中华大蟾蜍卵母细胞质膜存在钙依赖性的氯离子通道

Calcium-dependent chloride channels in plasma membrane of oocytes from toad,Bufo bufo gargarizans

本文采用双微电极电压钳方法研究了中华大蟾蜍卵母细胞内源性电压门控型离子通道的成分及其生理特性。卵母细胞去极化至 -30 mV 及更正电压时,有一持续的电压依赖性外向电流出现。钾离子通道拮抗剂四乙基氯化氨(tetraethy-lammonium chloride, TEA, 10 mmol/L)和 4- 氨基吡啶(4-aminopyridine, 4-AP, 10 mmol/L)协同作用时,该电流只能被抑制到最大电流幅度的(23.4±0.72)%。但是,上述浓度的TEA和4-AP 与氯离子通道拮抗剂5- 硝基-2, 3- 苯酚丙胺苯甲酸盐 (5-nitro-2,3-phenypropylamino benzoate, NPPB, 30 μmol/L)、无钙 Ringer 氏液或钙离子通道拮抗剂维拉帕米(40 μmol/L)协同作用时,可分别将此外向电流抑制到最大电流幅度的(2.1±0.08)%、(2.2±0.04)% 和(3.1±0.15)%。结果表明,中华大蟾蜍卵母细胞质膜上除有钾离子电流之外,还存在钙依赖性的氯离子电流。

In this paper, membrane current properties of the fully-grown oocytes from toad, Bufo bufo gargarizans, were studied by using two-microelectrode voltage clamp technique. Axion of adult female toad was destroyed, and then ovarian lobes containing oocytes in stage Ⅰ to Ⅵ were removed and incubated in Ca^2＋-free ND96 solution with collagenase （1.5 mg/ml） for 1 h. Subsequently, the oocytes were washed in Ca^2＋-free ND96 solution for 10 min to completely remove the follicular layer. For the experiments only the oocytes in stage V and VI were selected and used during 1 to 5 d. The membrane was depolarized from a holding potential of 80 mV to ＋60 mV in 10 mV step. It was found that a sustained outward current was elicited by depolarization. Potassium channel blockers （tetraethylammonium chloride, TEA, 10 mmol/L and 4-aminopyridine, 4-AP, 10 mmol/L） reduced the outward current to （23.4±0.72）% of the maximum. However, further addition of chloride channel blocker （5-nitro-2,3-phenypropylamino benzoate, NPPB, 30 μmol/L） could almost completely block the outward current to （2.1±0.08）% of the maximum. In the presence of TEA and 4-AP, removal of extracellular Ca^2＋ or adding verapamil （40 μmol/L）, could also reduce the outward current to （2.2±0.04）% and （3.1±0.15）% of the maximum, respectively. It is concluded that calcium-dependent chloride channels exist in plasma membrane of Bufo bufo gargarizans oocytes, besides potassium channels.