CORE--CELL CULTURE FACILITY

Na-K-Cl共转运体在跨上皮盐转运、细胞容量和电解质平衡中起着重要作用。肾脏共转运体NKCC2是哺乳动物肾上皮细胞在Henle环(TAL)粗大的升支中重吸收氯化钠的关键元件，在那里它是利尿剂如速尿的作用部位。因此，对NKCC2功能的充分了解将在许多电解质失衡疾病的诊断和治疗中发挥重要作用，尤其是高血压。最近的进展已经 阐明了共转运蛋白的三个选择性剪接变体之间的功能差异，展示了每个变体在钠和氯重吸收中的独特作用。本项目的目的是进一步了解哺乳动物肾脏中Na-K-Cl共转运体的功能机制，并了解该共转运体在肾功能调节中的意义。拟议的研究将使用重组NKCC2进行 在非洲爪哇卵母细胞和组织培养细胞以及小鼠肾脏中表达的蛋白质。具体地说：1)NKCC2的第二跨膜结构域的作用，进一步研究NKCC2的‘b’亚型所起的特殊作用，并研究跨膜结构域2和3之间高度保守的环的作用。2)通过突变N端特定的磷受体残基和绘制磷酸化残基的全谱来阐明磷酸化在NKCC2调控中的作用。3)将使用酵母双杂交和细胞表达方法来研究NKCC2与其调控和靶向伙伴的相互作用，特别关注候选调控激酶以及指导NKCCs顶端与基底侧向分选的机制。

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.