.2.4 Northern blotting . . . . . . . . . . . . . . . . . . . . . . . 27
2.2.2.5 Random{primed radioactive DNA labeling . . . . . . . . . 28
2.2.2.6 Northern hybridization . . . . . . . . . . . . . . . . . . . . 29
2.2.3 Protein biochemical analysis . . . . . . . . . . . . . . . . . . . . . . 29
2.2.3.1 Membrane protein extraction from mouse tissues . . . . . . 30
2.2.3.2 SDS{PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.3.3 Western blot . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.3.4 Co{immunoprecipitation . . . . . . . . . . . . . . . . . . . 31
vTABLE OF CONTENTS
2.2.3.5 Fluorescence resonance energy transfer (FRET) . . . . . . 32
2.2.4 Yeast genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.2.4.1 Yeast two hybrid screening . . . . . . . . . . . . . . . . . . 33
2.2.4.2 Transformation of DNA into competent yeast cells . . . . . 37
2.2.4.3 Preparation of yeast total DNA using glass beads . . . . . 37
2.2.4.4 Yeast protein extraction . . . . . . . . . . . . . . . . . . . . 38
2.2.4.5 Yeast colony{PCR . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.4.6 Colony{lift lter assay . . . . . . . . . . . . . . . . . . . . . 39
2.2.5 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.5.1 General cell culture techniques . . . . . . . . . . . . . . . . 39
2.2.5.2 Primary cell culture: melanocyte isolation from the mouse
dermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.5.3 Generation of stable mammalian cell lines using the GeneSwitch™
system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.2.6 Methods for histology and immunohistochemistry . . . . . . . . . . . 42
2.2.6.1 General histological techniques . . . . . . . . . . . . . . . . 42
2.2.6.2 -galactosidase (lacZ) assay . . . . . . . . . . . . . . . . . 42
2.2.6.3 Immuno uorescent labeling of adherent cells . . . . . . . . 44
2.2.6.4 Confocal and uorescence microscopy . . . . . . . . . . . . 44
2.2.6.5 Antigen design and antibody production . . . . . . . . . . 44
2+2.2.7 Measurement of the intracellular free Ca concentration . . . . . . 45
2.2.7.1 Fura-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.2.7.2 Aequorin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.2.8 LacZ transgenic TRPM3 mouse, mouse genotyping and phenotyping 48
2.2.8.1 LacZ transgenic TRPM3 mouse . . . . . . . . . . . . . . . 48
2.2.8.2 Genotyping of lacZ transgenic TRPM3 mice . . . . . . . . 48
=2.2.8.3 Phenotypic analysis of TRPM3 mice . . . . . . . . . . . 50
3 Results 52
3.1 Molecular cloning and functional analysis of mTRPM3 . . . . . . . . . . . . 52
3.1.1 Molecular cloning of mM3 and mM3 . . . . . . . . . . . . . . 521719 1337
3.1.2 Analysis of mM3 and mM3 protein expression . . . . . . . . 531719 1337
3.1.3 Functional analysis of mM3 . . . . . . . . . . . . . . . . . . . . . 561719
viTABLE OF CONTENTS
2+3.1.3.1 Quanti cation of intracellular Ca concentration increase
in HEK-293 cells expressing mM3 and mM3 . . . . 571719 1337
2+3.1.3.2 Effects of divalent cations on Ca entry in HEK-293 cells
expressing mM3 . . . . . . . . . . . . . . . . . . . . . . 591719
3.2 Identi cation of functional domains in mM3 . . . . . . . . . . . . . . . . 611719
3.2.1 Structure of the pore and C{terminus of mM3 . . . . . . . . . . 611719
3.2.1.1 Site{directed mutagenesis of amino acid residues relevant to
2+Ca entry in the mM3 pore . . . . . . . . . . . . . . . 611719
3.2.1.2 Characterization of the mM3 C{terminus . . . . . . . . 641719
3.2.2 Analysis of mM3 homomer formation . . . . . . . . . . . . . . . 691719
3.2.3 Identi cation of putative interaction partners of mM3 . . . . . . 741719
3.2.3.1 Identi cation of putative interaction partners of mM3 1719
using the yeast two hybrid system . . . . . . . . . . . . . . 74
3.2.3.2 Analysis of protein kinase inhibitor{gamma as an interaction
partner for mM3 . . . . . . . . . . . . . . . . . . . . . . 771719
3.3 Tissue distribution of mouse TRPM3 . . . . . . . . . . . . . . . . . . . . . . 79
3.3.1 Northern blot analysis of mouse TRPM3 tissue distribution . . . . . 79
3.3.2 Western blot of mouse expression . . . . . . . . . . 80
3.3.2.1 Characterization of mouse TRPM3 antibodies . . . . . . . 80
3.3.2.2 Analysis of the TRPM3 protein expression in the adult mouse
eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
3.3.3 Histochemical and immunohistochemical analysis of TRPM3 expres-
sion using a lacZ transgenic mouse model . . . . . . . . . . . . . . . 83
=3.3.3.1 LacZ gene expression in lacZ transgenic TRPM3 adult
mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.3.3.2 Tissue distribution of TRPM3 during mouse development . 90
=3.4 Phenotypic analysis of a TRPM3 mouse model . . . . . . . . . . . . . . 96
3.4.1 Genotyping and genetic background correction . . . . . . . . . . . . 96
=3.4.2 The viability of TRPM3 mice . . . . . . . . . . . . . . . . . . . . 98
=3.4.3 Morphological observations on TRPM3 mice . . . . . . . . . . . 99
=3.4.4 Behavioral analyses of the visual ability of TRPM3 mice . . . . . 99
4 Discussion 101
4.1 Molecular cloning and functional analysis of mM3 and mM3 . . . . . 1011719 1337
viiTABLE OF CONTENTS
4.1.1 Cloning and characterization of mM3 and mM3 . . . . . . . . 1011719 1337
4.1.2 Mutagenesis analysis of the pore . . . . . . . . . . . . . . . . . . . . 104
4.2 Molecular structure and functional relevance of the C{terminus . . . . . . . 106
4.2.1 C{terminus of mM3 and palmitoylation . . . . . . . . . . . . . . 1061719
4.2.2us of mM3 and homomultimerization . . . . . . . . . . 1081719
4.2.3 C{terminus of mM3 and its putative interaction partners . . . . 1091719
4.3 Expression pattern of mouse TRPM3 . . . . . . . . . . . . . . . . . . . . . . 110
4.3.1 Expression analysis of in mouse tissues using Northern blot,
antibodies and lacZ staining . . . . . . . . . . . . . . . . . . . . . . 110
4.3.2 Mouse TRPM3 and subcellular localization . . . . . . . . . . . . . . 111
4.3.3 Mouse expression and its biological roles . . . . . . . . . . . 112
4.4 Phenotype of the TRPM3 knockout mice . . . . . . . . . . . . . . . . . . . 113
5 Conclusions 115
References 139
Curriculum Vitae 141
Declaration 143
viii