NBC3 expression in rabbit collecting duct: colocalization with vacuolar H+-ATPase.
NBC3 expression in rabbit collecting duct: colocalization with vacuolar H+-ATPase.
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DOI：
10.1152/ajprenal.1999.277.6.f974
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发表时间：
1999-12
期刊：
影响因子：
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通讯作者：
A. Pushkin;K. Yip;I. Clark;N. Abuladze;T. Kwon;S. Tsuruoka;G. Schwartz;S. Nielsen;I. Kurtz
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文献类型：
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作者：
A. Pushkin;K. Yip;I. Clark;N. Abuladze;T. Kwon;S. Tsuruoka;G. Schwartz;S. Nielsen;I. Kurtz
我们最近克隆并鉴定了一种独特的碳酸氢钠协同转运蛋白NBC 3，它与NBC家族的其他成员不同，是乙基异丙基氨氯吡咪（EIPA）可降解的，DIDS不敏感的，并且是电中性的（A. Pushkin，N.阿布拉泽岛Lee，D.纽曼，黄哲伦和我。克兹274：16569-16575，1999）。在本研究中，一个特定的多克隆抗肽羧基端抗体，NBC 3-C1，产生并用于确定NBC 3蛋白在兔肾脏中的表达模式。兔肾免疫印迹检测到约200 kDa的主带。兔肾冰冻切片的免疫细胞化学显示，在皮质和外髓集合管的闰细胞的顶膜的特异性染色。收集管中NBC 3蛋白表达的模式与用针对液泡H+-ATP酶31-kDa亚基的抗体染色的相同切片几乎相同。此外，NBC 3-C1抗体共免疫沉淀液泡H+-ATP酶31-kDa亚基。在外髓集合管（内条纹）的功能研究表明，A型闰细胞有一个顶端Na+依赖的碱转运，是EIPA可识别和DIDS不敏感。这些数据表明，NBC 3参与收集管中的H+/碱运输。NBC 3和液泡H+-ATP酶在A型闰细胞中的密切联系表明这两种转运蛋白之间存在潜在的结构/功能相互作用。
We have recently cloned and characterized a unique sodium bicarbonate cotransporter, NBC3, which unlike other members of the NBC family, is ethylisopropylamiloride (EIPA) inhibitable, DIDS insensitive, and electroneutral (A. Pushkin, N. Abuladze, I. Lee, D. Newman, J. Hwang, and I. Kurtz. J. Biol. Chem.274: 16569-16575, 1999). In the present study, a specific polyclonal antipeptide COOH-terminal antibody, NBC3-C1, was generated and used to determine the pattern of NBC3 protein expression in rabbit kidney. A major band of ∼200 kDa was detected on immunoblots of rabbit kidney. Immunocytochemistry of rabbit kidney frozen sections revealed specific staining of the apical membrane of intercalated cells in both the cortical and outer medullary collecting ducts. The pattern of NBC3 protein expression in the collecting duct was nearly identical to the same sections stained with an antibody against the vacuolar H+-ATPase 31-kDa subunit. In addition, the NBC3-C1 antibody coimmunoprecipitated the vacuolar H+-ATPase 31-kDa subunit. Functional studies in outer medullary collecting ducts (inner stripe) showed that type A intercalated cells have an apical Na+-dependent base transporter that is EIPA inhibitable and DIDS insensitive. The data suggest that NBC3 participates in H+/base transport in the collecting duct. The close association of NBC3 and the vacuolar H+-ATPase in type A intercalated cells suggests a potential structural/functional interaction between the two transporters.