Structural and functional analysis of the Ca_1tnv1.4 L-type calcium channel from mouse retina [Elektronische Ressource] / Ludwig Baumann

Ludwig Baumann

Description

Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians Universität München Structural and functional analysis of the Ca 1.4 L-type calcium channel vfrom mouse retina Ludwig Baumann aus Eichendorf 2006 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Prof. Dr. Martin Biel betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, den 25.04.2006 _________________________ Ludwig Baumann Dissertation eingereicht am 27.04.2006 1. Gutachter: Prof. Dr. M. Biel 2. Gutachter: Prof. Dr. A. Pfeifer Mündliche Prüfung am 18.05.2006 CONTENTS 3CONTENTS 1 INTRODUCTION.............................................................................................................. 8 1.1 Nomenclature and structure of voltage-gated calcium channels................................. 8 1.2 L-type calcium channels ................................................................................................ 10 1.3 Structure and regulation of the α1 subunit ................................................................. 11 1.3.1 Pharmacological regulation of L-type calcium channels.......................................... 12 2+1.3.2 Regulation by voltage and Ca ions ..................

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

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 Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians Universität München  

     Structural and functional analysis of the Ca v 1.4 L-type calcium channel from mouse retina     Ludwig Baumann aus Eichendorf  2006

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Erklärung  Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Prof. Dr. Martin Biel betreut.     Ehrenwörtliche Versicherung  Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.  München, den 25.04.2006 München, den 25.04.2006              Dissertation eingereicht am 1. Gutachter: 2. Gutachter: Mündliche Prüfung am 

  _________________________  Ludwig Baumann

27.04.2006  Prof. Dr. M. Biel  Prof. Dr. A. Pfeifer  18.05.2006 

CONTENTS

CONTENTS

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1INTRODUCTION .............................................................................................................. 8

1.1 ................................. 8Nomenclature and structure of voltage-gated calcium channels

1.2L-type calcium channels ................................................................................................ 10

1.3Structure and regulation of theα1 subunit ................................................................. 11 1.3.1  Pharmacological regulation of L-type calcium channels.......................................... 12  1.3.2  Regulation by voltage and Ca 2+ ions .......................................................................... 12 

1.4Physiological impact of Ca v 1.4α1 ................................................................................. 14

1.5Purpose of the study....................................................................................................... 16

2MATERIALS AND METHODS..................................................................................... 17

2.1Calcium channel constructs .......................................................................................... 17 2.1.1   17Constructs for electrophysiology.................................................................................  2.1.1.1  Construction of Ca v 1.4α1 mutants ............................................................................ 17  2.1.1.2  Construction of Ca v 1.2bα1 / Ca v 1.4α1 chimeric channels....................................... 18  2.1.2   .................................................................................... 18Constructs for GST pull-down  2.1.3   19Constructs for coimmunoprecipitation....................................................................... 

2.2Amplification and purification of DNA........................................................................ 20 2.2.1   20Transformation of competent E. coli..........................................................................  2.2.2   21Mini-Prep DNA isolation from E. coli .......................................................................  2.2.3   21Maxi-Prep DNA isolation from E. coli....................................................................... 

2.3 21Cell culture......................................................................................................................

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2.3.1 ............................................................................................ 21Culture of HEK 293 cells

2.3.2 22Transient transfection of HEK 293 cells for coimmunoprecipitation.......................

2.3.3Transient transfection of HEK 293 cells for electrophysiology ................................ 22

2.4 ........................................................................................................ 23Analysis of proteins

2.4.1 23GST pull-down assay................................................................................................... 2.4.1.1  Purification of GST fusion proteins expressed in E. coli .......................................... 23  2.4.1.2  Expression of  236xHis/Flag tagged ICDI peptide in E. coli.........................................  2.4.1.3   24Measurement of GST fusion protein concentration ..................................................  2.4.1.4   ......................................................... 24Measurement of overall protein concentration  2.4.1.5  Interaction with calmodulin ...................................................................................... 24  2.4.1.6  Interaction with ICDI ................................................................................................ 24 

2.4.2Coimmunoprecipitation............................................................................................... 25 2.4.2.1   ................................................ 25Purification of proteins expressed in HEK 293 cells  2.4.2.2  Quantification of proteins ......................................................................................... 25  2.4.2.3  Coimmunoprecipitation of proteins .......................................................................... 25 

2.4.3Western blot analysis................................................................................................... 26 2.4.3.1  SDS-polyacrylamid gel electrophoresis (SDS-PAGE) .............................................. 26  2.4.3.2  Immunological detection of proteins......................................................................... 27 

2.5Electrophysiology ........................................................................................................... 29

2.5.1Performance ................................................................................................................ 29

2.5.2Protocols ...................................................................................................................... 30

2.5.3 ............................................................................................................... 30Data Analysis

3RESULTS.......................................................................................................................... 33

3.1Functional characterization of Ca v 1.4α1 ..................................................................... 33

3.1.1Electrophysiological properties of wild type Cav1.4α1.............................................. 33

3.1.2Pharmacological properties of wild type Ca v 1.4α1 .................................................... 39

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3.2The lack of calcium dependent inactivation (CDI) ..................................................... 40 3.2.1  Calmodulin binding of Ca v 1.4α1 ................................................................................ 40  3.2.2  Identification of an inhibitory channel domain......................................................... 42  3.2.3  Interaction of ICDI and Ca v 1.4α1.............................................................................. 43  3.2.4  Abolishing CDI in Ca v 1.2 channels............................................................................ 45 

4DISCUSSION ................................................................................................................... 48

4.1Functional characterization of Ca v 1.4α1 ..................................................................... 48 4.1.1  Electrophysiological properties of Ca v 1.4α1 .............................................................. 48  4.1.2  Pharmacological profile of Ca v 1.4α1 ......................................................................... 49 

4.2Proposed mechanism for block of CDI in Ca v 1.4 channels........................................ 50

4.3Physiological function of Ca v 1.4 calcium channels ..................................................... 53

5SUMMARY....................................................................................................................... 55

6APPENDIX ....................................................................................................................... 57

6.1Sequence of Ca v 1.4α1 cloned from mouse retinal cDNA ........................................... 57

6.2Alignment........................................................................................................................ 64

6.3Primers ............................................................................................................................ 65

7REFERENCES ................................................................................................................. 66

8PUBLICATIONS ............................................................................................................. 72

9

10

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ACKNOWLEDGEMENTS............................................................................................. 73

CURRICULUM VITAE .................................................................................................. 74

ABBREVIATIONS

ABBREVIATIONS ANOVA: Analysis of variance CDI: Calcium dependent inactivation CNG: Cyclic-nucleotide-gated cDNA: Cyclic desoxyribonucleic acid CSNB: Congenital stationary nightblindness DMEM: Dulbecco´s modified eagle medium DHP: Dihydropyridine DNA: Desoxyribonucleic acid DTT: 1,4-Dithiothreitol  E. coli: Escherichia coli EDTA: Ethylenediaminetetraacetic acid EGFP Enhanced green fluorescent protein EGTA Ethylene glycol bis(β-aminoethylether) tetraacetic acid FBS: Fetal bovine serum GST: Glutathione-S-transferase HEK: Human embryonal kidney HEPES: 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid HVA: High voltage-activated ICDI: Inhibitor of calcium dependent inactivation LB: Luria-Bertani LTCC: L-type calcium channel LVA: Low voltage-activated PAGE: Polyacrylamide gel electrophoresis PBS: Phosphate buffered saline PO: Pore occluder PVDF: Polyvinylidene difluoride SDS: Sodium dodecyl sulfate SEM: Standard error of the mean TEA: Tetraethylammonium chloride TEMED: N,N,N',N -Tetramethylethylenediamine ' TBS: Tris bufferd saline Tris: Tris(hydroxymethyl)aminomethane VDI: Voltage dependent inactivation

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