Chen X Z, Coady M J, Jackson F, Berteloot A, Lapointe J Y
Département de Physique, Université de Montréal, Québec, Canada.
Biophys J. 1995 Dec;69(6):2405-14. doi: 10.1016/S0006-3495(95)80110-4.
Phlorizin-sensitive currents mediated by a Na-glucose cotransporter were measured using intact or internally perfused Xenopus laevis oocytes expressing human SGLT1 cDNA. Using a two-microelectrode voltage clamp technique, measured reversal potentials (Vr) at high external alpha-methylglucose (alpha MG) concentrations were linearly related to In[alpha MG]o, and the observed slope of 26.1 +/- 0.8 mV/decade indicated a coupling ratio of 2.25 +/- 0.07 Na ions per alpha MG molecule. As [alpha MG]o decreased below 0.1 mM, Vr was no longer a linear function of In[alpha MG]o, in accordance with the suggested capacity of SGLT1 to carry Na in the absence of sugar (the "Na leak"). A generalized kinetic model for SGLT1 transport introduces a new parameter, Kc, which corresponds to the [alpha MG]o at which the Na leak is equal in magnitude to the coupled Na-alpha MG flux. Using this kinetic model, the curve of Vr as a function of In[alpha MG]o could be fitted over the entire range of [alpha MG]o if Kc is adjusted to 40 +/- 12 microM. Experiments using internally perfused oocytes revealed a number of previously unknown facets of SGLT1 transport. In the bilateral absence of alpha MG, the phlorizin-sensitive Na leak demonstrated a strong inward rectification. The affinity of alpha MG for its internal site was low; the Km was estimated to be between 25 and 50 mM, an order of magnitude higher than that found for the extracellular site. Furthermore, Vr determinations at varying alpha MG concentrations indicate a transport stoichiometry of 2 Na ions per alpha MG molecule: the slope of Vr versus In[alpha MG]o averaged 30.0 +/- 0.7 mV/decade (corresponding to a stoichiometry of 1.96 +/- 0.04 Na ions per alpha MG molecule) whenever [alpha MG]o was higher than 0.1 mM. These direct observations firmly establish that Na ions can utilize the SGLT1 protein to cross the membrane either alone or in a coupled manner with a stoichiometry of 2 Na ions per sugar, molecule.
利用表达人SGLT1 cDNA的完整或内部灌注的非洲爪蟾卵母细胞，测量由钠 - 葡萄糖共转运体介导的根皮苷敏感电流。采用双微电极电压钳技术，在高细胞外α - 甲基葡萄糖（α - MG）浓度下测得的反转电位（Vr）与ln[α - MG]o呈线性关系，观察到的斜率为26.1±0.8 mV/十倍浓度，表明每α - MG分子耦合2.25±0.07个钠离子。当[α - MG]o降至0.1 mM以下时，Vr不再是ln[α - MG]o的线性函数，这与SGLT1在无糖情况下转运钠的能力（“钠泄漏”）一致。SGLT1转运的广义动力学模型引入了一个新参数Kc，它对应于钠泄漏量与耦合的钠 - α - MG通量大小相等时的[α - MG]o。使用该动力学模型，如果将Kc调整为40±12 μM，则Vr作为ln[α - MG]o函数的曲线可以在整个[α - MG]o范围内拟合。使用内部灌注卵母细胞的实验揭示了SGLT1转运许多以前未知的方面。在双侧不存在α - MG的情况下，根皮苷敏感的钠泄漏表现出强烈的内向整流。α - MG对其内部位点的亲和力较低；Km估计在25至50 mM之间，比细胞外位点的Km高一个数量级。此外，在不同α - MG浓度下测定Vr表明，每α - MG分子的转运化学计量为2个钠离子：只要[α - MG]o高于0.1 mM，Vr与ln[α - MG]o的斜率平均为30.0±0.7 mV/十倍浓度（对应于每α - MG分子1.96±0.04个钠离子的化学计量）。这些直接观察结果确凿地证明，钠离子可以利用SGLT1蛋白单独或以每糖分子2个钠离子的化学计量耦合方式穿过膜。
