Kuwahara M, Shinbo I, Sato K, Terada Y, Marumo F, Sasaki S
Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, Japan.
Biochemistry. 1999 Dec 7;38(49):16340-6. doi: 10.1021/bi9916776.
Aquaporin-2 (AQP2), a vasopressin-regulated water channel, plays a major role in urinary concentration. AQP2 and the major intrinsic protein (MIP) of lens fiber are highly homologous (58% amino acid identity) and share a topology of six transmembrane helices connected by five loops (loops A-E). Despite the similarities of these proteins, however, the water channel activity of AQP2 is much higher than that of MIP. To determine the site responsible for this gain of activity in AQP2, several parts of MIP were replaced with the corresponding parts of AQP2. When expressed in Xenopus oocytes, the osmotic water permeability (P(f)) of MIP and AQP2 was 48 and 245 x 10(-)(4) cm/s, respectively. Substitutions in loops B-D failed to increase P(f), whereas substitution of loop E significantly increased P(f) 1.5-fold. A similar increase in P(f) was observed with the substitution of the front half of loop E. P(f) measurements taken in a yeast vesicle expression system also confirmed that loop E had a complementary effect, whereas loops B-D did not. However, P(f) values of the loop E chimeras were only approximately 30% of that of AQP2. Simultaneous exchanges of loop E and a distal half of transmembrane helix 5 just proximal to loop E increased P(f) to the level of that of AQP2. Replacement of helix 5 alone stimulated P(f) 2.7-fold. Conversely, P(f) was decreased by 73% when helix 5 of AQP2 was replaced with that of MIP. Moreover, P(f) was stimulated 2.6- and 3.3-fold after helix 5 of AQP1 and AQP4 was spliced into MIP, respectively. Our findings suggested that the distal half of helix 5 is necessary for maximum water channel activity in AQP. We speculate that this portion contributes to the formation of the aqueous pore and the determination of the flux rate.
水通道蛋白2（AQP2）是一种受血管加压素调节的水通道，在尿液浓缩过程中起主要作用。AQP2与晶状体纤维的主要内在蛋白（MIP）高度同源（氨基酸序列一致性为58%），并且具有由五个环（A - E环）连接的六个跨膜螺旋的拓扑结构。然而，尽管这些蛋白质存在相似性，但AQP2的水通道活性远高于MIP。为了确定AQP2中导致这种活性增加的位点，将MIP的几个部分替换为AQP2的相应部分。当在非洲爪蟾卵母细胞中表达时，MIP和AQP2的渗透水通透性（Pf）分别为48和245×10⁻⁴ cm/s。B - D环的替换未能增加Pf，而E环的替换使Pf显著增加了1.5倍。用E环前半部分进行替换时也观察到了类似的Pf增加。在酵母囊泡表达系统中进行的Pf测量也证实E环具有互补作用，而B - D环则没有。然而，E环嵌合体的Pf值仅约为AQP2的30%。同时交换E环和紧邻E环的跨膜螺旋5的后半部分可使Pf增加到AQP2的水平。单独替换螺旋5可使Pf增加2.7倍。相反，当AQP2的螺旋5被MIP的螺旋5替换时，Pf降低了73%。此外，将AQP1和AQP4的螺旋5分别拼接到MIP中后，Pf分别增加了2.6倍和3.3倍。我们的研究结果表明，螺旋5的后半部分对于AQP中最大水通道活性是必需的。我们推测这部分有助于水孔的形成和通量率的确定。
