CORE--CELL CULTURE FACILITY

核心--细胞培养设施

• 批准号: 6725905
• 负责人: BLISS FORBUSH
• 金额: $ 8.21万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6725905
• 关键词: biomedical facility; membrane transport proteins; renal tubular transport; tissue /cell culture

The Na-K-Cl cotransporter plays a vital role in transepithelial salt transport and cellular volume and electrolyte balance. The renal cotransporter, NKCC2, is a key element in the process of NaCl reabsorption across the mammalian renal epithelium in the thick ascending limb of the loop of Henle (TAL), where it is the site of action of diuretics such as furosemide. A full understanding of NKCC2 function will thus be of central importance in the diagnosis and treatment of many diseases of electrolyte imbalance, particularly hypertension. Recent progress has elucidated the functional differences among three alternatively spliced variants of the cotransporter, demonstrating a unique role for each in Na and Cl reabsorption. The goal of this project is to further understand the mechanisms that underlie the function of the Na-K-Cl cotransporter in the mammalian kidney, and to understand the significance of the cotransporter in regulation of renal function. The proposed studies will be carried out with recombinant NKCC2 protein expressed in Xenopus oocytes and tissue culture cells as well as with mouse kidney. Specifically: 1) The role of the second transmembrane domain of NKCC2 will be addressed, further investigating the specialized role played by the 'b' isoform of NKCC2, and examining the role of the highly-conserved loop between transmembrane domains 2 and 3. 2) The role of phosphorylation in the regulation of NKCC2 will be elucidated by mutating specific phosphoacceptor residues in the N-terminus and by mapping the full spectrum of phosphorylated residues. 3) The interaction of NKCC2 with its regulatory and targeting partners will be examined using yeast two-hybrid and cell expression approaches, with particular attention to candidate regulatory kinases and to the machinery that directs apical vs. basolateral sorting of the NKCCs.

Na-K-Cl 协同转运蛋白在跨上皮盐转运以及细胞体积和电解质平衡中起着至关重要的作用。肾协同转运蛋白 NKCC2 是哺乳动物肾上皮在亨利袢厚升肢 (TAL) 重吸收 NaCl 过程中的关键元件，也是速尿等利尿剂的作用部位。因此，充分了解 NKCC2 功能对于许多电解质失衡疾病（特别是高血压）的诊断和治疗至关重要。最近的进展有 阐明了协同转运蛋白的三种选择性剪接变体之间的功能差异，证明了每种变体在 Na 和 Cl 重吸收中的独特作用。该项目的目标是进一步了解哺乳动物肾脏中Na-K-Cl协同转运蛋白的功能机制，并了解协同转运蛋白在肾功能调节中的重要性。拟议的研究将使用重组 NKCC2 进行 爪蟾卵母细胞和组织培养细胞以及小鼠肾脏中表达的蛋白质。具体来说： 1) 将讨论 NKCC2 第二个跨膜结构域的作用，进一步研究 NKCC2 的“b”亚型所发挥的专门作用，并检查跨膜结构域 2 和 3 之间高度保守的环的作用。2 ) 磷酸化在 NKCC2 调节中的作用将通过突变 N 末端的特定磷酸受体残基并绘制全谱磷酸化残基。 3) 将使用酵母双杂交和细胞表达方法检查NKCC2与其调节和靶向伙伴的相互作用，特别注意候选调节激酶以及指导NKCC的顶端与基底外侧分选的机制。