The structure and function analysis of MATE transporter in Mammal.

人类多药毒性化合物排出物1(HMATE1)是一种电子中和H(+)/有机阳离子交换器，负责肾脏和肝脏结构无关的有毒有机阳离子的最终排泄步骤。为了阐明hMATE1识别底物的分子基础，我们将跨膜区保守的谷氨酸残基Glu273、Glu278、Glu300和Glu389替换为丙氨酸或天冬氨酸，并以四乙铵(TEA)和西咪替丁为底物在人胚胎肾293(HEK-293)细胞中表达后检测了突变蛋白的转运活性。除Glu273Ala外，其余突变体均在HEK-293细胞的细胞膜上得到充分表达和存在。突变体Glu278A1a的茶叶转运活性完全缺失。Glu300Ala和Glu389Ala及所有天冬氨酸突变体的活性均显著降低。Glu273Asp对西咪替丁的亲和力较高，而对茶叶的亲和力较低。Glu278Asp对西咪替丁的亲和力降低。Glu300Asp和Glu389Asp都降低了对茶叶的亲和力，而Glu389Asp对西咪替丁的亲和力比野生型转运蛋白高4倍，V(Max)值降低了约4倍。Glu273Asp和Glu300Asp均改变了茶树吸收的pH依赖性。这些结果表明，所有这些谷氨酸残基都参与了茶叶和西咪替丁的结合和/或运输，但它们各自的作用不同。

Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H(+)/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278A1a was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in V(max) value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different.