Abrami L, Berthonaud V, Deen P M, Rousselet G, Tacnet F, Ripoche P
Département de Biologie Cellulaire et Moléculaire, Service de Biologie Cellulaire, CEA Saclay, F-91191 Gif sur Yvette Cedex, France.
Pflugers Arch. 1996 Jan;431(3):408-14. doi: 10.1007/BF02207279.
In a recent work, we showed that the aquaporins 1 (AQP1) are permeable to certain small solutes such as glycerol. Here, we have further investigated the permeation pathway of glycerol through human AQP1 (hAQP1) by the use of mutants (C189S, H180A, H209A) and inhibitors such as P-chloromercuribenzene sulphonate (pCMBS), CuSO4 or phloretin, in comparison with other AQP-MIP (where MIP denotes major intrinsic protein) proteins: hAQP2, plant water channel gammaTIP and bacterial glycerol permease facilitator, GlpF. Glycerol movements were measured in Xenopus laevis oocytes. Apparent glycerol permeability coefficients (P'gly) were calculated from the rates of oocyte swelling upon exposure to an isoosmotic medium containing an inwardly directed gradient of glycerol and from [3H]glycerol uptake measurements. Similar P'gly values were obtained for hAQP1 and hAQP2 6 to 8 times greater than control indicating that hAQP2 also transports glycerol. P'gly of hAQP2-injected oocytes was pCMBS and CuSO4 sensitive. In contrast, the P'gly value of gammaTIP was close to that of control, indicating that gammaTIP does not transport glycerol. The hAQP1-C189S, -H180A and -H209A mutants gave P'gly values similar to those obtained for wild hAQP1, indicating that these mutations did not affect glycerol movements. However, the H209A mutant has an osmotic water permeability coefficient (Pf) value decreased by 50%. The inhibitory effect pCMBS on P'gly was maintained for the 2 His mutants and, more interestingly, was also conserved for the C189S mutant. CuSO4 significantly inhibited P'gly of oocytes expressing hAQP1, hAQP1-C189S, -H180A, and -H209A mutants and had no effect on P'gly of GlpF-injected oocytes. Phloretin was shown to inhibit by around 80% the glycerol fluxes of wild and mutant hAQP1, hAQP2 and to fully inhibit glycerol uptake in GlpF-injected oocytes.
在最近的一项研究中,我们发现水通道蛋白1(AQP1)对某些小分子溶质如甘油具有通透性。在此,我们通过使用突变体(C189S、H180A、H209A)和抑制剂如对氯汞苯磺酸盐(pCMBS)、硫酸铜或根皮素,进一步研究了甘油通过人水通道蛋白1(hAQP1)的渗透途径,并与其他水通道主要内在蛋白(MIP)进行了比较:hAQP2、植物水通道γTIP和细菌甘油通透酶促进剂GlpF。在非洲爪蟾卵母细胞中测量甘油的转运。根据卵母细胞在暴露于含有内向甘油梯度的等渗介质时的肿胀速率以及[³H]甘油摄取测量值,计算表观甘油渗透系数(P'gly)。hAQP1和hAQP2获得了相似的P'gly值,比对照高6至8倍,表明hAQP2也能转运甘油。注射hAQP2的卵母细胞的P'gly对pCMBS和硫酸铜敏感。相比之下,γTIP的P'gly值接近对照值,表明γTIP不转运甘油。hAQP1 - C189S、- H180A和- H209A突变体的P'gly值与野生hAQP1的相似,表明这些突变不影响甘油的转运。然而,H209A突变体的渗透水渗透系数(Pf)值降低了50%。pCMBS对P'gly的抑制作用在2个组氨酸突变体中得以维持,更有趣的是,在C189S突变体中也得以保留。硫酸铜显著抑制表达hAQP