Vayro S, Lo B, Silverman M
Medical Research Council (M.R.C.) Membrane Biology Group, Department of Medicine, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8 Canada.
Biochem J. 1998 May 15;332 ( Pt 1)(Pt 1):119-25. doi: 10.1042/bj3320119.
We have exploited two mutants of the rabbit intestinal Na+/glucose carrier SGLT1 to explore the structure/function relationship of this Na+/glucose transporter in COS-7 cells. A functional N-terminal myc-epitope-tagged SGLT1 protein was constructed and used to determine the plasma-membrane localization of SGLT1. The kinetic and specificity characteristics of the myc-tagged SGLT1 mutant were identical with those of wild-type SGLT1. Immunogold labelling and electron microscopy confirmed the topology of the N-terminal region to be extracellular. Expression of the SGLT1 A166C mutant in these cells showed diminished levels of Na+-dependent alpha-methyl-d-glucopyranoside transport activity compared with wild-type SGLT1. For SGLT1 A166C, Vmax was 0.92+/-0.08 nmol/min per mg of protein and Km was 0.98+/-0.13 mM; for wild-type SGLT1, Vmax was 1.98+/-0.47 nmol/min per mg of protein and Km was 0.36+/-0.16 mM. Significantly, phlorrhizin (phloridzin) binding experiments confirmed equal expression of Na+-dependent high-affinity phlorrhizin binding to COS-7 cells expressing SGLT1 A166C or wild-type SGLT1 (Bmax 1.55+/-0.18 and 1.69+/-0.57 pmol/mg of protein respectively); Kd values were 0.46+/-0.15 and 0.51+/-0.11 microM for SGLT1 A166C and wild-type SGLT1 respectively. The specificity of sugar interaction was unchanged by the A166C mutation. We conclude that the replacement of an alanine residue by cysteine at position 166 has a profound effect on transporter function, resulting in a decrease in transporter turnover rate by a factor of 2. Taken as a whole the functional changes observed by SGLT1 A166C are most consistent with the mutation having caused an altered Na+ interaction with the transporter.
我们利用兔肠道Na⁺/葡萄糖载体SGLT1的两个突变体,在COS-7细胞中探究该Na⁺/葡萄糖转运体的结构/功能关系。构建了一个带有功能性N端myc表位标签的SGLT1蛋白,并用于确定SGLT1在质膜上的定位。带有myc标签的SGLT1突变体的动力学和特异性特征与野生型SGLT1相同。免疫金标记和电子显微镜证实N端区域的拓扑结构为胞外。与野生型SGLT1相比,这些细胞中SGLT1 A166C突变体的表达显示出Na⁺依赖性α-甲基-D-吡喃葡萄糖苷转运活性水平降低。对于SGLT1 A166C,每毫克蛋白的Vmax为0.92±0.08 nmol/分钟,Km为0.98±0.13 mM;对于野生型SGLT1,每毫克蛋白的Vmax为1.98±0.47 nmol/分钟且Km为0.36±0.16 mM。值得注意的是,根皮素(根皮苷)结合实验证实,在表达SGLT1 A166C或野生型SGLT1的COS-7细胞中,Na⁺依赖性高亲和力根皮素结合的表达量相等(Bmax分别为1.55±0.18和1.69±0.57 pmol/毫克蛋白);SGLT1 A166C和野生型SGLT1的Kd值分别为0.46±0.15和0.51±0.11 μM。糖相互作用的特异性未因A166C突变而改变。我们得出结论,第166位的丙氨酸残基被半胱氨酸取代对转运体功能有深远影响,导致转运体周转率降低了2倍。总体而言,SGLT1 A166C观察到的功能变化最符合该突变导致Na⁺与转运体相互作用改变的情况。