Voltage gated potassium channels (Kv) play a key role in the nervous system- not only due to their involvement in the action potential. Vertebrates express four subtypes, which are termed Kv1, Kv2, Kv3 and Kv4, respectively. Tsha3 is a Kv1 channel which was originally isolated from brain tissue of rainbow trout (Oncorhynchus mykiss). This channel possesses an unique amino terminus and a characteristic amino acid sequence in the T1 domain, which is engaged in the oligomerization of Kv α-subunits and is thus involved into the segregation of subfamilies. The two major goals of this thesis were the structural and functional characterization of the N-terminal cytosolic domain of Tsha3 as well as the invention of a system to gain data about the functional dynamics of full length Kv channels. Molecular biological techniques were used to isolate mRNA from trout brains, to transcribe it into cDNA and clone it into vectors. DNA from such plasmids was ligated into expression vectors for heterologous expression in E. coli, P. pastoris and Sf21 cells, with concomitant fusion of marker proteins (GFP or DsRed) or tags (6 x HisTag or StrepTagII) due to the individual experiment. Protein was overexpressed in E. coli and affinity purified to analyze separated domains with biochemical (SDS-PAGE and Western Blot, Pull-Down-Assay or Dot-Blot-Assay) or biophysical (CD-spectroscopy, EPR spectroscopy) efforts. The P. pastoris system to express Tsha1 was established, to generate a system for future EPR-measurements of whole Kv channels. Heterologous expression of Kv1α (Tsha3 and Tsha1) and the core domain of Kvβ in Sf21 cells was performed to analyze the subcellular distribution of the respective subunits via fluorescence microscope and via subcellular fractionation of cell lysates with downstream biochemical analyses (SDS-PAGE and Western Blot). Furthermore the gating of diverse fusion constructs of Tsha3 in co-expressions and the gating of diverse cystein substitution mutants of Tsha1 were measured via path-clamp recordings in whole cell modus. The structural analyses of the N-terminal cytosolic domain (NCD) of Tsha3 revealed that the 128 amino acid containing part before the T1-domain (Tsha3-NT) can be structurally divided into three parts of different structure and mobility. The most outward part possesses a very high mobility and is putatively unfolded as random coil. This section is expected to express no tertiary contacts. The middle part of Tsha3-NT is structured in α-helices and β-sheets and thus slightly immobile. This folded part is also assumed to build no tertiary structure and to be exposed into the cytosol. The third, which is directly neighboring the T1 domain, has the most restricted mobility of Tsha3-NT. It consists predominantly of α-helices and exhibits a tertiary structure, putatively with the T1 domain. Tsha3-NCD self-tetramerizes and oligomerizes with Tsha1, although mutations exist in Tsha3 in conserved amino acids, which were reported to function in subfamily specific hetero-tetramerization. Thus it is proven, that Tsha3 takes part in the segregation into the Kv1 subfamily. Furthermore, Tsha3 interacts with the core domain of Kvβ2 although there are also mutations in the reported consensus sequence for interaction. Association of Kvβ2 in co-expression studies directs Tsha3-DsRed fusion constructs from internal vesicular structures into the cell membrane. But the fusion with DsRed is leading to a loss of function of Tsha3 which cannot be rescued by co-expression of the chaperone Kvβ2. But- without fusion of marker proteins- Tsha3 was identified as an outward rectifier in a cooperative Bachelor Thesis. These structural data lead to the assumption, that Tsha3-NT exhibits lateral interactions and especially the helical but mobile middle part of the N-terminus can play such a role. Due to the localization next to the membrane, interactions with membrane proteins- putatively with protein cascades are possible. Although Tsha3-NT contains no reported interaction domains for protein-protein interactions, follow-up experiments should be performed to shed light on this interesting question. Tsha1 C30S C31S C180S C224A C239S C389S C424S C476S is a complete cysteine free mutant, which was identified as a functional voltage-gated potassium channel. It was expressed in and purified from eukaryotic cells (P. pastoris) and therefore it can be assumed to be properly folded and modified. After a slight optimization of the features of expression, this system can be used to reconstitute Tsha1 channels into liposomes and use them for Freeze Quench EPR to gain structural information about a Kv1 channel in the open as well as in the closed state. This is the first report of the establishment of a full length Kv for studies of structure and functional dynamics experiments.
| Identifer | oai:union.ndltd.org:uni-osnabrueck.de/oai:repositorium.ub.uni-osnabrueck.de:urn:nbn:de:gbv:700-2014062012546 |
| Date | 20 June 2014 |
| Creators | Herrling, Regina |
| Contributors | Apl. Prof. Dr. Gunnar Jeserich, Prof. Dr. Hildgund Schrempf |
| Source Sets | Universität Osnabrück |
| Language | German |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/zip, application/pdf |
| Rights | Namensnennung - Nicht-kommerziell - Weitergabe unter gleichen Bedingungen 4.0 International, http://creativecommons.org/licenses/by-nc-sa/4.0/ |
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