Panayotova-Heiermann M, Eskandari S, Turk E, Zampighi G A, Wright E M
Department of Physiology, UCLA Medical Center, Los Angeles, California 90095-1751, USA.
J Biol Chem. 1997 Aug 15;272(33):20324-7. doi: 10.1074/jbc.272.33.20324.
To test the hypothesis that the C-terminal half of the Na+/glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C5) coding for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in Xenopus oocytes. Expression and function of C5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C5 protein was synthesized in 20-fold higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased 2-fold after C5-cRNA injection compared with noninjected oocytes. The diameters of the C5 particles were heterogeneous (4.8 +/- 0.3, 7.1 +/- 1.2, and 10.3 +/- 0.8 nm) in contrast to the endogenous particles (7.6 +/- 1.2 nm). C5 increased the alpha-methyl-D-glucopyranoside (alphaMDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K0.5alphaMDG of 50 mM and a turnover of approximately 660 s-1. Influx was independent of Na+ with transport characteristics similar to those of SGLT1 in the absence of Na+: 1) selective (alphaMDG > D-glucose > D-galactose >> L-glucose approximately D-mannose), 2) inhibited by phloretin, KiPT = approximately 500 microM, and 3) insensitive to phlorizin. These results indicate that C5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC5 (the human equivalent of C5), hC4 (human SGLT1 amino acids 407-648, helices 10-13), and hN13 (amino acids 1-648, helices 1-13), further suggest that helices 10-13 form the sugar permeation pathway for SGLT1.
为了验证钠/葡萄糖共转运蛋白（SGLT1）的C端半段包含糖通透途径这一假说，编码兔SGLT1第407 - 662位氨基酸（第10 - 14个螺旋）的cDNA构建体（C5）在非洲爪蟾卵母细胞中表达。通过蛋白质印迹法、电子显微镜、放射性示踪法和电生理方法对C5的表达和功能进行追踪。C5蛋白的合成水平比SGLT1高20倍。与未注射的卵母细胞相比，注射C5 - cRNA后卵母细胞质膜原生质面的颗粒密度增加了2倍。与内源性颗粒（7.6±1.2 nm）不同，C5颗粒的直径具有异质性（4.8±0.3、7.1±1.2和10.3±0.8 nm）。C5使α - 甲基 - D - 吡喃葡萄糖苷（αMDG）的摄取量比未注射的卵母细胞增加了20倍，其表观K0.5αMDG为50 mM，周转数约为660 s-1。在没有Na+的情况下，内流不依赖于Na+，其转运特性与SGLT1相似：1）具有选择性（αMDG＞D - 葡萄糖＞D - 半乳糖＞＞L - 葡萄糖≈D - 甘露糖），2）被根皮素抑制，KiPT≈500 μM，3）对根皮苷不敏感。这些结果表明C5表现为一种特异性低亲和力葡萄糖单向转运体。对另外三个构建体hC5（C5的人类等同物）、hC4（人类SGLT1第407 - 648位氨基酸，第10 - 13个螺旋）和hN13（第1 - 648位氨基酸，第1 - 13个螺旋）的初步研究进一步表明，第10 - 13个螺旋构成了SGLT1的糖通透途径。
