Iserovich P, Kuang K, Chun T, Fischbarg J
Department of Physiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Biol Cell. 1997 Aug;89(5-6):293-7.
Measurements of the cell membrane diffusional water permeability (Pd) are important to characterize water passage across water channels and across the lipid bilayer component of the membrane. Existing methods for those measurements are involved; however, we report here a simple procedure to estimate Pd in Xenopus laevis oocytes and similar large cells. Due to the different densities of H2O and D2O (heavy water), an oocyte transferred from normal medium to a D2O-based medium floats initially, but subsequently sinks when a certain amount of the water originally in them is replaced by the D2O that diffuses in. We describe how the 'flotation time' (time that oocytes float in a heavy water solution before they start sinking) yields the Pd of the plasma membrane. Determination of Pd by this procedure and by the rate of tritiated water (T2O) efflux give for Pd results which are very close: 2.2 +/- 0.2 (n = 8) and 2.0 +/- 0.1 (n = 6) microns/s, respectively (T = 10 degrees C). Furthermore, our method detects the increase in Pd elicited in oocytes by either expression of water channel proteins, or by treating them with the pore-forming antibiotic amphotericin B. This method appears useful to gauge the expression and function of pore-forming, water-permeable membrane proteins.
测量细胞膜的扩散水渗透率（Pd）对于表征水通过水通道以及穿过膜的脂质双分子层成分的过程很重要。然而，现有的测量这些参数的方法较为复杂；不过，我们在此报告一种在非洲爪蟾卵母细胞及类似的大细胞中估算Pd的简单方法。由于H2O和D2O（重水）的密度不同，从正常培养基转移到以D2O为基础的培养基中的卵母细胞最初会漂浮，但当其中原本的一定量水被扩散进来的D2O取代后，卵母细胞随后会下沉。我们描述了“漂浮时间”（卵母细胞在重水溶液中开始下沉之前漂浮的时间）如何得出质膜的Pd。通过此方法以及通过氚化水（T2O）外流速率测定Pd所得到的结果非常接近：分别为2.2±0.2（n = 8）和2.0±0.1（n = 6）微米/秒（T = 10摄氏度）。此外，我们的方法能够检测到通过水通道蛋白的表达或用成孔抗生素两性霉素B处理卵母细胞所引起的Pd增加。该方法似乎有助于评估成孔的、水可渗透的膜蛋白的表达和功能。
