MOLECULAR BIOLOGY OF BRUSH BORDER TRANSPORT PROTEINS

有机溶质的主动转运，如糖、氨基酸和+D 原核生物和真核生物细胞膜上的小肽 通过Na和H+偶联转运蛋白进行。在过去的几年里， 克隆、测序和表征了几种哺乳动物转运蛋白。这些 包括肠和肾Na+偶联葡萄糖协同转运蛋白SGLTI 和SGLT 2，Na+和K+依赖性神经元和上皮细胞高亲和力 谷氨酸转运蛋白EAAC 1，肠H+偶联寡肽 转运蛋白PepT1及其肾脏亚型hPepT2，以及肾脏易化的 尿素转运蛋白UT2。本建议的重点是如何 向上的溶质迁移与电化学离子梯度有关。我 选择了Na +-偶联葡萄糖转运蛋白和H +-偶联葡萄糖转运蛋白。 寡肽转运蛋白作为模型来解决这个问题。最 研究这些蛋白质的有用系统已经在非洲爪蟾中表达 卵母细胞，随后进行摄取研究和电生理学实验。 SGLT 1的先前研究和PepT 1的初步实验 提供了关于特定运输机制的有趣信息 由这些蛋白质显示，这些蛋白质不同于EAAC 1的蛋白质， GABA转运蛋白GAT-1，但具有某些共同的原理。它 似乎很可能，结合转运蛋白修饰的研究， 通过基因工程，这些结果将产生一个整体的分子， 转运蛋白如何将上坡溶质转运与电化学 离子梯度 具体地说，我建议构建嵌合体， SGLT 1和SGLT 2，以便将功能属性分配给特定的 SGLT 1中的区域，并使用定点突变来定位 参与Na+偶联机制的单个氨基酸残基。 为了研究PepT1和hPepT2的H +-偶联机制，我将首先 基于电生理学研究构建动力学模型， 同位素通量测量来了解这些蛋白质的功能。我 然后将这些模型与SGLT 1、GAT-1和EAAC 1进行比较， 评估轮胎是否是这些运输的共同原则 流程.接下来，我将研究单个氨基酸的影响， PepT1与两个远亲同源序列保守， 转运体的转运功能。最后，我将阐明 PepT1在蛋白质消化的肠跨上皮转运中发挥作用 最终产品。这些研究的结果应该提供信息， 对我们理解活性溶质 运输工作，信息将是无价的， 三级结构的解释，当它变得可用。

Active transport of organic solutes such as sugars, amino acids an+d small peptides across cell membranes of prokaryotes and eukaryotes proceeds via Na and H+-coupled transporters. In the past few years I have cloned, sequenced and characterized several-mammalian transporters. These include the intestinal and renal Na+-coupled glucose cotransporters SGLTI and SGLT2, the Na+ and K+-dependent neuronal and epithelial high affinity glutamate transporter EAAC1, the intestinal H+-coupled oligopeptide transporter PepT1 and its renal isoform hPepT2, and the renal facilitated urea transporter UT2. The present proposal focuses on the question of how uphill solute transport is linked to electrochemical ion gradients. I have chosen the Na+-coupled glucose transporters and the H+-coupled oligopeptide transporter as models to address this question. The most useful system to study these proteins has been expression in Xenopus oocytes followed by uptake studies and electrophysiological experiments. Previous studies of SGLT1 and preliminary experiments of PepT1 have provided interesting information on specific transport mechanisms displayed by these proteins which are distinct from those of EAAC1 and the GABA transporter GAT-1, yet have certain principles in common. It seems likely that, in combination with studies of transporters modified by genetic engineering, these results will yield an overall molecular view of how transporters link uphill solute transport to electrochemical ion gradients. Specifically, I propose to construct chimeras between SGLT1 and SGLT2 in order to assign functional properties to specific regions in SGLT1 and to use site-directed mutagenesis to pinpoint individual amino acid residues involved-in the Na+-coupling mechanism. To study the H+-coupling mechanism of PepT1 and hPepT2 I will first construct kinetic models based on electrophysiological studies and - isotopic flux measurements to understand how these proteins function. I will then compare these models with those of SGLT1, GAT-1 and EAAC1 and evaluate whether tire are common principles underlying these transport processes. Next, I will study the effect of individual amino acids of PepT1 which are conserved with two distantly related homologous transporters on transporter function. Finally, I will elucidate the role PepT1 plays in intestinal trans-epithelial transport of protein digestion end-products. The results from these studies should provide information of general importance to our understanding of how active solute transporters work, information which will be invaluable to the interpretation of tertiary structures when it becomes available.