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

Abstract

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.

The form and function of channelrhodopsin

“…Two other key developments—the fiberoptic neural interface and versatile targeting methodology (32–37)—would ultimately enable generalizable millisecond-precision genetically targeted neuronal control with light throughout the brain during behavior (3, 14). By 2007, optogenetic control of defined spiking patterns in specified neurons deep in the hypothalamus of freely moving adult mice had been achieved, along with resulting behavioral state transitions (33).…”

“…3) appears useful for algae in coping with high light intensities but poses challenges for neuroscience. This inactivation manifests as a probabilistic alternative photoisomerization around the ATR C15=NH bond (37), corresponding to transition from a syn-cycle to an anti-cycle (Fig. 3) as in animal rhodopsins during transitions from Meta-I to Meta-III (38) or in bacteriorhodopsin during dark adaptation (39).…”

“…3) (40). Under dark adaptation, the ATR exists as all-trans/15-anti, but shortly after illumination a mixture of two similar but distinguishable states appears (all-trans/15-anti and 13-cis/15-syn), leading to conducting states in 13-cis/15-anti (O1) and 13-trans/15-syn (O2) configurations, respectively (37, 41). The O1/O2 ratio also depends on membrane voltage and pH gradient, and differs substantially among ChR variants, contributing to diversity of photocurrent properties (1, 2, 42).…”

“…Chromophore configurations in RSB: all-trans, 15-anti (trans, anti) of dark-adapted state; 13-cis, 15-syn (cis, syn) of second dark state (increasingly occupied after blue illumination during photocurrent inactivation from initial I 0 to stationary I S ; inset) (1, 37). In corresponding open states occupied after 13 C= 14 C photoisomerization, RSB is in the 13-cis, 15-anti (cis, syn) configuration in the open conducting state O1 and 13-trans, 15-syn (trans, syn) in O2 (1, 37).…”

“…In corresponding open states occupied after 13 C= 14 C photoisomerization, RSB is in the 13-cis, 15-anti (cis, syn) configuration in the open conducting state O1 and 13-trans, 15-syn (trans, syn) in O2 (1, 37). Cis-trans isomerization is indicated by red arrows, anti-syn isomerization by blue arrows, photochemical conversions by green arrows, and thermal conversions by black arrows.…”