Molecular Basis and Physiological Roles of Xenobiotic Exporters

(1)构建了四环素/H+反转运体TetA (B)的完整的半胱氨酸扫描突变体，并通过位点定向化学修饰和氧化二硫交联形成双Cys突变体的详细拓扑结构进行了研究。结果表明，TetA (B)形成一个圆形，在分子中心有一个充满水的通道，在膜的中间有一个渗透性屏障。(2)建立了用荧光药物测量MRP2介导的药物外排的方法。然后，我们通过位点定向诱变确定了其转运功能和抑制剂结合所必需的跨膜基本残基。(3)我们首先成功地获得了外生菌出口商的晶体。现在，我们正在使用SPring8进行x射线晶体学分析。(4)从血小板中发现了一种新的可能的鞘氨醇-1-磷酸输出源，并克隆了其完整cDNA并在细胞系中表达。

(1) We constructed the complete set of cysteine-scanning mutants of tetracycline/H+ antiporter TetA (B) and investigated the detailed topology by means of site-directed chemical modification and oxidative disulfide cross-linking formation of double Cys mutants. As a result, it was revealed that TetA (B) forms a circular form and involves a water-filled channel at the center of the molecule with a permeability barrier in the middle of the membrane.(2) We established the method for measuring the drug efflux mediated by MRP2 by means of fluorescent drugs. Then, we determined the transmembrane basic residues essential for its transport function and the binding of the inhibitor by means of site-directed mutagenesis.(3) We first succeeded to obtain crystals of xenobiotic exporters. Now, we are undergoing the X-ray crystallographic analysis using SPring8.(4) We found a novel possible exporter for sphingosine-1-phosphate from platelete and its complete cDNA was cloned and expressed in a cell line.