Na+-H+ Transport in Renal Apical Membranes

描述（由申请人提供）：肾近端小管转运蛋白NHE 3（钠氢交换器3）和Npt 2（磷酸钠协同转运蛋白IIa）的调节涉及PDZ蛋白NHERF-1和NHERF-2（钠氢交换器调节因子），其与这些转运蛋白结合并抑制调节转运活性的激素信号。本申请探索了NHERF蛋白彼此相互作用以及与其它PDZ蛋白相互作用以调节NHE 3、Npt 2和其它蛋白的活性和/或细胞表面表达的假设。为此，开发了NHERF-1（-/-）小鼠，这将证明在定义哺乳动物肾脏中NHERF的功能方面非常有价值，并为与人类疾病相关的电解质代谢缺陷提供了新的见解。 具体目标1是确定NHERF在细胞中定位的结构基础及其对肾靶点识别的影响。凝胶覆盖和下拉试验将用于确定NHERF-1磷酸化、二聚化和靶点（如NHE 3和Npt 2）识别所需的区域。 共表达，共免疫沉淀和共聚焦显微镜将用于研究NHERF磷酸化，定位，靶点识别和生理效应之间的联系。 具体目标2是确定NHERF亚型在NHE 3信号复合物调节中的作用。将在野生型（WT）和NHERF-1（-/-）缺失小鼠中使用体内微灌注、小管悬浮液和原代细胞培养物中的荧光测量以及分离的刷状缘膜囊泡（BBM）中的22 Na+摄取来确定NHE 3的急性和慢性激素调节。将使用免疫细胞化学、细胞表面生物素化和亚细胞器的蛋白质免疫印迹研究WT和NHERF-1缺失小鼠中的NHE 3转运。 具体目标3将定义NHERF-1在Npt 2的运输和活性中的作用。清除和平衡实验以及使用培养细胞和分离的BBM的研究将用于检查cAMP、激素如PTH和维生素D以及饮食中磷摄入量的改变对WT和NHERF-1（-/-）动物中Npt 2分布和活性的影响。

DESCRIPTION (provided by applicant): Regulation of renal proximal tubule transporters, NHE3 (sodium hydrogen exchanger 3) and Npt2 (sodium phosphate cotransporter Ila) involve the PDZ proteins NHERF-1 and NHERF-2 (sodium hydrogen exchanger regulatory factors) that bind to these transporters and transduce hormonal signals that regulate transport activity. The current application explores the hypothesis that the NHERF proteins interact with one another and with other PDZ proteins to regulate the activity and/or cell surface expression of NHE3, Npt2 and other proteins. To this end, a NHERF-1 (-/-) mouse was developed that will prove extremely valuable in defining the functions of NHERF in the mammalian kidney and providing new insights into the defects in electrolyte metabolism associated with human disease. Specific Aim 1 is to define the structural basis for NHERF localization in cells and its impact on recognition of renal targets. Gel overlays and pulldowns assays will be used to define the regions required for NHERF-1 phosphorylation, dimerization and recognition of targets such as NHE3 and Npt2. Coexpression, coimmunoprecipitation and confocal microscopy will be used to investigate the link between NHERF phosphorylation, localization, target recognition and physiologic effects. Specific Aim 2 is to define the role of NHERF isoforms in signal complex regulation of NHE3. Acute and chronic hormonal regulation of NHE3 will be determined in wild-type (WT) and NHERF-1 (-/-) null mice using in vivo microperfusion, fluorescence measurements in tubule suspensions and in primary cell cultures, and 22Na+uptake in isolated brush border membrane vesicles (BBM). NHE3 trafficking in WT and NHERF-1 null mice will be studied using immunocytochemistry, cell surface biotinylation and western immunoblotting of subcellular organelles. Specific Aim 3 will define the role of NHERF-1 in the trafficking and activity of Npt2. Clearance and balance experiments as well as studies using cultured cells and isolated BBM will be used to examine the effects of cAMP, hormones such as PTH and vitamin D and alterations in the dietary intake of phosphorus on Npt2 distribution and activity in WT and NHERF-1 (-/-) animals.