Direct detection of the chloride release and uptake reactions of Natronomonas pharaonis halorhodopsin

Membrane transport proteins undergo multistep conformational changes to fulfill the transport of substrates across biological membranes. Substrate release and uptake are the most important events of these multistep reactions that accompany significant conformational changes. Thus, their relevant structural intermediates should be identified to better understand the molecular mechanism. However, their identifications have not been achieved for most transporters due to the difficulty of detecting the intermediates. Herein, we report the success of these identifications for a light-driven chloride transporter halorhodopsin (HR). We compared the time course of two flash-induced signals during a single transport cycle. One is a potential change of Cl^--selective membrane, which enabled us to detect tiny Cl^--concentration changes due to the Cl^- release and the subsequent Cl^--uptake reactions by HR. The other is the absorbance change of HR reflecting the sequential formations and decays of structural intermediates. Their comparison revealed not only the intermediates associated with the key reactions but also the presence of two additional Cl^--binding sites on the Cl^--transport pathways. The subsequent mutation studies identified one of the sites locating the protein surface on the releasing side. Thus, this determination also clarified the Cl^--transport pathway from the initial binding site until the release to the medium.