Sugar Binding Residue Affects Apparent Na+ Affinity and Transport Stoichiometry in Mouse Sodium/Glucose Cotransporter Type 3B
Sugar Binding Residue Affects Apparent Na+ Affinity and Transport Stoichiometry in Mouse Sodium/Glucose Cotransporter Type 3B
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DOI：
10.1074/jbc.m110.187880
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发表时间：
2011-03-11
影响因子：
4.8
通讯作者：
Barcelona, Stephanie
中科院分区：
文献类型：
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作者：
Diez-Sampedro, Ana;Barcelona, Stephanie
SGLT 1是一种钠/葡萄糖协同转运蛋白，在每个循环中随每个葡萄糖分子移动两个Na+离子。SGLT 3蛋白属于同一家族，被描述为葡萄糖传感器而不是葡萄糖转运蛋白。因此，人SGLT 3（hSGLT 3）不转运糖，但细胞外葡萄糖使表达它的细胞去极化。小鼠SGLT 3b（mSGLT 3b）虽然转运糖，但与SGLT 1相比，其表观糖亲和力较低，化学计量部分未偶联，这表明mSGLT 3b也是一种糖传感器。副溶血弧菌SGLT的晶体结构表明残基Gln（428）直接与糖相互作用。哺乳动物蛋白质中的相应氨基酸457在所有SGLT 1蛋白质中保守为谷氨酰胺。在SGLT 3蛋白中，谷氨酸是该位置最常见的残基，尽管它在mSGLT 3b中是甘氨酸，在大鼠SGLT 3b中是丝氨酸。为了测试该残基对SGLT 3蛋白功能的贡献，我们构建了SGLT 3b突变体，其分别重现SGLT 1和hSGLT 3中的残基457、谷氨酰胺和谷氨酸。谷氨酰胺在残基457的存在下增加了表观Na+和糖的亲和力，而谷氨酸降低了表观Na+的亲和力。此外，谷氨酸转运更多的阳离子每糖分子比野生型蛋白质。我们提出了一个模型，其中阳离子释放胞内没有释放糖从中间状态。该模型解释了与SGLT 1相比在野生型和G457 E-mSGLT 3b中观察到的解偶联电荷：糖转运表型，以及在hSGLT 3中发生的无糖转运的糖激活阳离子转运。
SGLT1 is a sodium/glucose cotransporter that moves two Na+ ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting that mSGLT3b is also a sugar sensor. The crystal structure of the Vibrio parahaemolyticus SGLT showed that residue Gln(428) interacts directly with the sugar. The corresponding amino acid in mammalian proteins, 457, is conserved in all SGLT1 proteins as glutamine. In SGLT3 proteins, glutamate is the most common residue at this position, although it is a glycine in mSGLT3b and a serine in rat SGLT3b. To test the contribution of this residue to the function of SGLT3 proteins, we constructed SGLT3b mutants that recapitulate residue 457 in SGLT1 and hSGLT3, glutamine and glutamate, respectively. The presence of glutamine at residue 457 increased the apparent Na+ and sugar affinities, whereas glutamate decreased the apparent Na+ affinity. Moreover, glutamate transported more cations per sugar molecule than the wild type protein. We propose a model where cations are released intracellularly without the release of sugar from an intermediate state. This model explains the uncoupled charge: sugar transport phenotype observed in wild type and G457E-mSGLT3b compared with SGLT1 and the sugar-activated cation transport without sugar transport that occurs in hSGLT3.