Direkt zum InhaltDirekt zur SucheDirekt zur Navigation
▼ Zielgruppen ▼
  Zielgruppenmenü

Humboldt-Universität zu Berlin - Experimentelle Biophysik

Dr. Jonas Wietek

 

Email:    jonas.wietek@gmail.com

Weizmann Institute, Israel

Ion Gating in Channelrhodopsins – Investigation of the Selectivity Mechanism

Channelrhodopsins (ChRs) are light-gated ion channels functioning as primary photoreceptors in motile green algae1. The majority of ChRs exhibit a general selectivity for mono- and divalent cations2–7. An exclusive selectivity for one cation specie or even anions has never been reported so far. Since ChRs are extensively used for depolarizing (activate) neuronal cells in vivo and in vitro in the emerging field of optogenetics8–12, it would be of fundamental interest to create a ChR of contrary function to silence specific neurons or neuronal networks. Nowadays only archaeal ion pumps can be used for light controlled neuronal inhibition13–15, sharing the drawback of demanding high light intensities to be activated. Especially for long term experiments this is achieved at the cost of exposure to very high light doses causing substantial damages in the tissue of interest.

To overcome the depicted problems my work focuses on design of light-gated ChRs that are highly selective for potassium (K+) or chloride (Cl-). In both cases, light gated channel opening would inhibit firing of action potentials in neuronal cells due to the respective ion flux across the cell membrane. The novel inhibiting ChRs could be combined with the already known variants exhibiting prolonged photocycles16,17, providing a powerful optogenetic tool for long time inhibition of neurons at low light intensities, which is superior to using ion pumps. Moreover, color shifted inhibitory ChR variants will be designed using them for instance in simultaneous in- and activation experiments of neurons in combination with common ChR actuators.

The methods used for my approach are molecular biology, electrophysiology (on HEK cells & Xenopus oocytes), cell culture, confocal microscopy and time resolved ion imaging.

 

References

1.        Hegemann, P. Planta 203, 265–74 (1997).

2.        Nagel, G. et al. Proc. Natl. Acad. Sci. U. S. A. 100, 13940–5 (2003).

3.        Ernst, O.P. et al. J. Biol. Chem. 283, 1637–43 (2008).

4.        Tsunoda, S.P. & Hegemann, P. Photochem. Photobiol. 85, 564–9 (2009).

5.        Lin, J.Y., Lin, M.Z., Steinbach, P. & Tsien, R.Y. Biophys. J. 96, 1803–14 (2009).

6.        Berndt, A., Prigge, M., Gradmann, D. & Hegemann, P. Biophys. J. 98, 753–61

           (2010).

7.        Schneider, F., Gradmann, D. & Hegemann, P. Biophys. J. 105, 91–100 (2013).

8.        Fiala, A., Suska, A. & Schlüter, O.M. Curr. Biol. 20, R897–903 (2010).

9.        Fenno, L., Yizhar, O. & Deisseroth, K. Annu. Rev. Neurosci. 34, 389–412 (2011).

10.      Bernstein, J.G. & Boyden, E.S. Trends Cogn. Sci. 15, 592–600 (2011).

11.      Rein, M.L. & Deussing, J.M. Mol. Genet. Genomics 287, 95–109 (2012).

12.      Mei, Y. & Zhang, F. Biol. Psychiatry 71, 1033–8 (2012).

13.      Chow, B.Y. et al. Nature 463, 98–102 (2010).

14.      Zhang, F. et al. Nature 446, 633–9 (2007).

15.      Gradinaru, V., Thompson, K.R. & Deisseroth, K. Brain Cell Biol. 36, 129–39

          (2008).

16.      Berndt, A., Yizhar, O., Gunaydin, L. a, Hegemann, P. & Deisseroth, K. Nat.

          Neurosci. 12, 229–34 (2009).

17.      Yizhar, O. et al. Nature 477, 171–8 (2011).

 

 

Journal Publications

Wietek, J., Rodriguez-Rozada, S., Tutas, J., Tenedini, F., Grimm, C., Oertner, T., Soba, P., Hegemann, P., and Wiegert, J. S.# (2017) Artificial anion-conducting channelrhodopsins with tuned spectra, modified kinetics and enhanced light sensitivity. bioRxiv. 10.1101/156422 

 

Grimm, C.*, Vierock, J.*, Hegemann, P., and Wietek, J.# (2017) Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins. J. Vis. Exp. 10.3791/55497, C

 

Wietek J.#, Broser M., Krause B.S., Hegemann P.: Identification of a Natural Green Light Absorbing Chloride Conducting Channelrhodopsin from Proteomonas sulcata. Journal of Biological Chemistry 01/2016; 291(8). DOI:10.1074/jbc.M115.699637

 

Berndt A.*, Lee S.Y.*, Wietek J.*, Ramakrishnan C., Steinberg E.E., Rashid A.J., Kim H., Park S., Santoro A., Frankland P.W., Iyer S.M., Pak S., Ährlund-Richter S., Delp S.L., Malenka R.C., Josselyn S.A., Carlén M., Hegemann P., Deisseroth K.: Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity. Proceedings of the National Academy of Sciences 12/2015; 113(4). DOI:10.1073/pnas.1523341113

 

Wietek, J., and Prigge, M.‡ (2016) Enhancing Channelrhodopsins: An Overview. in Methods in Molecular Biology, 1408, 141–165

 

Wietek J., Beltramo R, Scanziani M, Hegemann P#, Oertner TG#, Wiegert JS (2015) An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo.  Scientific Reports  doi:10.1038/srep14807 

 

Wietek, J.*, Wiegert, J.S.*, Adeishvili, N., Schneider, F., Watanabe, H., Tsunoda, S.P., Vogt, A., Elstner, M., Oertner, T.G., Hegemann, P. (2014) Conversion of Channelrhodopsin into a light-gated chloride channel. Science 344, 409 – 412. http://www.sciencemag.org/content/344/6182/409.full.pdf

 

Comment: Hayashi, S. (2014) Silencing Neurons with Light. Science 369 – 370. http://www.sciencemag.org/content/344/6182/369.full.pdf

Daniel Evanko (2014), Nature Methods, 11, 608. http://www.nature.com/nmeth/journal/v11/n6/full/nmeth.2988.html

 

Other publications:

F. Richter, U. S. Scheib, J. Mehlhorn, R. Schubert, J. Wietek, O. Gernetzki, P. Hegemann, T. Mathes and A. Möglich# (2014), Upgrading a Microplate Reader for Photobiology and All-Optical Experiments. Photochem. Photobiol. Sci., 2014, DOI: 10.1039/C4PP00361F. http://pubs.rsc.org/en/content/articlepdf/2014/PP/C4PP00361F?page=search

 

Wietek, J., Haralampiev, I., Amoussouvi, A., Herrmann, A., & Stöckl, M.# (2013). Membrane bound α-synuclein is fully embedded in the lipid bilayer while segments with higher flexibility remain. FEBS letters, 587(16), 2572–7.

 

Vogt, A.*, Wietek, J.*, & Hegemann, P.# (2013). Gloeobacter rhodopsin, limitation of proton pumping at high electrochemical load. Biophysical journal, 105(9), 2055–63.

 

Prigge, M.*, Schneider, F.*, Tsunoda, S. P., Shilyansky, C., Wietek, J., Deisseroth, K., & Hegemann, P. (2012). Color-tuned channelrhodopsins for multiwavelength optogenetics. The Journal of biological chemistry, 287(38), 31804–12.

 

 

Book Chapters:

Wietek J., Prigge M.: Enhancing Channelrhodopsins: An Overview. Optogenetics, 1408 edited by Arash Kianianmomeni, 03/2016: chapter 10: pages 141-165; Springer New York., ISBN: 978-1-4939-3510-9 

 

* Equal contribution

# Corresponding Author