Funktionelle Rekonstitution von Connexonen in artifizielle Membranen: Expression, Reinigung und Charakterisierung von Connexin 43

Functional reconstitution of connexons in artificial membranes: expression, purification and characterization of connexin 43

by Christian Carnarius
Doctoral thesis
Date of Examination:2012-06-11
Date of issue:2012-11-19
Advisor:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Christian Griesinger
Persistent Address: http://dx.doi.org/10.53846/goediss-2189

 

 

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Abstract

English

Connexin 43 (Cx43) is the most prominent member of the connexin family in mammalian tissues. In biological lipid membranes, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, forming gap junction channels. Membrane channel proteins are pivotal for communication between cells. Dysfunctional ion channels are associated to diseases like osteoporosis, epilepsy, ataxia, myotonia and cardiac arrhythmia. Therefore, there is an interest in studying these channel proteins to elucidate their functional properties and determine how to manipulate their functionality. This is commonly carried out by ion channel recordings, which require invasive contacting by voltage clamp methods in cells. The alternative approach taken here was to utilize connexon-doped artificial membranes for electrical coupling of connexon-expressing cells via gap junction channels. In this work, an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (Cx43
Keywords: connexin 43; Cx43; green fluorescent protein; GFP; pore spanning membranes; porous alumina; artificial membranes; planar patch clamp; melittin; Pichia pastoris; Dictyostelium discoideum
Schlagwörter: Connexin 43; Cx43; Grün fluoreszierendes Protein; GFP; Porenüberspannende Membranen; Poröses Aluminiumoxid; Artifizielle Membranen; Planar Patch Clamp; Melittin; Pichia pastoris; D. discoideum
 
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