FUNCTION AND REGULATION OF NKCC2 IN THE MAMMALIAN KIDNEY

NKCC2 在哺乳动物肾脏中的功能和调节

• 批准号: 6725895
• 负责人: BLISS FORBUSH
• 金额: $ 18.67万
• 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/6725895
• 关键词: Xenopus oocyte; chimeric proteins; chloride ion; complementary DNA; fluorescence microscopy; immunofluorescence technique; ion transport; laboratory mouse; laboratory rabbit; laboratory rat; membrane permeability; membrane transport proteins; molecular cloning; phosphorylation; potassium ion; protein isoforms; protein localization; protein structure function; recombinant proteins; renal tubular transport; sodium ion; tissue /cell culture; yeast two hybrid system

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 rabbit and 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共转运蛋白在跨盐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。肾脏共转运蛋白NKCC2是NaCl重吸收过程中跨哺乳动物肾上皮的过程中的关键要素，在Henle（TAL）的较厚上升的升节中，它是源自源自雌二胺的利尿剂的作用位置。因此，对NKCC2功能的全面了解将在诊断和治疗许多电解质失衡疾病（尤其是高血压）中至关重要。最近的进展已有 阐明了共转运蛋白的三个剪接变体之间的功能差异，表明了Na和Cl重吸附中每个变体的独特作用。该项目的目的是进一步了解Na-k-Cl共转运蛋白在哺乳动物肾脏中的功能的基础，并了解共转运蛋白在调节肾功能方面的重要性。拟议的研究将使用重组NKCC2进行 蛋白质在异爪蟾卵母细胞和组织培养细胞以及兔和小鼠肾脏中表达。具体来说： 1）将解决NKCC2的第二个跨膜结构域的作用，进一步研究NKCC2的“ B”同工型的专业作用，并检查了NKCC2的“ B”同工型的作用，并检查了跨膜域2和3之间高度保守的循环的作用。绘制磷酸化的全光谱 残留物。 3）NKCC2与其调节和靶向伴侣的相互作用将使用酵母两杂交和细胞表达方法进行检查，特别注意候选调节性激酶以及指导NKCC的顶端与基底外侧分类的机械。