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J. Biol. Chem., Vol. 283, Issue 3, 1637-1643, January 18, 2008

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Photoactivation of Channelrhodopsin^*

Oliver P. Ernst¹, Pedro A. Sánchez Murcia², Peter Daldrop, Satoshi P. Tsunoda, Suneel Kateriya^¶, and Peter Hegemann³

From the Institut für medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany, the Institut für Biologie, Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany, and the ^¶Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India

Channelrhodopsins (ChRs) are light-gated ion channels that control photomovement of microalgae. In optogenetics, ChRs are widely applied for light-triggering action potentials in cells, tissues, and living animals, yet the spectral properties and photocycle of ChR remain obscure. In this study, we cloned a ChR from the colonial alga Volvox carteri, VChR. After electrophysiological characterization in Xenopus oocytes, VChR was expressed in COS-1 cells and purified. Time-resolved UV-visible spectroscopy revealed a pH-dependent equilibrium of two dark species, D₄₇₀/D₄₈₀. Laser flashes converted both with 200 µs into major photointermediates P₅₁₀/P₅₃₀, which reverted back to the dark states with 15-100 ms. Both intermediates were assigned to conducting states. Three early intermediates P₅₀₀/P₅₁₅ and P₃₉₀ were detected on a ns to µs time scale. The spectroscopic and electrical data were unified in a photocycle model. The functional expression of VChR we report here paves the way toward a broader structure/function analysis of the recently identified class of light-gated ion channels.

Received for publication, September 26, 2007 , and in revised form, November 6, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants Sfb 449 and Sfb 740 (to O. P. E.) and Sfb 498 (to P. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data.

² Recipient of an Erasmus fellowship from the European Commission, Spanish Ministerio Educación y Ciencia, and Universidad Complutense Madrid.

¹ To whom correspondence may be addressed. Tel.: 49-30-450-524-111; Fax: 49-30-450-524-952; E-mail: oliver.ernst{at}charite.de.

³ To whom correspondence may be addressed. Tel.: 49-30-2093-8681; E-mail: hegemape{at}rz.hu-berlin.de.

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