Light-Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-trans and 13-cis Retinal Isomers.

Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named "light adaptation". The main objective of this spectroscopic study was to elucidate the molecular processes associated with light-dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all-trans configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all-trans,15-anti and 13-cis,15-syn configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13)═C(14) and C(15)═N bonds. Both isomers of this DAapp state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13)═C(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DAapp) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-trans isomer than for the 13-cis isomer, possibly because of different chromophore-water interactions. Our data demonstrating two complementary photocycles of the DAapp isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light-dark adaptation, as suggested previously by electrical measurements.

Fast, repetitive light-activation of CaV3.2 using channelrhodopsin 2.

Channelrhodopsin-2 (ChR2) is a light-gated ion channel that is successfully used in neurosciences to depolarize cells with blue light. In this regard control of membrane voltage with light opens new perspectives for the characterization of ion channels and the search for inhibitors or modulators. Here, we report a control of membrane potential with ChR2 and the potassium channel mTrek for the purpose of screening for ion channel specific drugs. To verify principle we have chosen the voltage gated calcium channel Ca(V)3.2 as potential drug target. For this purpose we transfected the ChR2 gene into a HEK293T-cell line that permanently expresses Ca(V)3.2 and the K-channel mTrek. The resting potential was adjusted with low concentration of extracellular potassium ions whereas transient depolarization was achieved by activation of ChR2 with short pulses of blue light. Calcium ion influx through Ca(V)3.2 was monitored by observing fura-2 fluorescence. This approach allowed a repetitive activation of Ca(V)3.2. The Ca(2+) influx was specifically blocked by the inhibitor mibefradil. Since this assay is genetically-encoded, it may be employed for a variety of voltage-gated calcium channels and should be applicable to multi-well reader formats for high-throughput screening.

Channelrhodopsin-1: a light-gated proton channel in green algae.

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.