Elucidation of Cl--pumping mechanism and significance of trimer formation of halorhodopsin

Halorhodopsin （HR）是盐细菌膜中表达的一种光驱动Cl^-泵。我们成功地开发了一种新的方法：在非洲爪蟾卵母细胞中表达HR，并测量由于Cl^-运输引起的光诱导膜电流。这种方法是高度定量的。利用大肠杆菌表达系统制备了多种HR突变体。对突变体的光化学和转运活性进行了仔细的研究。我们发现Arg123是一个非常重要的氨基酸残基。在从N到O中间体的转变过程中，Cl被释放到细胞质中，而在O到HR的转变过程中，Cl被从细胞外空间吸收。我们发现，即使在洗涤剂的存在下，HR也会形成三聚体，并且三聚体比单体更坚固。

Halorhodopsin (HR) is a light-driven Cl^- pump expressed in the membrane of Halobacteria. We succeeded in developing a new method : HR was expressed in Xenopus laevis Oocytes, and photo-induced membrane current was measured due to the Cl^- transport. This method is highly quantitative. We prepared various HR mutants using E. coli expression system. Careful investigation on both the photochemistry and transport activities of the mutants has been done. We found Arg123 that is an essentially important amino acid residue. During the transition from N to O intermediate, Cl is released to cytoplasm while Cl is uptaken from extracellular space during O to HR' transition. We showed that HR forms trimer even in the presence of detergents, and that the trimer is more robust than the monomer.