Coupled sodium/glucose cotransport by SGLT1 requires a negative charge at position 454.
Coupled sodium/glucose cotransport by SGLT1 requires a negative charge at position 454.
复制标题
SGLT1 耦合的钠/葡萄糖共转运需要 454 位有负电荷。
DOI：
10.1021/bi048652d
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发表时间：
2004
期刊：
影响因子：
2.9
通讯作者：
Hirayama,BruceA
中科院分区：
文献类型：
--
作者：
Díez-Sampedro,Ana;Loo,DonaldDF;Wright,ErnestM;Zampighi,GuidoA;Hirayama,BruceA
SGLT1 的 Na+/葡萄糖共转运是一个紧密耦合的过程，由穿过质膜的 Na+ 电化学梯度驱动。我们之前提出SGLT1包含单独的Na+-和葡萄糖结合域，A166（在Na+域中）接近D454（在糖域中），并且这些残基之间的相互作用影响糖特异性和转运。我们现在已经在非洲爪蟾细胞中表达了突变体 D454C，并通过用 Cys 或 His 替换 Asp 以及用不同电荷的烷基化试剂（MTSES-、MTSET+、MMTS0、MTSHE0 和碘乙酸盐-）对 D454C 进行化学突变来检查残基 454 上电荷的作用。通过测量糖转运和协同转运蛋白电流来检查功能特性。此外，D454C 用荧光染料标记，标记转运蛋白的荧光被记录为电压和配体浓度的函数。数据表明（1）天冬氨酸454对于蛋白质正常运输至质膜至关重要； (2) D454C的功能特性发生显着变化，即周转数减少和电压敏感性丧失，但糖选择性或糖与Na+亲和力没有变化； (3) 残基 454 上的负电荷增加了 Na+ 和糖的转运，正常化学计量为 2 Na+:1 糖。相反，残基 454 上的正电荷使 Na+ 和糖转运解偶联，表明负电荷在协调共转运机制中的重要性。
Na+/glucose cotransport by SGLT1 is a tightly coupled process that is driven by the Na+electrochemical gradient across the plasma membrane. We have previously proposed that SGLT1 contains separate Na+- and glucose-binding domains, that A166 (in the Na+domain) is close to D454 (in the sugar domain), and that interactions between these residues influence sugar specificity and transport. We have now expressed the mutant D454C inXenopus laevisoocytes and examined the role of charge on residue 454 by replacing the Asp with Cys or His, and by chemical mutation of D454C with alkylating reagents of different charge (MTSES-, MTSET+, MMTS0, MTSHE0, and iodoacetate-). Functional properties were examined by measuring sugar transport and cotransporter currents. In addition, D454C was labeled with fluorescent dyes and the fluorescence of the labeled transporter was recorded as a function of voltage and ligand concentration. The data shows that (1) aspartate 454 is critically important for the normal trafficking of the protein to the plasma membrane; (2) there were marked changes in the functional properties of D454C, i.e., a reduction in turnover number and a loss of voltage sensitivity, although there were no alterations in sugar selectivity or sugar and Na+affinity; (3) a negative charge on residue 454 increased Na+and sugar transport with a normal stoichiometry of 2 Na+:1 sugar. A positive charge on residue 454, in contrast, uncoupled Na+and sugar transport, indicating the importance of the negative charge in the coordination of the cotransport mechanism.