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

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En savoir plus
112 pages

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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....................................................................

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Publié le 01 janvier 2008
Nombre de lectures 38
Poids de l'ouvrage 2 Mo

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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

3.1 The amino acid sequence is conserved in the amino terminal part of barttin............... 47

3.2 Barttin mutants enable ClC-Kb exit from the endoplasmic reticulum.......................... 48
3.2.1 WT and mutant barttin enable complex glycosylation of ClC-Kb................. 48
3.2.2 Q32X and G47R barttin decrease ClC-Kb channel expression...................... 50

3.3 Most barttin mutants transport ClC-Kb to the plasma membrane................................. 51

IV Contents
3.3.1 G10S, E88X and WT barttin stimulate ClC-Kb insertion into the plasma
membrane....................................................................................................... 51
3.3.2 Only Q32X barttin is inable to induce ClC-Kb membrane insertion............. 56
3.3.3 E88X barttin lacks the sorting mechanism of WT barttin.............................. 58

3.4 Loss of protopore activation by most barttin mutants................................................... 59
3.4.1 Only E88X induces currents like WT barttin in human ClC-K isoforms....... 59
3.4.2 All barttin mutants but Q32X invert the voltage dependence of activation
of rat V166E ClC-K1...................................................................................... 61


4. Discussion....................................................................................................................... 67

4.1 Effect on endoplasmatic reticulum exit and channel stability....................................... 69

4.2 Insertion into the surface membrane............................................................................. 71
4.2.1 Intracellular versus Membrane localisation.................................................... 71
4.2.2 Apical versus basolateral membrane insertion............................................... 72

4.3 Channel activation by WT and mutant barttin.............................................................. 73
4.3.1 Human isoforms.............................................................................................. 73
4.3.2 Rat ClC isoforms............................................................................................ 74

4.4 Conclusions................................................................................................................... 75

4.5 Further research............................................................................................................. 75


5. Abstract ......................................................................................................................... 79

5.1 Abstract and keywords, Englisch.................................................................................. 81
5.2 Zusammenfassung und Stichwörter, German................................................................ 82

Contents V
6. References..................................................................................................................... 85

7. Acknowledgements........................................................................................................ 93

8. Curriculum vitae........................................................................................................... 95

9. List of publications........................................................................................................ 97














VI List of Figures and Tables





































List of Figures and Tables VII
List of Figures and Tables

Figures

1.1 Membrane proteins in a cell from the thick ascending limb of Henle.......................... 4
1.2 Dendrogram of human ClC genes................................................................................. 7
1.3 Representation of a ClC protein and the position of its 4 conserved regions................ 12

2.1 Scheme of the Quikchange reaction.............................................................................. 23
2.2 Scheme of apical biotinylation of MDCKII cells grown on filters............................... 41

3.1 Structure and conservation of barttin............................................................................ 47
3.2 ClC-Kb is complex glycosylated only in the presence of barttin.................................. 48
3.3 All barttin mutants induce complex glycosylation of ClC-Kb...................................... 49
3.4 G47R and Q32X lower the amount of ClC-Kb opposed to WT barttin........................ 51
3.5 Confocal images of fixed MDCK cells expressing WT or mutant CFP tagged barttin.51
3.6 Confocal images of fixed MDCK cells expressing YFP tagged ClC-Kb alone or
with WT or mutant barttin CFP..................................................................................... 52
3.7 Confocal images of living MDCK cells........................................................................ 52
3.8 Horizontal and vertical confocal images of living MDCK cells grown on dishes....... 53
3.9 Confocal images and vertical slides of stably WT or E88X barttin CFP transfected
polarized MDCK cells................................................................................................... 55
3.10 Q32X does not colocalise in the membrane with rClC-K1......................................... 55
3.11 Only Q32X barttin is impaired in ClC-Kb membrane insertion................................. 57
3.12 E88X barttin lacks epithelial sorting........................................................................... 58
3.13 Only E88X barttin induces currents in human ClC-K channels as WT barttin........... 59
3.14 Activation of the ClC-Kb protopore by WT, E88X and G47R barttin....................... 60
V3.15 GK / GP motif in ClC-K channels............................................................................. 61 L
3.16 All barttin mutants but Q32X invert rat V166E ClC-K1 voltage dependence of
activation..................................................................................................................... 62
3.17 Barttin mutations, except G10S and E88X, affect the current amplitude of
V166E ClC-K1............................................................................................................ 63
VIII List of Figures and Tables

3.18 Non-stationary noise analysis of V166E rClC-K1 alone or in the presence of WT,
R8L, G10S, G47R or E88X barttin............................................................................. 64


Tables

1.1 Mutations found in the BSND gene resulting in Bartter Syndrome type IV................. 16

2.1 Oligonucleotides used for Quikchange or pcr mutagenesis.......................................... 22
2.2 Consistence of running and stacking gel of denaturing SDS gels................................. 36
2.3 Reaction conditions for a deglycosylation assay........................................................... 38