Glu 87 of Channelrhodopsin‐1 Causes pH‐dependent Color Tuning and Fast Photocurrent Inactivation†

Channelrhodopsins (ChR1 and ChR2) are directly light‐gated ion channels acting as sensory photoreceptors in the green alga Chlamydomonas reinhardtii. These channels open rapidly after light absorption and both become permeable for cations such as H⁺, Li⁺, Na⁺, K⁺ and Ca²⁺. K_m for Ca²⁺ is 16.6 mm in ChR1 and 18.3 mm in ChR2 whereas the K_m values for Na⁺ are higher than 100 mm for both ChRs. Action spectra of ChR1 peak between 470 and 500 nm depending on the pH conditions, whereas ChR2 peaks at 470 nm regardless of the pH value. Now we created two chimeric ChRs possessing helix 1–5 of ChR1 and 6, 7 of ChR2 (ChR1/2_5/2), or 1, 2 from ChR1 and 3–7 from ChR2 (ChR1/2_2/5). Both ChR‐chimera still showed pH‐dependent action spectra shifts. Finally, a mutant ChR1E87Q was generated that inactivated only slowly in the light and showed no spectral shift upon pH change. The results indicate that protonation/deprotonation of E87 in helix 1 alters the chromophore polarity, which shifts the absorption and modifies channel inactivation accordingly. We propose a trimodal counter ion complex for ChR1 but only a bimodal complex for ChR2.