Klonieren und Charakterisieren von P/Q-Typ-Calciumkanälen für Mikroskopie an lebenden Zellen

Cloning and characterization of P/Q-type calcium channels for live cell imaging

by Martin Juha
Doctoral thesis
Date of Examination:2013-09-03
Date of issue:2013-09-03
Advisor:Prof. Dr. Erwin Neher
Referee:Prof. Dr. Fred Wouters
Referee:Prof. Dr. Martin Oppermann
Persistent Address: http://dx.doi.org/10.53846/goediss-4030

 

 

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Abstract

English

In all excitable cells voltage-dependent calcium channels can be found. They mediate the entry of calcium into cells following depolarization. Calcium contributes to a wide variety of cellular response like muscle contraction, neurotransmitter release, gene expression and also synaptogenesis. For the neurotransmitter release vesicles are needed and the release is supported from a variety of different proteins. Research in the field of neurotransmitter release revealed the probable existence of two different vesicle populations within the RRP which differ in their release probability, the velocity of recruitment and their fusion competence. These different characteristics might be caused by a significant heterogeneity concerning the distance between the vesicle and the controlling calcium channels. To approximate these presumptions and to specify the exact location of P/Q-type calcium channels a specific labeling of the α1-subunit of these channels was performed. The labeling technique included the covalent labeling of the α1-subunit using a short 11- residue -long ACP-ybbR-Tag and an adenoviral expression system under the requirements of live cell imaging. Besides this, experiments using the STED-microscope were carried out. Although the specific labeling of P/Q-type calcium channels could neither be successfully performed in HEK293 cells, nor in hippocampal neurons nor by using the STED-microscope, it could be demonstrated that the ACP labeling procedure works in general using the chosen methods. A specific membrane labeling was performed. It could also be proven that the inserted tag did not disturb the function of the calcium channel.
Keywords: calcium channels; vesicle pool; ACP-labeling; ybbR; transmitter release; SNARE; calcium channelopathies; labeling methods; slow-releasing vesicles; fast-releasing vesicles; calcium cluster; live cell imaging
Schlagwörter: Calciumkanäle; Vesikelpool; Transmitterfreisetzung; Calciumkanal-bedingte Erkrankungen; Färbemethoden; schnell-freisetzende Vesikel; langsam-freisetzende Vesikel
 
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