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

Halorhodopsin （HR）是一种光驱动的向内定向的Cl^-泵。为了更详细地阐明Cl^-转运的分子机制，我们利用各种物理化学技术精确地测定了通过HR的转运活性，得到了以下结果。(1)晶体结构清楚地表明，初生Cl^-接近质子化希夫碱（PSB），并通过与带正电荷的PSB的静电相互作用而稳定[PDB: 1E12]。根据晶体结构，我们突变了可能参与氯转运的氨基酸残基。确定了几个重要的氨基酸残基；Arg123， Ser130和Asp252。(2)特别是，两个氨基酸残基Ser130和Asp252被证明在Cl^-在视网膜细胞外（EC）通道中远离结合位点的易位中起重要作用。(3)为了实现载体易位，HR蛋白必须在HR中具有一个或多个分子阀，以阻止泵送周期中Cl^-的泄漏或回流（从细胞质（CP）到EC通道）。Ser130与PBS配合，在阴离子传输循环中作为分子阀阻止Cl^-的内部泄漏和回流。

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.