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Microbial Na+‐pumping rhodopsin (NaR) is a promising optogenetic tool due to its unique ability to transport Na+. Like most rhodopsin‐based tools, NaR is limited to light‐based control. In this study, our objective was to develop a novel mode of modulation for NaR beyond light control. By introducing a potential Cl− binding site near the putative Na+ release cavity, we engineered Nonlabens dokdonensis rhodopsin 2 (NdR2) to be modulated by Cl−, an essential chemical in organisms. The engineered NdR2 demonstrated an approximately two‐fold increase in Na+ pump activity in the presence of 100 mM Cl− compared to Cl‐‐free solution. Increasing Cl− concentration decreased the lifetimes of the M and O intermediates accordingly. The analysis of competitive ion uptake suggested the bound Cl− may increase the Na+ affinity and selectivity. This chemical modulation allows for more diverse and precise control over cellular processes, advancing the development of next‐generation optogenetic tools. Notably, our Cl‐‐modulated NdR2 establishes an innovative mechanism for linking Cl− to Na+‐related processes, with potential applications in optogenetic therapies for related diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This article, titled "Cover Feature: Engineering a Cl-Modulated Light-Driven Na+ Pump," discusses the development of a new chemical modulation mode for a microbial Na+-pumping rhodopsin (NaR) protein. The researchers introduced a potential chloride (Cl-) binding site in the protein, which increased the uptake and transport activity of sodium ions (Na+). This Cl--modulated NaR has potential applications in optogenetic therapies for neurological diseases. The article provides more information on this research and can be found in the Chemistry - A European Journal. [Extracted from the article]

Temperature responses of asphalt pavements are significant, because of their potential influence on pavement structure design and distress prevention, particularly in areas that experience large temperature difference. In this study, a 3D finite element model is developed based on transient heat transfer. The aim is to analyse the temperature responses of the Karamay–Altay Highway in northern Xinjiang, which tends to experience large temperature differences and low temperatures. First, the climatic characteristics and pavement distresses in northern Xinjiang were investigated, and the condition defining a large temperature difference was established. Then, to guide the design of the Karamay–Altay Highway pavement structure, the temperature fields of seven pavement structures were determined. The temperature field of the Str-m structure in the Karamay–Altay area was compared to that in Xi'an. In the Karamay–Altay area, it was observed that Str-4 exhibited the minimum temperature variation at the bottom of the asphalt course and base course. It was clear that the degree of downward temperature transfer was closely related to the structure combination type. The results also suggested that a thicker asphalt layer would effectively reduce temperature effects and diminish distresses resulting from temperature. By comparing of the temperature fields of the Str-m structure in the Karamay–Altay area and in Xi'an, it was observed that pavement temperature fields changed drastically in the case study area, and hence revealed that severe pavement cracks in this area were closely related to abrupt temperature changes, low temperatures and large temperature differences. Hence, it was concluded that in northern Xinjiang, pavement designers should consider the large temperature difference condition very carefully. [ABSTRACT FROM AUTHOR]

Microbial Na + -pumping rhodopsin (NaR) is a promising optogenetic tool due to its unique ability to transport Na + . Like most rhodopsin-based tools, NaR is limited to light-based control. In this study, our objective was to develop a novel mode of modulation for NaR beyond light control. By introducing a potential Cl - binding site near the putative Na + release cavity, we engineered Nonlabens dokdonensis rhodopsin 2 (NdR2) to be modulated by Cl - , an essential chemical in organisms. The engineered NdR2 demonstrated an approximately two-fold increase in Na + pump activity in the presence of 100 mM Cl - compared to Cl - -free solution. Increasing Cl - concentration decreased the lifetimes of the M and O intermediates accordingly. The analysis of competitive ion uptake suggested the bound Cl - may increase the Na + affinity and selectivity. This chemical modulation allows for more diverse and precise control over cellular processes, advancing the development of next-generation optogenetic tools. Notably, our Cl - -modulated NdR2 establishes an innovative mechanism for linking Cl - to Na + -related processes, with potential applications in optogenetic therapies for related diseases., (© 2023 Wiley-VCH GmbH.)