Loo D D, Hirayama B A, Gallardo E M, Lam J T, Turk E, Wright E M
Department of Physiology, University of California Los Angeles School of Medicine, Center for the Health Sciences, Los Angeles, CA 90095-1751, USA.
Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7789-94. doi: 10.1073/pnas.95.13.7789.
The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady-state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady-state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.
协同转运蛋白跨细胞膜偶联其底物的机制尚不清楚。一个普遍提出的模型是，协同转运是由配体诱导的构象转变导致的，这种转变改变了配体结合位点从膜的一侧到另一侧的可及性。为了验证这个模型，我们测量了共价探针与置于钠/葡萄糖协同转运蛋白（SGLT1）假定的糖转运结构域中的一个半胱氨酸残基（Q457C）的可及性。突变蛋白Q457C能够转运糖，但在被甲硫基磺酸盐试剂烷基化后转运被消除。烷基化阻断了糖的转运但不影响糖的结合。Q457C对烷基化试剂的可及性需要外部的钠离子，并且被外部的糖和根皮苷阻断。可及性的电压依赖性与SGLT1的稳态前电荷移动直接相关。用罗丹明-6-马来酰亚胺标记的Q457C进行的电压跳跃实验表明，荧光变化的时间进程和水平紧密跟随稳态前电荷移动。我们得出结论，构象变化是钠和糖转运偶联的原因，并且Q457在SGLT1介导的糖转运中起关键作用。
