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

Abstract

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 (kH) to that of K+ uptake (kK) is >108. Compared to the WT, the G263W/N61P mutant exhibited a decrease of approximately 40-fold in kH/kK. 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.