Wright E M, Loo D D, Panayotova-Heiermann M, Lostao M P, Hirayama B H, Mackenzie B, Boorer K, Zampighi G
Department of Physiology, UCLA School of Medicine 90024-1751.
J Exp Biol. 1994 Nov;196:197-212. doi: 10.1242/jeb.196.1.197.
Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps. The facilitative and H(+)-driven sugar transporters belong to a gene family with a distinctive secondary structure profile. The Na(+)-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na(+)- and H(+)-driven sugar cotransporters share common transport mechanisms. To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques. For SGLT1, we have developed a six-state ordered model that accounts for the experimental data. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural studies have confirmed these estimates of SGLT1 density. (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues.(ABSTRACT TRUNCATED AT 250 WORDS)
原核生物和真核生物中的糖转运蛋白属于一个包含12个跨膜α螺旋的膜蛋白大家族。它们分为两类：一类是易化型（单向转运体），另一类是浓缩型（协同转运体或同向转运体）。浓缩型转运体由离子泵产生和维持的H⁺或Na⁺梯度提供能量。易化型和H⁺驱动的糖转运蛋白属于一个具有独特二级结构特征的基因家族。Na⁺驱动的转运蛋白属于一个单独的小基因家族，在一级或二级结构水平上均无同源性。Na⁺和H⁺驱动的糖协同转运体可能具有共同的转运机制。为了探究这些机制，我们在非洲爪蟾卵母细胞中表达了克隆的真核生物Na⁺/糖协同转运体（SGLT），并使用双电极电压钳技术测量了糖转运的动力学。对于SGLT1，我们建立了一个六态有序模型来解释实验数据。为了验证该模型，我们进行了以下实验。（i）我们使用电压阶跃技术测量了SGLT1的预稳态动力学。在无糖情况下，SGLT1表现出瞬时载体电流，反映了蛋白质的电压依赖性构象变化。载体电流的时间常数给出了构象变化速率常数的估计值，而电荷移动（瞬时电流的积分）给出了质膜中SGLT1蛋白数量和价态的估计值。超微结构研究证实了这些SGLT1密度的估计值。（ii）我们通过对选定残基进行定点诱变来干扰协同转运体的动力学。（摘要截短于250字）
