RELAXATION KINETICS OF THE NA+/GLUCOSE COTRANSPORTER
RELAXATION KINETICS OF THE NA+/GLUCOSE COTRANSPORTER
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DOI：
10.1073/pnas.90.12.5767
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发表时间：
1993-06-15
影响因子：
11.1
通讯作者：
WRIGHT, EM
中科院分区：
文献类型：
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作者：
LOO, DDF;HAZAMA, A;WRIGHT, EM
一类重要的整合膜蛋白，共转运蛋白，将溶质转运与电化学电位梯度偶联;例如，Na+/葡萄糖协同转运蛋白利用Na+电化学电位梯度在细胞中积累糖。到目前为止，共转运蛋白的动力学分析大多局限于稳态参数。在这项研究中，我们研究了前稳态动力学Na+/葡萄糖共转运。克隆的人转运蛋白（hSGLT 1）在非洲爪蟾卵母细胞中表达，电压钳技术用于监测膜电位阶跃变化后的电流瞬变。瞬态表现出电压依赖性时间常数（tau）范围在2和10 ms之间。电荷移动Q符合玻尔兹曼关系，最大电荷Q（max）几乎等于20 nC，表观价态z为1，50% Q（max）时电位V0.5为-39 mV。 将外部Na+从100 mM降低到10 mM，Q（max）降低了40%，V0.5从-39 mV偏移到-70 mV，对z没有影响，并降低了tau的电压依赖性。 Q（max）与温度无关，但tau依赖于温度（在-50 mV下，温度升高10 ℃，tau增加几乎等于2.5倍）。添加糖或根皮苷降低Q（max）。对hSGLT 1预稳态动力学的分析表明，在不存在糖的情况下，膜电位一步后的电荷转移是由于反应循环中的两个步骤：Na+结合/解离（30%）和蛋白质在膜场中的重定向（70%）。速率限制步骤似乎是Na+结合/解离。Q（max）提供了转运蛋白密度的量度（几乎等于10（4）/mum 2）。电荷转移测量提供了深入了解部分反应的Na+/葡萄糖协同转运蛋白，并结合基因工程的蛋白质，提供了一个强大的工具，研究运输机制。
An important class of integral membrane proteins, cotransporters, couple solute transport to electrochemical potential gradients; e.g., the Na+/glucose cotransporter uses the Na+ electrochemical potential gradient to accumulate sugar in cells. So far, kinetic analysis of cotransporters has mostly been limited to steady-state parameters. In this study, we have examined pre-steady-state kinetics of Na+/glucose cotransport. The cloned human transporter (hSGLT1) was expressed in Xenopus oocytes, and voltage-clamp techniques were used to monitor current transients after step changes in membrane potential. Transients exhibited a voltage-dependent time constant (tau) ranging between 2 and 10 ms. The charge movement Q was fitted to a Boltzmann relation with maximal charge Q(max) of almost-equal-to 20 nC, apparent valence z of 1, and potential V0.5 of -39 mV for 50% Q(max). Lowering external Na+ from 100 to 10 mM reduced Q(max) 40%, shifted V0.5 from -39 to -70 mV, had no effect on z, and reduced the voltage dependence of tau. Q(max) was independent of, but tau was dependent on, temperature (a 10-degrees-C increase increased tau by a factor of almost-equal-to 2.5 at -50 mV). Addition of sugar or phlorizin reduced Q(max). Analyses of hSGLT1 pre-steady-state kinetics indicate that charge transfer upon a step of membrane potential in the absence of sugar is due to two steps in the reaction cycle: Na+ binding/dissociation (30%) and reorientation of the protein in the membrane field (70%). The rate-limiting step appears to be Na+ binding/dissociation. Q(max) provides a measure of transporter density (almost-equal-to 10(4)/mum2). Charge transfer measurements give insight into the partial reactions of the Na+/glucose cotransporter, and, combined with genetic engineering of the protein, provide a powerful tool for studying transport mechanisms.