Multiple photocycles of channelrhodopsin.

Two rhodopsins with intrinsic ion conductance have been identified recently in Chlamydomonas reinhardtii. They were named "channelrhodopsins" ChR1 and ChR2. Both were expressed in Xenopus laevis oocytes, and their properties were studied qualitatively by two electrode voltage clamp techniques. ChR1 is specific for H+, whereas ChR2 conducts Na+, K+, Ca2+, and guanidinium. ChR2 responds to the onset of light with a peak conductance, followed by a smaller steady-state conductance. Upon a second stimulation, the peak is smaller and recovers to full size faster at high external pH. ChR1 was reported to respond with a steady-state conductance only but is demonstrated here to have a peak conductance at high light intensities too. We analyzed quantitatively the light-induced conductance of ChR1 and developed a reaction scheme that describes the photocurrent kinetics at various light conditions. ChR1 exists in two dark states, D1 and D2, that photoisomerize to the conducting states M1 and M2, respectively. Dark-adapted ChR1 is completely arrested in D1. M1 converts into D1 within milliseconds but, in addition, equilibrates with the second conducting state M2 that decays to the second dark state D2. Thus, light-adapted ChR1 represents a mixture of D1 and D2. D2 thermally reconverts to D1 in minutes, i.e., much slower than any reaction of the two photocycles.

Evidence for a light-induced H(+) conductance in the eye of the green alga Chlamydomonas reinhardtii.

Rhodopsin-mediated photoreceptor currents, I(P), of the unicellular alga Chlamydomonas reinhardtii were studied under neutral and acidic conditions. We characterized the kinetically overlapping components of the first, flash-induced inward current recorded from the eye, I(P1), as a low- and high-intensity component, I(P1a) and I(P1b), respectively. They peak between 1 and 10 ms after the light-flash and are both likely to be carried by Ca(2+). I(P1a) and I(P1b) exhibit half-maximal photon flux densities, Q(1/2), of approximately 0.14 and 58 microE m(-2), and maximal amplitudes of approximately 4.9 and 38 pA, respectively. At acidic extracellular pH values (pH 3-5), both I(P1) currents are followed by distinct H(+) currents, I(P2a) and I(P2b), with maxima after approximately 5 and 100 ms, respectively. Because the Q(1/2) values of I(P1b) and I(P2b) virtually coincide with Q(1/2) of rhodopsin bleaching, we suggest that the respective conductances G(1b) and G(2b) are closely coupled to the rhodopsin, whereas the low light-saturating conductances G(1a) and G(2a) reflect transducer-activated states of a second rhodopsin photoreceptor system.