Engineering a Cl‐‐Modulated Light‐Driven Na+ Pump

Abstract

AbstractMicrobial 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.