Investigation of voltage- and light-sensitive ion channels

by Ulrich Fromme
Doctoral thesis
Date of Examination:2016-02-29
Date of issue:2016-08-09
Advisor:Prof. Dr. Christoph F. Schmidt
Referee:Dr. Andreas Neef
Referee:Prof. Dr. Tobias Moser
Referee:Prof. Dr. Florentin Wörgötter
Referee:Prof. Dr. Stefan Klumpp
Referee:Prof. Dr. Marc Timme
Persistent Address: http://dx.doi.org/10.53846/goediss-5783

 

 

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Abstract

English

In this work, the properties of ion channels in biological membranes are characterized. Two systems are investigated: The conductance changes of the membrane of the axon initial segment (AIS) due to fast sodium channels, and the recently discovered light sensitive ion channel Chronos. The latter investigation focused purely on the properties of the light gated channels and not, for example, their distribution in the membrane. As vectors holding the Chronos gene can be inserted into simple, small, round cells such as HEK cells, the electrophysiological measurements can be performed with standard patch-clamp equipment and techniques. The evaluation of the data needed to extract single molecule parameters is highly complex however, and was performed by optimizing newly developed Markov models using an elaborate optimization routine. Using this approach, the currents mediated by Chronos can be reproduced with high precision, and several interesting properties of the molecular transitions were discovered. The most notable being the relation of Chronos' fast kinetics at low intensities to the short lifetimes of its conducting states as compared to the widely used Channelrhodopsin-2 (ChR2). This leads to a lower opening probability for Chronos than for ChR2. To achieve similar depolarization, more channels or higher single-channel conductance are thus necessary for Chronos. Another interesting effect is the relatively weak light adaptation of Chronos. This reduces the light intensities needed for stimulation shortly after prior illumination. For the AIS investigation, the electrophysiological measurements are highly complicated, as they have to be performed in living neurons and not only need to discern single molecule properties but also the distribution of these molecules in the membrane. This requires the determination of the geometrical structure of the cell as well as highly sensitive measurements at numerous positions. A setup capable of performing these tasks has been created and a proof of principle has been conducted for every step, including the topographical imaging at acceptable speed, the high-precision positioning of electrodes, the stimulation via Chronos and low-noise potential measurements.
Keywords: Channelrhodopsin; Chronos; axon initial segment; scanning ion conductance microscopy; SICM; ChR2; genetic algorithm
 
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