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共转运体在上皮盐转运和细胞体积电解质平衡中起着至关重要的作用。肾共转运蛋白NKCC2是哺乳动物肾上皮在亨氏袢（TAL）厚升肢上盐重吸收过程中的一个关键因素，它是利尿剂如速尿的作用部位。因此，充分了解NKCC2的功能将对许多电解质失衡疾病的诊断和治疗至关重要，特别是高血压。最近的进展