MOLECULAR PHYSIOLOGY OF BAND 3-LIKE PROTEINS OF KIDNEY

肾脏带 3 样蛋白的分子生理学

• 批准号: 3244860
• 负责人: SETH Leo ALPER
• 金额: $ 20.11万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-15 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3244860
• 关键词: Xenopus oocyte; acidity /alkalinity; band 3 protein; binding proteins; calcium flux; cell growth regulation; chimeric proteins; chloride channels; clone cells; cytoskeletal proteins; gene deletion mutation; intracellular transport; kidney cell; protein structure function; site directed mutagenesis; tissue /cell culture; transfection; transport proteins

This proposal forms the center of the applicant's long-term objectives. They are to elucidate the structure and function of band 3-related proteins of the kidney, and to define their roles in normal and pathologic renal physiology at the levels of the individual cell, the local epithelium, and the entire kidney. These objectives, as met through the proposal's specific aims, will contribute to an understanding of how the renal tubular epithelial cell uses mechanisms of pHi, volume, and intracellular solute regulation present in most or all cells to modify the composition of the urine, thereby regulating pH, volume, and solute concentrations of the entire body. Towards these ends, the specific aims are to: 1. Characterize by isotopic flux studies the anion transport properties of heterologous KB3/AE1 and B3RP/AE2 expressed in Xenopus oocytes. 2. Characterize by fluorometric pHi studies the anion transport properties of heterologous KB3/AE1 and B3RP/AE2 expressed transiently in COS cells, using novel methods for supravital identification of the transfected cells. 3. Define by construction of deletions and hybrid proteins and by site-directed mutagenesis the amino acid residues of KB3/AE1 and of B3RP/AE2 responsible for ion binding and/or translocation, for pHi sensitivity of transport, and for binding of cytoskeletal proteins. 4. Develop a epithelial and nonepithelial cell lines which stably overexpress or underexpress KB3/AE1 or B3RP/AE2, or their subdomains. 5. Compare pHi regulation in the above cell lines and their parental lines. Test the hypothesis that B3RP/AE protein expression can suppress cell proliferation and/or transformation by lowering pHi. 6. Test the hypothesis that B3RP/AE protein expression is required for acute regulatory volume increase (RVI). 7. Test the hypothesis that overexpression of N-terminal cytoplasmic domains of B3RP/AE proteins will disrupt normal cytoskeletal assembly and/or polarization of plasma membrane in MDCK cells. Fulfillment of the specific aims should enhance our understanding of renal acid/base handling, concentration mechanisms, and Na+ reabsorption, and of the diverse disorders of renal cytoskeletal organization. It will provide clues to the nature of progressive renal disease, as embodied by the question of why renal epithelial cells respond to proliferative stimuli only by hypertrophy and not by hyperplasia. Finally, it will provide a avenues for rational design of new diuretic drugs, and for new cytoprotection protocols for organs at risk of ischemic damage.

该提案构成了申请人长期 目标. 目的是阐明能带的结构和功能 3-肾脏相关蛋白，并确定其在正常 以及在单个细胞水平上的病理性肾脏生理学， 局部上皮和整个肾脏。 这些目标， 通过该提案的具体目标，将有助于 了解肾小管上皮细胞如何利用机制 pHi，体积和细胞内溶质调节存在于大多数或 所有的细胞来改变尿液的组成，从而调节 整个身体的pH值、体积和溶质浓度。 朝向 为实现这些目标，具体目标是： 1. 通过同位素通量表征研究了阴离子的迁移 表达的异源KB 3/AE 1和B3 RP/AE 2的性质 非洲爪蟾卵母细胞。 2. 通过荧光pHi表征研究阴离子转运 表达的异源KB 3/AE 1和B3 RP/AE 2的性质 瞬时在COS细胞，使用新的方法， 鉴定转染的细胞。 3. 通过构建缺失和杂合蛋白来定义， 通过定点诱变， KB 3/AE 1和B3 RP/AE 2负责离子结合和/或 易位，用于转运的pHi敏感性，以及用于 细胞骨架蛋白的结合。 4. 开发上皮和非上皮细胞系， 稳定过表达或低表达KB 3/AE 1或B3 RP/AE 2，或 他们的子域。 5. 比较上述细胞系及其细胞系中的pHi调节， 父母线 检验B3 RP/AE蛋白 表达可以抑制细胞增殖和/或 通过降低pHi进行转化。 6. 检验B3 RP/AE蛋白表达是 急性调节性血容量增加（RVI）。 7. 检验N-末端的过度表达 B3 RP/AE蛋白质的胞质结构域将破坏正常的 细胞骨架组装和/或质膜极化 在 MDCK细胞。 具体目标的实现应加强我们对以下方面的理解： 肾脏酸/碱处理、浓缩机制和Na+ 重吸收，以及各种肾脏细胞骨架疾病 organization. 它将为进步的本质提供线索 肾脏疾病，具体表现为为什么肾上皮细胞 细胞对增殖刺激的反应仅为肥大，而不是 增生 最后，为合理设计提供了途径 新的利尿药物，以及新的器官细胞保护方案 有缺血性损伤的危险