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.

钠-钾-氯协同转运蛋白在跨上皮盐转运和细胞容积及电解质平衡中起着重要作用。肾脏协同转运蛋白NKCC 2是跨亨利袢（TAL）厚升支中的哺乳动物肾上皮重吸收NaCl过程中的关键元件，在那里它是利尿剂（如呋塞米）的作用部位。因此，对NKCC 2功能的充分理解将在许多电解质失衡疾病，特别是高血压的诊断和治疗中具有核心重要性。最近的进展是 阐明了协同转运蛋白的三种可变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。本研究的目的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意义。将使用重组NKCC 2进行拟定研究 在非洲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。具体而言：1）NKCC 2的第二跨膜结构域的作用将被解决，进一步研究NKCC 2的“B”同种型所起的专门作用，并检查跨膜结构域2和3之间的高度保守环的作用。2)磷酸化在NKCC 2调控中的作用将通过突变N-末端的特异性磷酸受体残基和绘制磷酸化残基的全谱来阐明。3)将使用酵母双杂交和细胞表达方法检查NKCC 2与其调控和靶向伙伴的相互作用，特别注意候选调控激酶和指导NKCC的顶侧与基底侧分选的机制。