K(+)-Dependent Photocycle and Photocurrent Reveal the Uptake of K(+) in Light-Driven Sodium Pump

Engineering light-controlled K(+) pumps from Na(+)-pumping rhodopsins (NaR) greatly expands the scope of optogenetic applications. However, the limited knowledge regarding the kinetic and selective mechanism of K(+) uptake has significantly impeded the modification and design of light-controlled K(+) pumps, as well as their practical applications in various fields, including neuroscience. In this study, we presented K(+)-dependent photocycle kinetics and photocurrent of a light-driven Na(+) pump called Nonlabens dokdonensis rhodopsin 2 (NdR2). As the concentration of K(+) increased, we observed the accelerated decay of M intermediate in the wild type (WT) through flash photolysis. In 100 mM KCl, the lifetime of the M decay was approximately 1.0 s, which shortened to around 0.6 s in 1 M KCl. Additionally, the K(+)-dependent M decay kinetics were also observed in the G263W/N61P mutant, which transports K(+). In 100 mM KCl, the lifetime of the M decay was approximately 2.5 s, which shortened to around 0.2 s in 1 M KCl. According to the competitive model, in high KCl, K(+) may be taken up from the cytoplasmic surface, competing with Na(+) or H(+) during M decay. This was further confirmed by the K(+)-dependent photocurrent of WT liposome. As the concentration of K(+) increased to 500 mM, the amplitude of peak current significantly dropped to approximately ~60%. Titration experiments revealed that the ratio of the rate constant of H(+) uptake (k(H)) to that of K(+) uptake (k(K)) is >10(8). Compared to the WT, the G263W/N61P mutant exhibited a decrease of approximately 40-fold in k(H)/k(K). Previous studies focused on transforming NaR into K(+) pumps have primarily targeted the intracellular ion uptake region of Krokinobacter eikastus rhodopsin 2 (KR2) to enhance K(+) uptake. However, our results demonstrate that the naturally occurring WT NdR2 is capable of intracellular K(+) uptake without requiring structural modifications on the intracellular region. This discovery provides diverse options for future K(+) pump designs. Furthermore, we propose a novel photocurrent-based approach to evaluate K(+) uptake, which can serve as a reference for similar studies on other ion pumps. In conclusion, our research not only provides new insights into the mechanism of K(+) uptake but also offers a valuable point of reference for the development of optogenetic tools and other applications in this field.