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

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

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Published 01 January 2006
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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 Cav1.4 L-type calcium channel from 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 München, den 25.04.2006 Dissertation eingereicht am 1. Gutachter: 2. Gutachter: Mündliche Prüfung am
_________________________  Ludwig Baumann
27.04.2006Prof. Dr. M. BielProf. Dr. A. Pfeifer18.05.2006
CONTENTS
CONTENTS
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1INTRODUCTION .............................................................................................................. 8
1.1 ................................. 8Nomenclature and structure of voltage-gated calcium channels
1.2L-type calcium channels ................................................................................................ 10
1.3Structure and regulation of theα1 subunit ................................................................. 111.3.1Pharmacological regulation of L-type calcium channels.......................................... 121.3.2Regulation by voltage and Ca2+ions .......................................................................... 12
1.4Physiological impact of Cav1.4α1 ................................................................................. 14
1.5Purpose of the study....................................................................................................... 16
2MATERIALS AND METHODS..................................................................................... 17
2.1Calcium channel constructs .......................................................................................... 172.1.1 17Constructs for electrophysiology.................................................................................2.1.1.1Construction of Cav1.4α1 mutants ............................................................................ 172.1.1.2Construction of Cav1.2bα1 / Cav1.4α1 chimeric channels....................................... 182.1.2 .................................................................................... 18Constructs for GST pull-down2.1.3 19Constructs for coimmunoprecipitation.......................................................................
2.2Amplification and purification of DNA........................................................................ 202.2.1 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.......................
2.3.3Transient transfection of HEK 293 cells for electrophysiology ................................ 22
2.4 ........................................................................................................ 23Analysis of proteins
2.4.1 23GST pull-down assay...................................................................................................2.4.1.1Purification of GST fusion proteins expressed in E. coli .......................................... 232.4.1.2Expression 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 concentration2.4.1.5Interaction with calmodulin ...................................................................................... 242.4.1.6Interaction with ICDI ................................................................................................ 24
2.4.2Coimmunoprecipitation............................................................................................... 252.4.2.1 ................................................ 25Purification of proteins expressed in HEK 293 cells2.4.2.2Quantification of proteins ......................................................................................... 252.4.2.3Coimmunoprecipitation of proteins .......................................................................... 25
2.4.3Western blot analysis................................................................................................... 262.4.3.1SDS-polyacrylamid gel electrophoresis (SDS-PAGE) .............................................. 262.4.3.2Immunological detection of proteins......................................................................... 27
2.5Electrophysiology ........................................................................................................... 29
2.5.1Performance ................................................................................................................ 29
2.5.2Protocols ...................................................................................................................... 30
2.5.3 ............................................................................................................... 30Data Analysis
3RESULTS.......................................................................................................................... 33
3.1Functional characterization of Cav1.4α1 ..................................................................... 33
3.1.1Electrophysiological properties of wild type Cav1.4α1.............................................. 33
3.1.2Pharmacological properties of wild type Cav1.4α1 .................................................... 39
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3.2The lack of calcium dependent inactivation (CDI) ..................................................... 403.2.1Calmodulin binding of Cav1.4α1 ................................................................................ 403.2.2Identification of an inhibitory channel domain......................................................... 423.2.3Interaction of ICDI and Cav1.4α1.............................................................................. 433.2.4Abolishing CDI in Cav1.2 channels............................................................................ 45
4DISCUSSION ................................................................................................................... 48
4.1Functional characterization of Cav1.4α1 ..................................................................... 484.1.1Electrophysiological properties of Cav1.4α1 .............................................................. 484.1.2Pharmacological profile of Cav1.4α1 ......................................................................... 49
4.2Proposed mechanism for block of CDI in Cav1.4 channels........................................ 50
4.3Physiological function of Cav1.4 calcium channels ..................................................... 53
5SUMMARY....................................................................................................................... 55
6APPENDIX ....................................................................................................................... 57
6.1Sequence of Cav1.4α1 cloned from mouse retinal cDNA ........................................... 57
6.2Alignment........................................................................................................................ 64
6.3Primers ............................................................................................................................ 65
7REFERENCES ................................................................................................................. 66
8PUBLICATIONS ............................................................................................................. 72
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ACKNOWLEDGEMENTS............................................................................................. 73
CURRICULUM VITAE .................................................................................................. 74
ABBREVIATIONS
ABBREVIATIONSANOVA: 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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