Bacteriorhodopsin-like cation channelrhodopsin based optogenetic tools for the future optical cochlear implant
by Eva Wüstenhagen
Date of Examination:2025-06-03
Date of issue:2025-05-07
Advisor:Prof. Dr. Tobias Moser
Referee:Prof. Dr. Tobias Moser
Referee:Prof. Dr. Luis A. Pardo
Sponsor:Else-Kröner-Fresenius-Promotionskolleg für Medizinstudierende Göttingen
Persistent Address:
http://dx.doi.org/10.53846/goediss-11252
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Abstract
English
Deafness is a widespread and severe condition. Electrical cochlear implants, which use electrical stimulation of the auditory nerve, enable speech comprehension in those patients suffering from sensorineural hearing loss. However, the frequency resolution of these implants is limited due to the broad spread of electrical current. To address this issue, the use of light, which can be more precisely confined in space, is being developed as the basis for an optical cochlear implant. The introduction of light-gated ion channels, known as channelrhodopsins, into spiral ganglion neurons enables the necessary light-driven excitation. However, channelrhodopsins exhibit significant variation in their electrophysiological properties. None of the published variants meets all the criteria for use in an optical cochlear implant. This doctoral thesis primarily focuses on the criteria redshifted action spectrum, fast channel kinetics, large current densities, and minimal desensitization. This doctoral thesis aims to expand the optogenetic toolbox and contribute to designing a channelrhodopsin variant suitable for optical cochlear implants. This aim was pursued through three different approaches: investigating representatives of the newly described Bacteriorhodopsin-like channelrhodopsin family, improving the characteristics of the published variant ChRmine, and contributing to the establishment of automation for channelrhodopsin characterization. NG cells were used to express channelrhodopsin variants, which were then characterized through whole-cell patch-clamp experiments. Plasma membrane expression was assessed by analyzing line profiles from single-stack confocal images. The effectiveness of the automated patch clamp system SyncroPatch 384 in determining light dependence was investigated by comparing it to the manual patching set-up. The investigation of Bacteriorhodopsin-like channelrhodopsins revealed a significant variability within the subfamily. One representative, DN22769-c0-g2-i1, demonstrated fast channel kinetics and reduced desensitization, making it a valuable candidate for in vivo applications. Attempts to increase the current density of this variant by optimizing plasma membrane expression were unsuccessful. However, a more detailed examination of plasma membrane expression uncovered additional variability within the Bacteriorhodopsin-like channelrhodopsin subfamily. The investigation of various ChRmine variants revealed that mutations within a random coil region between transmembrane helix two and three affect closing kinetics. Further investigation of this region could result in the development of a ChRmine variant with faster closing kinetics. Two variants, ChReef-H33R and ChReef-T119A, demonstrated accelerated closing kinetics while maintaining current densities similar to wild-type values. Combining both mutations in the variant ChReef-H33R/T119A did not potentiate the accelerating effects on closing kinetics. This thesis deepens the understanding of the mechanisms behind ChRmines’ photocycle by characterizing its variants. Furthermore, it demonstrates the reproducibility of a light dependence measurement obtained manually using the automated patch clamp system SyncroPatch 384. Additional work is required to establish this system, but the results suggest the potential for future automation in characterizing channelrhodopsins. This could significantly accelerate the investigation of new variants and the optimization of known ones. Based on these results, it is recommended to utilize automated patch-clamp to examine numerous variants of Bacteriorhodopsin-like channelrhodopsin and ChRmine. These variants can be obtained from mutant libraries based on partially advantageous variants, such as ChReef-H33R, and hot spot regions, such as the random coil region between transmembrane helix two and three.
Keywords: channelrhodopsin; Optogenetics; Optical cochlear implant; Neuroprosthetic; Cochlear implant; ChReef; ChRmine; SyncroPatch 384; patch-clamp; Bacteriorhodopsin-like channelrhodopsin; Plasma membrane expression
