Modulation of Ca_1hn2_1hn+ dependent inactivation of Ca_1hn2_1hn+ channels by intracellular signalling [Elektronische Ressource] / von Vladan Rankovic

Rankovicv

Sujets

• Biologie

Informations

• Publié par: otto-von-guericke-universitat_magdeburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 19 Mo

Extrait

2+ 2+“Modulation of Ca dependent inactivation of Ca
channels by intracellular signalling”


Dissertation
zur Erlangung des akademischen Grades

doctor rerum naturalium
(Dr. rer. nat.)


genehmigt durch die Fakultät für Naturwissenschaften
der Otto-von-Guericke-Universität Magdeburg


von
Dipl. Biol. Vladan Rankovic

geb.am
05.07.1975 in Smederevska Palanka, Serbia



Gutachter: Prof. Dr. Oliver Stork

Prof. Dr. Peter Lipp



eingericht am: 22.06.2009

vorteidigt am: 15.12.2009














































































To my parents and my family!


Vladan Rankovic


















































































Acknowledgment




I would like to express all my gratitude to Prof. Dr. Oliver Stork, Prof. Dr. Thomas Budde and Dr.
Thomas Munsch for supervising my work. They helped me to develop my scientific attitude through
fruitful discussions and inspiring suggestions. Under their guidance I gained the knowledge and the
confidence that enabled me to compile my PhD thesis successfully. During my PhD work they were
my bosses but in the same time they were my very good friends. I am very thankful to them for being
available whenever I needed help.

I am deeply grateful to Dr. Peter Landgraf for being a very good friend and for his excellent teaching
in the field of molecular biology and protein biochemistry. His assistance and advices helped me in
carrying out successfully many of the experiments presented in this study, and his support and
friendliness always made our discussions both educational and enjoyable.

Moreover, I got valuable experience in protein biochemistry as well as help and support from Dr.
Michael R. Kreutz and Prof. Dr. Eckart D. Gundelfinger which was also my supervisor in graduate
school (GRK1167). To Dr. Michael R. Kreutz and Marina Mikhaylova I am also very thankful for
kindly providing me with antibody and construct for caldendrin. Many thanks also to Xenia Gorny for
giving me construct for AKAP 79.

During my PhD work I got help and support in molecular biology techniques, namely RT-PCR and
quantitative Real-Time PCR, from Dr. Tatyana Kanyshkova. Moreover, I am very thankful to Petra
Ehling for helping me with immunohistochemistry.

I am thankful to Prof. Dr. Volkmar Lessmann for supporting my work in Institute of Physiology as
well as for inspiring discussions and helpful suggestions. My thanks also goes to Dr. Tanja Brigadski
for helping me with German-English translations, and being available whenever I needed help.

I would also like to acknowledge the excellent technical assistance and constant support of Regina
Ziegler, Angela Jahn, Monika Marunde and Corinna Borutzki. Their kind help allowed me to cross lot
of difficulties.

The time I spent at the Institute of Physiology was a pleasant, eventful and exciting experience due to
all my colleagues and co-workers.

Above all, this work was achieved thanks to the support, love and encouragement of my wife Marija,
my family and all my friends, who were always present to help me to overcome difficult moments.
Their support gave me the strength to look ahead.


2+ 2+Modulation of Ca dependent inactivation of Ca channels by intracellular signalling
- Table of contents and figure index -
__________________________________________________________________________________________________________________________________________

Table of Contents:

English summary ..................................................................................................................... 1
Zusammenfassung (German summary) ................................................................................ 2

1. Introduction ........... 3
1.1 Introduction of the thalamocortical system ................................................................................... 3
2+1.2 Voltage-gated Ca channels and their classification ................................................................. 4
2+1.3 Ca signalling network in thalamus ............................................................................................ 6
1.4 Calcium dependent inactivation (CDI) of VGCCs ........................................................................ 7
1.4.1 Identification of CDI .............................................................................................................. 7
1.4.2 Mechanisms of CDI ............................................................................................................... 8
2+ 2+1.4.2.1 Ca - induced Ca release (CICR) .................................................................................... 8
1.4.2.2 β-adrenergic signalling cascade ......................................................................................... 9
1.4.2.2.1 cAMP-dependent PKA and A kinase anchoring protein member family (AKAPs) ........ 11
1.4.2.3 Cytoskeleton, calcium binding proteins and dephosphorylation processes in CDI
modulation .................................................................................................................................... 13
1.5 Aim of the project ........................................................................................................

2. Materials ............................................................................................................................. 16
2.1 Lab instruments and equipment .................................................................................................. 16
2.2 Kits, enzymes and molecular biology reagents ........................................................................... 16
2.2.1 Molecular weight markers ................................................................................................... 17
2.2.2 Plasmids ............................................................................................................................... 17
2.2.3 Bacteria ................ 17
2.2.4 Mammalian cells .................................................................................................................. 17
2.2.5 Cell culture media and reagents for mammalian cells ........................................................ 18
2.2.6 Culture medium and additives for bacteria ......................................................................... 18
2.3 Drugs ........................................................................................................................................... 18
2.4 Antibodies ................... 20
2.4.1 Primary antibodies for Western blot and immunostaining .................................................. 20
2.4.2 Secondary antibodies for Western blot and immunostaining .............................................. 21
2.5 Buffers and solvents used in biochemical or molecular biology work ........................................ 21
2.6 Animals ....................................................................................................................................... 22

3. Methods ............................................................................................................................... 23
3.1 Tissue Preparation ...................................................................................................................... 23
3.2 Patch clamp recordings .............................................................................................................. 23
2+ 2+Modulation of Ca dependent inactivation of Ca channels by intracellular signalling
- Table of contents and figure index -
__________________________________________________________________________________________________________________________________________

2+ 2+3.3 Fluorimetric Ca measurement (Ca imaging) ........................................................................ 24
3.4 Reverse transcription-polymerase chain reaction (RT-PCR) assays .......................................... 24
3.5 Cell Type Specific RT - PCR ....................................................................................................... 25