CLONING AND EXPRESSION OF AQP3, A WATER CHANNEL FROM THE MEDULLARY COLLECTING DUCT OF RAT-KIDNEY
CLONING AND EXPRESSION OF AQP3, A WATER CHANNEL FROM THE MEDULLARY COLLECTING DUCT OF RAT-KIDNEY
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DOI：
10.1073/pnas.91.23.10997
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发表时间：
1994-11-08
影响因子：
11.1
通讯作者：
FRINDT, G
中科院分区：
文献类型：
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作者：
ECHEVARRIA, M;WINDHAGER, EE;FRINDT, G
内髓集合管的末端部分表现出高度的水渗透性，其不依赖于细胞内cAMP的增加，并且不被已知的肾上皮水通道CHIP 28（28-kDa通道形成整合蛋白）和WCH-CD（集合管水通道蛋白）的活性所解释。从大鼠肾乳头mRNA开始，用简并引物进行逆转录PCR，假设推定的通道是主要内在蛋白（MIP）家族蛋白质的成员。鉴定了一个cDNA片段，并用于筛选大鼠肾脏cDNA文库。分离到一个1.9kb的cDNA克隆。该基因的开放阅读框为876 bp，编码292个氨基酸，M（r）为31，431。水通道蛋白3（AQP 3; 31.4-kDa水通道蛋白）是MIP家族的新成员。北方印迹分析显示，肾髓质中存在一个约1.9 kb的AQP 3单一转录本，主要存在于内髓质中。原位杂交结果显示，在延髓集合管细胞中发现了丰富的信息。将AQP 3互补RNA注入非洲爪蟾卵母细胞，可显著增加卵母细胞的渗透水通透性。这种渗透性的活化能为3.0千卡/摩尔（1卡= 4.184 J），它是完全封闭的1 mM的对氯汞苯磺酸盐，这种抑制被逆转的5 mM二硫苏糖醇。cAMP不增加这种水渗透性。AQP 3不允许单价离子（Na、K、Cl）通过;然而，它对尿素具有轻微渗透性。本研究表明，存在一个额外的水通道，AQP 3，在髓质集合管上皮细胞。
The terminal part of the inner medullary collecting duct exhibits a high degree of water permeability that is independent of increased intracellular cAMP and not accounted for by the activity of the known renal epithelial water channels CHIP28 (28-kDa channel-forming integral protein) and WCH-CD (collecting duct water channel protein). Starting with rat kidney papilla mRNA, reverse transcription PCR was performed with degenerate primers assuming that the putative channel would be a member of the major intrinsic protein (MIP) family of proteins. A cDNA fragment was identified and used to screen a rat kidney cDNA library. A 1.9-kb cDNA clone was isolated. The open reading frame of 876 bp coded for a protein of 292 amino acids (M(r) 31,431). Aquaporin 3 (AQP3; 31.4-kDa water channel protein) is a newly discovered member of the MIP family. Northern blot analysis showed a single transcript for AQP3 of approximate to 1.9 kb present in the renal medulla, predominantly in the inner medulla. With in situ hybridization, abundant message was found in the cells of the medullary collecting ducts. Injection of the complementary RNA of AQP3 into Xenopus oocytes markedly increased the osmotic water permeability. This permeability had an energy of activation of 3.0 kcal/mol (1 cal = 4.184 J), it was fully blocked by 1 mM p-chloromercuriphenylsulfonate, and this inhibition was reversed by 5 mM dithiothreitol. cAMP did not increase this water permeability. AQP3 did not permit passage of monovalent ions (Na, K, Cl); however, it is slightly permeable to urea. The present study demonstrates the existence of an additional water channel, AQP3, in epithelial cells of the medullary collecting duct.