(A) The halophilic unicellular alga Dunaliella salina.
(B) Sequence homology between the algal ChRs and BR within the third TM helix. The typically conserved E123 position has been replaced with an Ala in DChR1 (and is shown on a yellow background), conserved residues are shown on a blue background, and amino acids likely interacting with the chromophore are shown in red.
(C) Lack of a proton acceptor in DChR1 (A178), compared with BR (D85) and Chlamydomonas ChR2 (CChR2; E123). ASR (Anabaena sensory rhodopsin) has been crystallized with a mixture of all-trans and 13-cis retinal seen as an overlay ().
(D) DChR1 photocurrents are unaffected by changes in the extracellular cation composition (sole cation present in each condition shown on category x axis). Cation exchange was performed in 5 mM Mops-NMG, 0.1 mM MgCl2 with 100 mM LiCl, KCl, NaCl, guanidium chloride, or NMG chloride (pH 7.5). We used a human codonadapted DChR sequence (amino acid residues 1–339) as a template for capped RNA synthesis by T7 RNA polymerase (mMessage mMachine, Ambion). Oocyte preparation and injection of capped RNA were carried out as described previously (), and two-electrode voltage clamp was performed with a Turbo Tec-05 (NPI Electronic) or a GeneClamp 500 (Molecular Devices) amplifier on an oocyte after 3–7 days of the capped RNA injection. Continuous light was provided by a75-W Xenon lamp (Jena Instruments) and delivered to the oocytes via a 3 mm light guide. The light passed through a 500 ± 25 nm broadband filter (Balzers) with an intensity of 46 mW/cm2.
(E) In contrast, DChR1 photocurrent is highly sensitive to changes in the pH environment. Solutions contained 100 mM NMG chloride, 0.1 mM MgCl2, 0.1 mM CaCl2 with 5 mM glycine (pH 9.0), 5 mM Mops-NMG (pH 7.5), 5 mM citrate (pH 6, 5.5, 5.0, 4.6, 4.2).
(F) Introduction or alteration of a proton acceptor (A178E or E309D) in the DChR1 retinal-binding pocket causes a pronounced red-shift in the absorption spectrum. We applied 10 ns laser flashes as described previously (); solutions for action spectra recording contained 100 mM NaCl, 0.1 mM MgCl2, 0.1 mM CaCl2, and 5 mM citrate (pH 4.2).