Untersuchung TRPC-modulierender Gestagene und Proteine [Elektronische Ressource] / von Susanne Miehe

Susanne Miehe

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Untersuchung TRPC-modulierender Gestagene und
Proteine



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften





vorgelegt beim Fachbereich für Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main




von
Susanne Miehe
aus Rochlitz


Frankfurt am Main 2008
(D30)





















vom Fachbereich für Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität als Dissertation angenommen.








Dekan: Prof. Dr. Harald Schwalbe
1. Gutachter: Prof. Dr. Dieter Steinhilber
2. Gutachter: Prof. Dr. Andreas Busch
Datum der Disputation: 04. Juli 2008






Investigation of TRPC channel-modulating progestins and
proteins



Dissertation
for the Achievement of the Doctor’s Degree
of Natural Sciences





submitted to the Faculty of Biochemistry, Chemistry and Pharmacy
of the Johann Wolfgang Goethe-University
Frankfurt am Main




by
Susanne Miehe
from Rochlitz


Frankfurt am Main 2008
(D30)

Table of contents
Table of contents
1 Introduction....................................................................................................................1
1.1 Calcium signalling.....................................................................................................1
2+1.1.1 Store- and receptor-operated Ca influx.............................................................2
1.1.2 Activation of store-operated channels .................................................................4
1.2 The TRP channel superfamily ..................................................................................6
1.3 The TRPC family ......................................................................................................9
1.3.1 Structural features of TRPCs...............................................................................9
1.3.2 TRPC-interacting proteins .................................................................................11
1.3.3 Activation mechanisms......................................................................................12
1.3.4 TRPC subfamilies..............................................................................................14
1.4 Aims........................................................................................................................20
2 Materials and methods................................................................................................22
2.1 Materials .................................................................................................................22
2.1.1 Chemicals, enzymes, consumables ..................................................................22
2.1.2 Kits.....................................................................................................................24
2.1.3 Antibodies..........................................................................................................25
2.1.4 Bacterial strains .................................................................................................25
2.1.5 Yeast strains......................................................................................................25
2.1.6 Cell lines and primary cells................................................................................25
2.1.7 Primers ..............................................................................................................26
2.1.8 siRNA27
2.1.9 Genetic constructs.............................................................................................27
2.1.10 Apparatus28
2.1.11 Buffers, media and solutions .............................................................................29
2.2 Molecular biological methods .................................................................................32
2.2.1 Determination of nucleic acid concentrations and cell density ..........................32
2.2.2 Primer construction............................................................................................32
2.2.3 Polymerase chain reaction (PCR) .....................................................................32
2.2.4 DNA restriction digest........................................................................................32
2.2.5 Dephosphorylation of linearized vectors............................................................33
2.2.6 DNA gel electrophoresis....................................................................................33
2.2.7 Ligation ..............................................................................................................33
2.2.8 TOPO cloning ....................................................................................................33
2.2.9 Gateway cloning ................................................................................................34
2.2.10 Transformation of chemically competent bacteria .............................................34
2.2.11 Electroporation of bacteria.................................................................................34
2.2.12 Plasmid amplification and purification................................................................34
2.2.13 DNA sequencing35
2.2.14 Analysis of nucleotide and protein sequences...................................................35
2.2.15 Expression and purification of GST fusion proteins...........................................35
2.3 Yeast two-hybrid (Y2H) system ..............................................................................36
2.3.1 cDNA library titering and amplification...............................................................37
2.3.2 Transformation of yeast.....................................................................................38
2.3.3 ß-galactosidase assay.......................................................................................39
2.3.4 Plasmid preparation from yeast .........................................................................39
2.4 Culture of mammalian cells ....................................................................................40
2.4.1 Transfection of mammalian cells .......................................................................42
2.4.2 Generation of a HM1 cell line stably expressing mTRPC5-YFP........................42
Table of contents
2.5 Protein biochemical methods .................................................................................42
2.5.1 Preparation of cell lysates..................................................................................42
2.5.2 Determination of protein content........................................................................43
2.5.3 SDS-PAGE ........................................................................................................43
2.5.4 Western blot.......................................................................................................43
2.5.5 GST pulldown assay..........................................................................................44
2.5.6 Co-immunoprecipitation.....................................................................................44
2.5.7 Surface expression analysis..............................................................................44
2.5.8 Peptidyl-prolyl cis-trans isomerization assay.....................................................45
2.5.9 Phospholipid overlay assay ...............................................................................46
2.5.10 Cova-PIP specificity plate assay46
2.5.11 Immunofluorescence .........................................................................................46
2+2.6 Fluorometric [Ca ] measurements ........................................................................47 i
2.7 Patch clamp recordings ..........................................................................................49
2.8 In vitro vascular function50
2.9 Statistics .................................................................................................................51
3 Results..........................................................................................................................52
3.1 Differential inhibition of TRPC channels by norgestimate.......................................52
3.1.1 FLIPR measurements........................................................................................52
3.1.2 Patch clamp recordings .....................................................................................58
3.1.3 Isometric tension recording of aortic rings.........................................................61
3.2 Physical interaction of SESTD1 and TRPC channels.............................................63
3.2.1 Y2H results ........................................................................................................63
3.2.2 Mapping of the TRPC4-SESTD1 interaction site..