Heidelberg, Univ., Diss., 2008.

Techn. University, Diss., 2004--Darmstadt.

Das Membranstrommuster von Oligodendrozyten verändert sich während der Entwicklung dieser zellen sehr stark. Während die Membranleitfähigkeit von Oligodendrozyten-Vorläuferzellen von auswärts rektifizierenden Kaliumkanälen geprägt ist, exprimieren reife Oligodendrozyten passive, nicht spannungsabhängige Kaliumkanäle. Die Aktivität dieser Kanäle beeinflußt die Proliferation und Differenzierung dieser Zellen. In der vorliegenden Arbeit wurde die Expression von spannungsaktivierbaren Kaliumkanälen des Kv1-Typs (Shaker-Typ) in kultivierten Oligodendrozyten-Vorläuferzellen anhand der Transkriptexpression, der Expression von Kv1-Proteinen und der elektrophysiologischen und pharmakologischen Analyse der Membranströme untersucht. Auf mRNA Ebene wurden unterschiediche Kombinationen von Kv1.1, Kv1.4; Kv1.5 und Kv1.6 Transkripten gefunden. Ebenfalls wurde in einigen Zellen eine signifikante Menge an Kv1.2 und Kv1.3 Transkripten gefunden. Die Heterogenität der Transkriptexpression konnte nicht mit Unterschieden in den elektrophysiologischen Eigenschaften korrelliert werden. Die Expression der Kv1 Proteine wurde mit Hilfe von immunozytochemischen Färbungen mit spezifischen polyklonalen Antikörpern gegen die Kanäle Kv1.1 bis Kv1.6 untersucht. Alle Oligodendrozyten-Vorläuferzellen exprimierten die Kanäle Kv1.4 (85% der Zellen), Kv1.5 (99 %) und Kv1.6 (99 %), Kv1.1 Proteine wurden von 10 % der Zellen gebildet. Um den funktionellen Beitrag der Kv1 Kanäle zum Gesamtzellstrom zu bestimmen, wurde die Stromaktivierung und -inaktivierung sowie die Sensitivität der Ströme gegen die spezifischen Kaliumkanalblocker getestet. Dabei wurden durch TEA (1-100 mM), 4-AP (0,125-1 mM) und Chinidin (5-100 mM) jeweils ein großer Teil der Ströme gehemmt, durch CTX, DTX und MCDP wurde die Kanalaktivität nicht beeinflußt. Um den Beitrag der Kanalproteine Kv1.4 bzw. Kv1.1 zu den elektrophysiologischen Eigenschaften des Gesamtzellstromes zu testen, wurden an einzelnen Oligodendrozyten-Vorläuferzellen kombinierte elektrophysiologische Untersuchungen und immunozytochemische Färbungen durchgeführt. Dabei konnten keine signifikanten Unterschiede zwischen Kv1./Kv1.4 positiven und Kv1.1/Kv1.4 negativen Zellen festgestellt werden. Aus den Untersuchungen ergeben sich folgende Schlußfolgerungen: Oligodendrozyten exprimieren eine Vielzahl unterschiedlicher Kv1 Transkripte.Die überwiegende Mehrzahl der Oligodendrozyten-Vorläuferzellen exprimieren die Kv1 Proteine Kv1.4, Kv1.5 und Kv1.6.Der Gesamtzellstrom kann vorwiegend durch Kv1.5 Kanäle oder durch eine Kombination von Kv1.4/Kv1.6 Kanälen sowie durch Mitglieder anderer Familien spannungsabhängiger Kaliumkanäle getragen werden. Um zu untersuchen, ob spannungsabhängige Kaliumkanäle durch die Aktivierung von inhibitorischen Neurotransmitterrezeptoren beeinflußt werden, wurden kultivierte Körnerzellen als Modellsystem verwendet, da diese eine hohe Dichte an Kv Kanälen sowie an GABA Rezeptoren exprimieren. Im "cell-attached" Modus der Patch-Clamp-Technik wurde die Reaktion von einzelnen auswärts rektifizierenden Kaliumkanälen während der GABA-Antwort untersucht. Mit diesem Ansatz konnte gezeigt werden, daß die Öffnungswahrscheinlichkeit dieser Kanäle während der Reaktion der Zelle auf GABA stark zurückgeht. Da Oligodendrozyten-Vorläuferzellen ebenfalls GABAA-Rezeptoren exprimieren, ist anzunehmen, daß deren Aktivierung über einen analogen Mechanismus zur Blockierung von Kaliumkanälen führt. / The membrane current pattern of oligodendrocytes changes dramatically during cell development. In oligodendrocyte precursor cells the membrane conductance is dominated by outwardly rectifying potassium channels, mature oligodendrocytes on the other hand express passive, not voltage-gated potassium channels. The activity of these channels influences the proliferation and differentiation of the cells. In the present work the expression of outwardly-rectifying potassium channels of the Kv1-type (Shaker-type) was analysed in oligodendrocyte precursor cells in culture. Expression of Kv1 transcripts, Kv1 proteins as well as electrophysiological and pharmacological properties of these channels were tested. Different combinations of Kv1.1, Kv1.4, Kv1.5 and Kv1.6 transcripts were detected at mRNA level. In some cells also a significant amount of Kv1.2 and Kv1.3 transcripts was found. The heterogeneity of transcript expression could not be correlated with differences in electrophysiological properties. The expression of Kv1 channel proteins was analysed using immunocytochemical stainings with specific monoclonal antibodies against the channel molecules Kv1.1 to Kv1.6. All oligodendrocyte precursor cells expressed the channel molecules Kv1.4 (85 % of the cells), Kv1.5 (99 %) and Kv1.6 (99 %), Kv1.1 proteins were detected in 10 % of the cells. To find out the functional contribution of Kv1 channels to the whole-cell current of the cells the activation and inactivation characteristics as well as the sensitivity of the potassium current to different potassium channel specific antagonists was tested. Parts of the current were inhibited by TEA (1-100 mM), 4-AP (0,125-1 mM) and Chinidin (5-100 mM), CTX, DTX and MCDP had no effect on the channel activity. To isolate the contribution of the channel molecules Kv1.1 and Kv1.4 the electrophysiological properties of the whole cell current electrophysiological analysis of single cells using whole-cell patch-clamp technique and immunocytochemical stainings were combined. With this method no significant differences between Kv1.1/Kv1.4-positive and Kv1.1/Kv1.4 negative cells could be detected. From these findings the following conclusions could be drawn: Oligodendrocyte precursors express various different Kv1 transcripts.The majority of oligodendrocyte precursor cells expresses the Kv1 proteins Kv1.4, Kv1.5 and Kv1.6.The total current (whole-cell current) most likely is carried through Kv1.5 channels or a combination of Kv1.4/Kv1.6 channels and probably another type of voltage-gated potassium channels. To find out if voltage-gated potassium channels are related to the activation of inhibitory neurotransmitter receptors a model system of cultured granule cells was used. This cell type was selected because they are known to express a high density of Kv channels as well as GABAA receptors as well. The activity of single outwardly rectifying potassium channels was detected using the cell-attached mode of patch-clamp technique. With this method it could be demonstrated that the open probability of voltage-gated potassium channels is markedly decreased during GABAA response. It could be concluded that the activation of GABAA receptors on oligodendrocyte precursor cells leads to the inhibition of potassium channels in the same way as in cultured granule cells.

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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.

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