Charakterisierung von Modulationsmechanismen der Kv1-Ionenkanalfamilie: Lipidsensitivität, RNA Editierung und Inaktivierungspartikel

Spannungsgesteuerte Kaliumkanäle (Kv-Kanäle) sind an der Regulation neuronaler und kardialer Aktivität beteiligt. Sie öffnen bei Depolarisation der Zelle und leiten unter physiologischen Bedingungen Kaliumauswärtsströme, wodurch sie zur Repolarisation beitragen. Kv-Kanäle modulieren die Dauer von Ak...

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Bibliographic Details
Main Author: Streit, Anne-Kathrin
Contributors: Decher, Niels (Prof. Dr. ) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2012
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Voltage-gated potassium channels (Kv-channels) participate in the regulation of neuronal and cardiac activity. They open upon depolarization of the cell and conduct potassium outward currents under physiological conditions, thus contributing to repolarization. Kv-channels modulate action potential duration and cell excitability. Different regulatory mechanisms can influence channel function. Amongst others, the physiological regulatory mechanisms include inactivation of Kv-channels induced by endogenous lipids, the RNA editing of the Kv1.1-channel, which leads to an exchange of isoleucine for valine within the central pore, as well as inactivation induced by accessory subunits. The underlying mechanism for lipid-induced inactivation and the consequences of Kv1.1 RNA editing are only partly understood and were examined in more detail in the present study. Furthermore, the structure of the inactivation particle of the accessory Kvβ1.3-subunit and the regulation of inactivation by this subunit were analyzed. First, the mechanism of channel inhibition by endogenous lipids like arachidonic acid, docosahexaenoic acid and anandamide was analyzed with the help of different methods. The characteristics of the inactivation such as kinetics, competition with the pore blocker TEA and influence by mutations in the central pore as well as the in silico models suggest that a physical occlusion of the open channel pore is the most likely mechanism for channel inhibition by lipids. Examination of the functional consequences of Kv1.1 RNA editing showed that edited channels exhibit a reduced sensitivity to pore blockers. This applies to the substances 4-Aminopyridine (4-AP) and Psoralen-4, which are used for experimental and clinical purposes, as well as to the lipids and related substances that have been newly identified as pore blockers in this study. Furthermore, it could be demonstrated that edited Kv1.1 subunits can modulate the pharmacology and lipid sensitivity of all Kv1-channels through heteromerization with other Kv1 channel subunits. In the course of the functional characterization of edited Kv1.1-channels a reduced current amplitude due to the editing was observed in different expression systems. Thereupon, the molecular correlates of this current reduction were investigated. Recordings on single-channel and whole-cell level and quantification of protein amount revealed that the editing does not cause changes neither in gating nor in the total amount of channel protein. However, a significantly diminished surface expression of edited Kv1.1-channel subunits could be detected, which is likely to be responsible for the reduced total current. These results describe for the first time a modulation of intracellular transport of Kv1.1-channel subunits by RNA editing. A pathophysiological relevance of Kv1.1 RNA editing is assumed in the context of epilepsy. A quantification of the extent of Kv1.1 editing in the entorhinal cortex of chronic epileptic rats from the kainic acid induced animal model showed an increased editing ratio in comparison to healthy control animals. Characteristic of epileptic animals is a resistance to 100 µM of the ictogenic Kv-channel blocker 4-AP, a concentration that induces epileptiform potentials in healthy control animals. In the present work, the effects of the increased editing ratio on this 4-AP concentration were examined in electrophysiological experiments in an expression system. While any influence of the observed increase in editing ratio on the time course or voltage-dependence of 4-AP block could be ruled out, a significant reduction of 4-AP sensitivity was detected. This reduced 4-AP sensitivity of Kv1-channels which is caused by the elevated editing ratio is presumably the basis of the altered 4-AP sensitivity of epileptic animals. Furthermore, aspects of the inactivation of Kv1-channels were examined in the course of this study. Kvβ1-subunits can induce fast A-type inactivation when attached to Kv1-channels. The conformation (or rather the binding mode) of the inactivation particle of the Kvβ1.3-subunit was analyzed by means of electrophysiological experiments and molecular modelling. The results suggest that the Kvβ1.3 inactivation particle most probably adopts a hairpin conformation when bound within the central pore of the Kv1.5-channel. Moreover, I could show that the Kvβ1.3-induced inactivation can be regulated by phosphoinositides.