Evidence for the involvement of Ala 166 in coupling Na(+) to sugar transport through the human Na(+)/glucose cotransporter.
Evidence for the involvement of Ala 166 in coupling Na(+) to sugar transport through the human Na(+)/glucose cotransporter.
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Ala 166 参与通过人 Na( )/葡萄糖协同转运蛋白将 Na( ) 与糖转运偶联的证据。
DOI：
10.1021/bi011026l
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Wright,EM
中科院分区：
文献类型：
--
作者：
Meinild,AK;Loo,DD;Hirayama,BA;Gallardo,E;Wright,EM
我们将位于人 Na+/葡萄糖协同转运蛋白 (hSGLT1) 跨膜片段 4 和 5 之间的推定 Na+ 转运途径中的残基 166 从丙氨酸突变为半胱氨酸 (A166C)。 A166C在非洲爪蟾细胞中表达，并采用电生理方法测定其功能。与 hSGLT1 相比，对 Na+ 的亲和力没有变化，而糖亲和力降低且糖特异性发生改变。转运蛋白的周转率降低，与hSGLT1相比，周转率取决于糖分子。将 A166C 暴露于 MTSEA 和 MTSET，但不暴露于 MTSES，会消除糖转运。 A166C 对烷基化试剂的可及性与蛋白质构象无关，表明残基始终可从细胞外表面接近。糖和根皮苷不能保护残基不被烷基化，表明残基166不位于糖途径中。 MTSEA、MTSET 和 MTSES 都改变了 A166C 的前稳态动力学，与 pH 无关，并且糖改变了这些动力学。如果通过将外部 pH 值提高到 9.0 来中和 MTSEA 上的正电荷，则 MTSEA 标记的 A166C 转运糖的能力会被逆转（Na + 和糖亲和力没有重大变化）。这些研究表明，166 位残基参与了 Na+ 和糖转运途径之间的相互作用。
We mutated residue 166, located in the putative Na+transport pathway between transmembrane segments 4 and 5 of human Na+/glucose cotransporter (hSGLT1), from alanine to cysteine (A166C). A166C was expressed inXenopus laevisoocytes, and electrophysiological methods were used to assay function. The affinity for Na+was unchanged compared to that of hSGLT1, whereas the sugar affinity was reduced and sugar specificity was altered. There was a reduction in the turnover rate of the transporter, and in contrast to that of hSGLT1, the turnover rate depended on the sugar molecule. Exposure of A166C to MTSEA and MTSET, but not MTSES, abolished sugar transport. Accessibility of A166C to alkylating reagents was independent of protein conformation, indicating that the residue is always accessible from the extracellular surface. Sugar and phlorizin did not protect the residue from being alkylated, suggesting that residue 166 is not located in the sugar pathway. MTSEA, MTSET, and MTSES all changed the pre-steady-state kinetics of A166C, independent of pH, and sugars altered these kinetics. The inability of MTSEA-labeled A166C to transport sugar was reversed (with no major change in Na+and sugar affinity) if the positive charge on MTSEA was neutralized by increasing the external pH to 9.0. These studies suggest that the residue at position 166 is involved in the interaction between the Na+and sugar transport pathways.