Department of Molecular Oral Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima-shi, Tokushima 770-8504, Japan.
Biol Cell. 2011 Feb;103(2):69-86. doi: 10.1042/BC20100086.
AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0-12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly103 > Asp103) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291, G1081-G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild-type) animals.
Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP-AQP5s) and expressed in polarized MDCK-II (Madin-Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP-AQP5 being translocated less efficiently. Thapsigargin and H-89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP-AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co-localized with LAMP2 (lysosome-associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co-localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation.
Replacement of highly conserved hydrophobic Gly103 with strongly hydrophilic Asp103 in rat AQP5, though it did not affect water permeability, may possibly have resulted in less efficient membrane trafficking and increased lysosomal degradation, leading to its lower expression in the apical membrane of the acinar cells in the SMG.
水通道蛋白（AQPs）是几乎所有生物中表达的水通道蛋白。在哺乳动物中，迄今已鉴定出 13 种 AQP（AQP0-12）。已知 AQP5 主要表达在外分泌细胞中，包括唾液腺腺泡细胞。早些时候，在大鼠 AQP5 基因中发现了一种天然存在的点突变（G308A，甘氨酸 103 突变为天冬氨酸 103）[Murdiastuti、Purwanti、Karabasil、Li、Yao、Akamatsu、Kanamori 和 Hosoi（2006 年）美国生理学杂志 291，G1081-G1088]；在这种突变体中，刺激和非刺激条件下初始唾液分泌的速率低于野生型（wt）动物。
这里详细描述了突变体分子。我们使用非洲爪蟾卵母细胞系统证明，突变 AQP5 的水通透性几乎与 wt 分子相同。标记有 GFP（绿色荧光蛋白；GFP-AQP5）并在极化的 MDCK-II（犬肾二型）细胞中表达的突变和 wt AQP5 首先出现在细胞质中的囊泡结构中，并在培养过程中被转运到上质膜或顶膜，突变 GFP-AQP5 的转运效率较低。他普西卡丁和 H-89 都能诱导两种分子的体外转运，而秋水仙碱则抑制了这种活性；在用任何实验条件处理的细胞中，显示突变 GFP-AQP5 顶端定位的细胞比例均小于显示 wt 分子顶端定位的细胞比例。在突变的颌下腺（SMG）组织中，AQP5 在腺泡细胞顶膜中的定位大大减少。在突变腺体的腺泡细胞质中，AQP5 阳性的囊泡结构与 LAMP2（溶酶体相关膜蛋白 2）或组织蛋白酶 D 共定位，而在 wt 腺体中，这种共定位非常罕见，这表明突变分子大量进入溶酶体进行降解。
在大鼠 AQP5 中，高度保守的疏水性甘氨酸 103 被强亲水性天冬氨酸 103 取代，尽管这不会影响水通透性，但可能导致膜运输效率降低和溶酶体降解增加，导致其在 SMG 腺泡细胞顶膜中的表达降低。
