Kuwahara M, Asai T, Sato K, Shinbo I, Terada Y, Marumo F, Sasaki S
Department of Homeostasis Medicine and Nephrology, School of Medicine, Tokyo Medical and Dental University, Japan.
Biochim Biophys Acta. 2000 Dec 15;1517(1):107-12. doi: 10.1016/s0167-4781(00)00268-2.
A genome project for the species Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein (MIP) family. We previously characterized one of these cDNAs known as C01G6.1. C01G6.1 was confirmed to be a water channel and newly designated as AQP-CE1 [Am. J. Physiol. 275 (1998) C1459-C1464]. In this paper, we examined the function of another MIP protein encoded by F40F9.9. This cDNA encodes a 274-amino acid protein showing a high sequence identity with mammalian aquaporin-8 (AQP8) water channel (35%) and d-TIP (34%), an AQP of Arabidopsis. The expression of F40F9.9 in Xenopus oocytes increased the osmotic water permeability (P(f)) 10.4-fold, and the activation energy for P(f) from Arrhenius plot was 4.7 kcal/mol, suggesting that F40F9.9 is a water channel (AQP-CE2). AQP-CE2 was not permeable to glycerol or urea. Oocyte P(f) was reversibly inhibited by 58% after an incubation with 0.3 mM HgCl(2). To identify the mercury-sensitive site, four individual cysteine residues in AQP-CE2 (at positions 47, 132, 149, 259) were altered to serine by site-directed mutagenesis. Of these mutants, only C132S had a P(f) similar to that of the wild-type together with an acquired mercury resistance, suggesting that Cys-132 is the mercury-sensitive site. Similar results were obtained by the mutation of Cys-132 to alanine (C132A). Replacement of Cys-132 with tryptophan decreased P(f) by 64%, but P(f) was still 2.5 times higher than that of the control. Cys-132 is located in the transmembrane helix 3, close to the transition to the extracellular loop C. These results suggest that the transmembrane helix 3, including Cys-132, might participate in the aqueous pore formation, or, alternatively, that Cys-132 might contribute to the construction of the AQP protein.
秀丽隐杆线虫的基因组计划已证明存在八个属于主要内在蛋白（MIP）家族的cDNA。我们之前对其中一个名为C01G6.1的cDNA进行了表征。C01G6.1被确认为水通道，并被重新命名为AQP-CE1[《美国生理学杂志》275(1998)C1459 - C1464]。在本文中，我们研究了由F40F9.9编码的另一种MIP蛋白的功能。该cDNA编码一种274个氨基酸的蛋白，与哺乳动物水通道蛋白8（AQP8）水通道（35%）和拟南芥的一种水通道蛋白d-TIP（34%）具有高度的序列同一性。F40F9.9在非洲爪蟾卵母细胞中的表达使渗透水通透性（P(f)）增加了10.4倍，并且根据阿伦尼乌斯曲线得出的P(f)的活化能为4.7千卡/摩尔,这表明F40F9.9是一种水通道（AQP-CE2）。AQP-CE2对甘油或尿素不通透。在用0.3 mM HgCl₂孵育后，卵母细胞的P(f)可逆性地被抑制了58%。为了确定汞敏感位点，通过定点诱变将AQP-CE2中的四个单个半胱氨酸残基（位于第47、132、149、259位）改变为丝氨酸。在这些突变体中，只有C132S具有与野生型相似的P(f)以及获得的汞抗性，这表明Cys-132是汞敏感位点。将Cys-132突变为丙氨酸（C132A）也得到了类似的结果。用色氨酸取代Cys-132使P(f)降低了64%，但P(f)仍比对照高2.5倍。Cys-132位于跨膜螺旋3中，靠近向细胞外环C的过渡处。这些结果表明，包括Cys-132在内的跨膜螺旋3可能参与水孔的形成，或者，Cys-132可能有助于AQP蛋白的构建。
