Fushimi K, Sasaki S, Yamamoto T, Hayashi M, Furukawa T, Uchida S, Kuwahara M, Ishibashi K, Kawasaki M, Kihara I
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
Am J Physiol. 1994 Oct;267(4 Pt 2):F573-82. doi: 10.1152/ajprenal.1994.267.4.F573.
Vasopressin-regulated water permeability of the kidney collecting duct is a key component of the urine concentration machinery. Recently, a cDNA for AQP-CD, the vasopressin-regulated water channel, initially reported as WCH-CD, has been isolated (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). AQP-CD was expressed in oocyte membrane using a Xenopus expression vector, and functional characteristics of AQP-CD were examined. Osmotic water permeability (Pf) of oocytes expressing AQP-CD was 138 +/- 19 microns/s (mean +/- SE), 12 times greater than the control (11 +/- 3 microns/s), 90% inhibited by 0.3 mM HgCl2, and weakly temperature dependent (energy of activation for Pf was 4.0 kcal/mol). Urea influx measured from 15-min [14C]urea uptake by oocytes injected with AQP-CD/expression vector 1 cRNA was 86 +/- 17% of the control. Two-electrode voltage-clamp experiments revealed insignificant ion conductance of AQP-CD. Immunoblots of membranes from rat kidney medulla and oocytes expressing AQP-CD using anti-AQP-CD COOH-terminal antibody showed a 29-kDa protein and 35- to 50-kDa high-molecular-mass forms. Immunohistochemistry showed apical and subapical localization of AQP-CD in the collecting duct principal cells. Our results indicated that AQP-CD is a 29-kDa protein, a selective water channel, distinct from a urea channel, and localized to the membranes of vasopressin-sensitive components in kidney collecting duct principal cells.
血管加压素调节的肾集合管水通透性是尿液浓缩机制的关键组成部分。最近，一种血管加压素调节的水通道AQP-CD的cDNA（最初报道为WCH-CD）已被分离出来（K. Fushimi、S. Uchida、Y. Hara、Y. Hirata、F. Marumo和S. Sasaki。《自然》伦敦361: 549 - 552, 1993）。使用非洲爪蟾表达载体在卵母细胞膜中表达AQP-CD，并检测了AQP-CD的功能特性。表达AQP-CD的卵母细胞的渗透水通透性（Pf）为138 +/- 19微米/秒（平均值 +/- 标准误），比对照（11 +/- 3微米/秒）高12倍，0.3 mM HgCl2可抑制90%，且对温度的依赖性较弱（Pf的活化能为4.0千卡/摩尔）。通过注射AQP-CD/表达载体1 cRNA的卵母细胞15分钟[14C]尿素摄取量测得的尿素流入量为对照的86 +/- 17%。双电极电压钳实验显示AQP-CD的离子电导不显著。使用抗AQP-CD羧基末端抗体对大鼠肾髓质和表达AQP-CD的卵母细胞膜进行免疫印迹，显示出一种29 kDa的蛋白质和35至50 kDa的高分子质量形式。免疫组织化学显示AQP-CD在集合管主细胞的顶端和顶端下定位。我们的结果表明，AQP-CD是一种29 kDa的蛋白质，是一种选择性水通道，与尿素通道不同，定位于肾集合管主细胞中血管加压素敏感成分的膜上。
