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J Mol Biol. 2008 Jan 18;375(3):686-94. Epub 2007 Nov 1.

Spectral characteristics of the photocycle of channelrhodopsin-2 and its implication for channel function.

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  • 1Max-Planck-Institut für Biophysik, Max-von-Laue Strasse 3, 60438 Frankfurt, Germany.

Abstract

In 2003, channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii was discovered to be a light-gated cation channel, and since that time the channel became an excellent tool to control by light neuronal cells in culture as well as in living animals with high temporal and spatial resolution in a noninvasive manner. However, little is known about the spectral properties and their relation to the channel function. We have expressed ChR2 in the yeast Pichia pastoris and purified the protein. Flash-photolysis data were combined with patch-clamp studies to elucidate the photocycle. The protein absorbs maximally at approximately 480 nm before light excitation and shows flash-induced absorbance changes with at least two different photointermediates. Four relaxation processes can be extracted from the time course that we have analysed in a linear model for the photocycle leading to the kinetic intermediates P(1) to P(4). A short-lived photointermediate at 400 nm, suggesting a deprotonation of the retinal Schiff base, is followed by a red-shifted (520 nm) species with a millisecond lifetime. The first three kinetic intermediates in the photocycle, P(1) to P(3), are described mainly by the red-shifted 520-nm species. The 400-nm species contributes to a smaller extent to P(1) and P(2). The fourth one, P(4), is spectroscopically almost identical with the ground state and lasts into the seconds time region. We compared the spectroscopic data to current measurements under whole-cell patch-clamp conditions on HEK 293 cells. The lifetimes of the spectroscopically and electrophysiologically determined intermediates are in excellent agreement. The intermediates P(2) and P(3) (absorbing at 520 nm) are identified as the cation permeating states of the channel. Under stationary light, a modulation of the photocurrent by green light (540 nm) was observed. We conclude that the red-shifted spectral species represents the open channel state, and the thermal relaxation of this intermediate, the transition from P(3) to P(4), is coupled to channel closing.

PMID:
18037436
DOI:
10.1016/j.jmb.2007.10.072
[PubMed - indexed for MEDLINE]
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