Griffith D A, Pajor A M
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641, USA.
Biochemistry. 1999 Jun 8;38(23):7524-31. doi: 10.1021/bi990076b.
The role of acidic amino acid residues in cation recognition and selectivity by the Na+/dicarboxylate cotransporter, NaDC-1, was investigated by site-directed mutagenesis and expression in Xenopus oocytes. Four of the residues tested, Asp-52, Glu-74, Glu-101, and Glu-332, were found to be unimportant for transport activity. However, substitutions of Asp-373 and Glu-475, conserved residues found in transmembrane domains M8 and M9, respectively, altered transport kinetics. Replacements of Asp-373 with Ala, Glu, Asn, and Gln resulted in changes in sodium affinity and cation selectivity in NaDC-1, indicating that the carbonyl oxygen at this position may play a role in the topological organization of the cation-binding site. In contrast, substitutions of Glu-475 led to dramatic reductions in transport activity and changes in transport kinetics. Substitution with Gln led to a transporter with increased substrate and sodium affinity, while the E475D mutant was inactive. The E475A mutant appeared to have poor sodium binding. Substrate-induced currents in the E475A mutant exhibited a strong voltage dependence, and a reversal of the current was seen at -30 mV. The results suggest that Glu-475 may play a role in cation binding and possibly also in mediating anion channel activity. Remarkably, mutations of both Asp-373 and Glu-475 affected the Km for succinate in NaDC-1, suggesting dual roles for these residues in determining the affinity for substrate and cations. We propose that at least one of the cation-binding sites and the substrate-binding site are close together in the carboxy-terminal portion of NaDC-1, and thus transmembrane domains M8 and M9 are candidate structures for the formation of the translocation pathway.
通过定点诱变以及在非洲爪蟾卵母细胞中的表达，研究了酸性氨基酸残基在Na⁺/二羧酸盐共转运蛋白NaDC-1对阳离子的识别和选择性中的作用。所测试的四个残基，即天冬氨酸-52、谷氨酸-74、谷氨酸-101和谷氨酸-332，被发现对转运活性不重要。然而，分别位于跨膜结构域M8和M9中的保守残基天冬氨酸-373和谷氨酸-475的替换，改变了转运动力学。用丙氨酸、谷氨酸、天冬酰胺和谷氨酰胺替换天冬氨酸-373导致NaDC-1中钠亲和力和阳离子选择性的变化，表明该位置的羰基氧可能在阳离子结合位点的拓扑结构中起作用。相比之下，谷氨酸-475的替换导致转运活性显著降低和转运动力学改变。用谷氨酰胺替换导致转运体对底物和钠的亲和力增加，而E475D突变体无活性。E475A突变体似乎钠结合能力较差。E475A突变体中底物诱导的电流表现出强烈的电压依赖性，并且在-30 mV时观察到电流反转。结果表明谷氨酸-475可能在阳离子结合中起作用，也可能在介导阴离子通道活性中起作用。值得注意的是，天冬氨酸-373和谷氨酸-475的突变都影响了NaDC-1中琥珀酸的Km，表明这些残基在决定对底物和阳离子的亲和力方面具有双重作用。我们提出，至少一个阳离子结合位点和底物结合位点在NaDC-1的羧基末端部分彼此靠近，因此跨膜结构域M8和M9是形成转运途径的候选结构。
