Preference of Proteomonas sulcata anion channelrhodopsin for NO₃^- revealed using a pH electrode method

Ion channel proteins are physiologically important molecules in living organisms. Their molecular functions have been investigated using electrophysiological methods, which enable quantitative, precise and advanced measurements and thus require complex instruments and experienced operators. For simpler and easier measurements, we measured the anion transport activity of light-gated anion channelrhodopsins (ACRs) using a pH electrode method, which has already been established for ion pump rhodopsins. Using that method, we successfully measured the anion transport activity and its dependence on the wavelength of light, i.e. its action spectra, and on the anion species, i.e. its selectivity or preference, of several ACRs expressed in yeast cells. In addition, we identified the strong anion transport activity and the preference for NO₃^- of an ACR from a marine cryptophyte algae Proteomonas sulcata, named PsuACR_353. Such a preference was discovered for the first time in microbial pump- or channel-type rhodopsins. Nitrate is one of the most stable forms of nitrogen and is used as a nitrogen source by most organisms including plants. Therefore, PsuACR_353 may play a role in NO₃^- transport and might take part in NO₃^--related cellular functions in nature. Measurements of a mutant protein revealed that a Thr residue in the 3^rd transmembrane helix, which corresponds to Cys102 in GtACR1, contributed to the preference for NO₃^-. These findings will be helpful to understand the mechanisms of anion transport, selectivity and preference of PsuACR_353.