A developmental approach to clarify a molecular mechanism of transporter utilizing a model transporter as a light-driven ion pump

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Halorhodopsin (HR) is a light-driven inward-directed Cl^- pump. To clarify the molecular mechanism of Cl^- transport in detail, we precisely determined the transport activities through HR with various physicochemical techniques, and the following results were obtained.(1) The crystal structure clearly shows that the primary Cl^- is proximal to the protonated Schiff base (PSB) and is stabilized by electrostatic interactions with the positive charged PSB [PDB : 1E12]. Based on the crystal structure, we mutated amino acid residues, which might be involved in Cltransport. Several important amino acid residues were identified ; Arg123, Ser130 and Asp252.(2) Especially, two amino acid residues Ser130 and Asp252 were proved out to play an important role in Cl^- translocation away from the binding site in the extracellular (EC) channel across the retinal.(3) For the sake of the vectorial translocation, HR protein must possess one or several molecular valves in HR to hinder the Cl^- leakage, or backflow (from cytoplasmic (CP) to EC channel) during pumping cycles. Ser130 coordinates with PBS and functions as a molecular valve to hinder the internal leak, backflux of Cl^- during anion-transport cycle.