Disease-causing dysfunctions of barttin, an accessory subunit of ClC-K chloride channels [Elektronische Ressource] / von Audrey Gertruda Huberdina Janssen

Audrey Janssen

Sujets

• Gesundheit

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2008
• Nombre de lectures: 38
• Poids de l'ouvrage: 2 Mo

Extrait

Disease-causing dysfunctions of barttin,
an accessory subunit of ClC-K chloride channels









Von der Naturwissenschaftlichen Fakultät
der Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades

Doktorin der Naturwissenschaften

-Dr. rer. nat.-





genehmigte Dissertation
von


Audrey Gertruda Huberdina Janssen, M.Sc.
geboren am 8. Juli 1980, in Venray, Niederlande














2008











































Referent: Prof. Dr. Christoph Fahlke
Korreferent: Prof. Dr. Anaclet Ngezahayo
Prüfer: Prof. Dr. Symeon Papadopoulos

Tag der Promotion: 15-August-2008 Contents I
Contents


1. Introduction................................................................................................................... 1

1.1 Membrane proteins in the kidney and the inner ear...................................................... 3
1.1.1 Kidney.................................................................................................................. 3
1.1.2 Inner ear................................................................................................................ 4

1.2 Bartter Syndrome........................................................................................................... 5

1.3 ClC chloride channel / transporter family..................................................................... 6
1.3.1 ClC-1, ClC-2, ClC-Ka, ClC-Kb and barttin................................................... 7
1.3.2 ClC-3, ClC-4 and ClC-5................................................................................. 9
1.3.3 ClC-6, ClC-7 and ostm1................................................................................. 10

1.4 ClC protein structure..................................................................................................... 10
1.4.1 Structure of the transmembrane core.............................................................. 10
1.4.2 Structure of the cytoplasmic domain.............................................................. 13

1.5 Barttin............................................................................................................................ 14
1.5.1 The PY motif.................................................................................................. 14
1.5.2 Disease-causing barttin mutations.................................................................. 15

1.6 Aim of this thesis........................................................................................................... 17


2. Materials and methods.................................................................................................. 19

2.1 Chemicals and materials................................................................................................ 21

2.2 Alignment...................................................................................................................... 21
II Contents
2.3 Molecular biology......................................................................................................... 21
2.3.1 Vectors............................................................................................................ 21
2.3.2 Mutagenesis.................................................................................................... 22
2.3.2.1 Primers and constructs.....................................................................
2.3.2.2 Quikchange: site directed mutagenesis............................................ 23
2.3.2.3 Polymerase Chain Reaction............................................................. 24
2.3.3 Transformation............................................................................................... 24
2.3.4 Plasmid recovery............................................................................................ 25
2.3.4.1 Plasmid recovery.............................................................................. 25
2.3.4.2 DNA concentration measurement....................................................
2.3.5 Agarose gel electrophoresis............................................................................ 25
2.3.6 DNA restriction.............................................................................................. 26
2.3.7 Gel extraction.................................................................................................. 26
2.3.7.1 Gel extraction................................................................................... 26
2.3.7.2 Glassmilk preparation......................................................................
2.3.8 Ligation........................................................................................................... 27
2.3.9 DNA sequencing.............................................................................................
2.3.9.1 DNA sequencing.............................................................................. 27
2.3.9.2 Ethanol precipitation of sequenced DNA........................................ 28
2.3.10 LB medium, agar plates and antibiotics....................................................... 28
2.3.11 Competent bacteria....................................................................................... 28

2.4 Cell culture of mammalian cells.................................................................................... 29
2.4.1 Cell lines, growth and splitting....................................................................... 29
2.4.2 Transfection.................................................................................................... 29
2.4.2.1 Lipofectamine.................................................................................. 30
2.4.2.1 Calcium phosphate precipitation......................................................
2.4.3 Stable transfected MDCK cells...................................................................... 30
2.4.4 Freezing cells.................................................................................................. 31
2.4.5 Thawing cells.................................................................................................. 31

2.5 Electrophysiology.......................................................................................................... 31
2.5.1 Cells................................................................................................................ 31
2.5.2 Setup............................................................................ ................................ 31 Contents III
2.5.3 Microelectrodes.............................................................................................. 32
2.5.4 Measure and reference electrode....................................................................
2.5.5 Gigaseal.......................................................................................................... 32
2.5.6 Whole cell configuration and solutions...........................................................33
2.5.7 Data analysis................................................................................................... 33

2.6 Fluorescence.................................................................................................................. 34

2.7 Confocal imaging.......................................................................................................... 34
2.7.1 Experiments on fixed cells.............................................................................. 34
2.7.2 Experiments on living cells............................................................................ 35

2.8 Protein Biochemistry..................................................................................................... 36
2.8.1 SDS polyacrylamide gel electrophoresis........................................................ 36
2.8.2 2x SDS loading buffer.................................................................................... 36
2.8.3 Western blotting.............................................................................................. 37
2.8.4 Deglycosylation..............................................................................................
2.8.5 Scanning of fluorescent proteins on SDS gels................................................ 38
2.8.6 Biotinylation................................................................................................... 39
2.8.6.1 Complete cell biotinylation..............................................................
2.8.6.2 Basolateral or apical cell membrane biotinylation........................... 40


3. Results............................................................................................................................. 45