Sequential absorption of two photons creates a bistable form of RubyACR responsible for its strong desensitization.

Channelrhodopsins with red-shifted absorption, rare in nature, are highly desired for optogenetics because light of longer wavelengths more deeply penetrates biological tissue. RubyACRs (Anion ChannelRhodopsins), a group of four closely related anion-conducting channelrhodopsins from thraustochytrid protists, are the most red-shifted channelrhodopsins known with absorption maxima up to 610 nm. Their photocurrents are large, as is typical of blue- and green-absorbing ACRs, but they rapidly decrease during continuous illumination (desensitization) and extremely slowly recover in the dark. Here, we show that long-lasting desensitization of RubyACRs results from photochemistry not observed in any previously studied channelrhodopsins. Absorption of a second photon by a photocycle intermediate with maximal absorption at 640 nm (P640) renders RubyACR bistable (i.e., very slowly interconvertible between two spectrally distinct forms). The photocycle of this bistable form involves long-lived nonconducting states (Llong and Mlong), formation of which is the reason for long-lasting desensitization of RubyACR photocurrents. Both Llong and Mlong are photoactive and convert to the initial unphotolyzed state upon blue or ultraviolet (UV) illumination, respectively. We show that desensitization of RubyACRs can be reduced or even eliminated by using ns laser flashes, trains of short light pulses instead of continuous illumination to avoid formation of Llong and Mlong, or by application of pulses of blue light between pulses of red light to photoconvert Llong to the initial unphotolyzed state.