The Voltage Dependent Sidedness of the Reprotonation of the Retinal Schiff Base Determines the Unique Inward Pumping of Xenorhodopsin

The new class of microbial rhodopsins, called xenorhodopsins (XeRs),([1]) extends the versatility of this family by inward H(+) pumps.([2–4]) These pumps are an alternative optogenetic tool to the light‐gated ion channels (e.g. ChR1,2), because the activation of electrically excitable cells by XeRs is independent from the surrounding physiological conditions. In this work we functionally and spectroscopically characterized XeR from Nanosalina (NsXeR).([1]) The photodynamic behavior of NsXeR was investigated on the ps to s time scale elucidating the formation of the J and K and a previously unknown long‐lived intermediate. The pH dependent kinetics reveal that alkalization of the surrounding medium accelerates the photocycle and the pump turnover. In patch‐clamp experiments the blue‐light illumination of NsXeR in the M state shows a potential‐dependent vectoriality of the photocurrent transients, suggesting a variable accessibility of reprotonation of the retinal Schiff base. Insights on the kinetically independent switching mechanism could furthermore be obtained by mutational studies on the putative intracellular H(+) acceptor D220.