MOLECULAR BIOLOGY OF BRUSH BORDER TRANSPORT PROTEINS

糖、氨基酸等有机溶质的主动运输 穿透原核生物和真核生物细胞膜的小肽 通过Na+和H+偶联转运体获得。在过去的几年里，我已经 克隆、测序并鉴定了几种哺乳动物转运蛋白。这些 包括肠道和肾脏的钠离子偶联葡萄糖共转运体SGLTI 和SGLT2，Na+和K+依赖的神经元和上皮的高亲和力 肠道H+偶联寡肽谷氨酸转运体EAAC1 转运蛋白PepT1及其肾脏异构体hPepT2与肾脏易化 尿素运输车UT2。本提案的重点是如何 上坡溶质运移与电化学离子梯度有关。我 选择了Na+偶联的葡萄糖转运蛋白和H+偶联的葡萄糖转运蛋白 寡肽转运体作为模型来解决这个问题。最多的 研究这些蛋白的有用系统已在非洲爪蛙中表达 卵母细胞随后进行摄取研究和电生理实验。 先前对SGLT1的研究和PepT1的初步实验已经 提供了有关特定传输机制的有趣信息 由这些不同于EAAC1和EAAC1的蛋白显示 GABA转运体GAT-1，但有某些共同的原则。它 似乎有可能，结合对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 运载器如何将上坡溶质运移与电化学联系起来 离子梯度。具体地说，我建议构建嵌合体 SGLT1和SGLT2，以便将功能属性分配给特定 并使用定点突变技术来精确定位 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 根据电生理研究建立动力学模型-- 同位素通量测量以了解这些蛋白质是如何发挥作用的。我 然后将这些模型与SGLT1、GAT-1和EAAC1进行比较，并 评估轮胎是否是这些运输的共同原则 流程。接下来，我将研究单独的氨基酸对 与两个远亲同源序列保守的PepT1 传送器上的传送器功能。最后，我将阐明这一角色 PepT1在蛋白质消化的肠道跨上皮转运中的作用 最终产品。这些研究的结果应该会提供信息 对我们理解溶质如何活泼具有普遍重要性 传送员的工作，这些信息对 当三级结构可用时对其进行解释。

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.