Functional analysis of the potassium channel beta subunit KCNE3 [Elektronische Ressource] / Patricia Preston Ferrer. Gutachter: T.J Jentsch ; W. Lockau ; D. Günzel

Patricia Preston Ferrer

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 72
• Langue: Deutsch
• Poids de l'ouvrage: 20 Mo

Extrait

Functional Analysis of the Potassium Channel Beta
Subunit KCNE3

Dissertation

zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)

im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Diplom-Biologin Patricia Preston Ferrer


Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. C. Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. A. Herrmann


Gutachter: 1. Prof. Dr. T.J Jentsch
2. Prof. Dr. W. Lockau
3. PD Dr. D. Günzel

Tag der mündlichen Prüfung: 22.October 2010


Table of Contents
1 ZUSAMMENFASSUNG................................................................................................1
2 INTRODUCTION ..........................................................................................................3
2.1 Ion Channels ............................................................................................................................................3
2.1.1 Resting Membrane Potential and NERNST Equilibrium Potential........................................................3
2.2 Potassium Channels.................................................................................................................................5
2.3 Voltage Gated Potassium Channels .......................................................................................................6
2.4 KCNQ Family..........................................................................................................................................7
2.5 KCNQ1.....................................................................................................................................................8
2.5.1 Electrophysiological Properties of KCNQ1 ...........................................................................................9
2.5.2 Pharmacology.......................................................................................................................................10
2.5.3 Intracellular Modulation of KCNQ1 ....................................................................................................10
2.6 KCNE Accessory Subunits ...................................................................................................................12
2.6.1 KCNE Family Members.......................................................................................................................12
2.6.2 Glycosylation of KCNE Proteins .........................................................................................................18
2.7 KCNQ1/KCNE Complexes...................................................................................................................19
2.7.1 Intracellular Trafficking of KCNQ1/KCNE Complexes ......................................................................19
2.7.2 KCNQ1 and KCNE Assembly and Modulation...................................................................................20
2.7.3 Functional Roles of KCNQ1/KCNE1 ..................................................................................................21
2.7.4 FuKCNQ1/KCNE2........25
2.8 Putative Functional Roles of KCNE3 ..................................................................................................27
2.8.1 KCNE3 in Intestinal Tract....................................................................................................................27
2.8.2 KCNE3 in Heart ...................................................................................................................................29
2.8.3 KCNE3 in Airways...............................................................................................................................30
2.8.4 KCNE3 in Central Nervous System...........32
2.8.5 3 in Skeletal Muscle...................................................................................................................32
2.9 Aim of the Study.........................33
3 MATERIALS................................................................................................................35
3.1 Chemicals and Enzymes........................................................................................................................35
3.2 Buffers and Solutions ............................................................................................................................35
3.3 Bacterial Culture ...................................................................................................................................37
3.3.1 Media....................................................................................................................................................37
3.4 Cell Culture of Embryonic Stem Cells.................................................................................................37
3.4.1 Media and Solutions.......................37
3.5 Plasmids..................................................................................................................................................38
3.6 Oligonucleotides.....................................................................................................................................38

3.7 Antibodies ..............................................................................................................................................39
3.7.1 Primary Antibodies...............................................................................................................................39
3.8 Electrophysiology ..........................................................................................................39
3.8.1 Chemicals .............................................................................................................................................39
3.8.2 Solutions...............................39
4 METHODS....................................................................................................................41
4.1 Microbiological Methods ......................................................................................................................41
4.1.1 Electroporation of Plasmid DNA into Competent Bacteria..................................................................41
4.2 Molecular Biological Methods..............................................................................................................41
4.2.1 DNA Plasmid “Mini” Preparation........................................................................................................41
4.2.2 Preparation of Plasmid DNA from 50 ml Cultures (Midiprep) ............................................................41
4.2.3 Isolation of Genomic DNA from ES Cells ...........................................................................................42
4.2.4 Genomic DNA Isolation from Mouse Tail Samples ............................................................................42
4.2.5 Digestion of DNA by Restriction Endonucleases.....42
4.2.6 DNA Agarose Gel Electrophoresis.......................................................................................................43
4.2.7 Isolation of DNA Fragments from Agarose Gels .................................................................................43
4.2.8 DNA Sequencing..................................................................................................................................43
4.2.9 Measuring DNA Concentrations ..........................................................................................................44
4.2.10 Polymerase Chain Reaction (PCR).....................................................................................................44
4.2.11 RNA Preparation ................................................................................................................................45
4.2.12 Purification of RNA ...........................................................................................................................46
4.2.13 cDNA Synthesis ..........................................................................................................46
4.2.14 Southern Blot...........................46
4.2.15 Northern Blot47
4.2.16 Radioactive Labeling of DNA Samples .............................................................................................47
4.2.17 Hybridization of Genomic DNA ........................................................................................................47
4.3 Protein Biochemical Methods...............................................................................................................48
4.3.1 Membrane Preparation....................48
4.3.2 Lysates of Transfected Cells.............................................................................................48
4.3.3 Deglycosylation of Membrane Bound Proteins....................................................................................48
4.3.4 Determination of Protein Concentrations .............................................................................................49
4.3.5 Sodium-Dodecyl-Sulfate-Polyacrylamide Gel Electrophresis (SDS-PAGE).......................................49
4.3.6 SDS-PAGE gel Comp