Effect of cell swelling on membrane and cytoplasmic distribution of pICln.

Effect of cell swelling on membrane and cytoplasmic distribution of pICln.
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细胞肿胀对 pICln 膜和细胞质分布的影响。

DOI:
10.1152/ajpcell.1998.274.6.c1545
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发表时间:
1998
期刊:
The American journal of physiology
影响因子:
--
通讯作者:
Strange,K
Strange,K
中科院分区:
--
文献类型:
--
作者:
Emma,F;Breton,S;Morrison,R;Wright,S;Strange,K

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pIClnis广泛存在于哺乳动物细胞中,并被认为在细胞体积调节中起关键作用。突变研究提出pICl是一种膨胀激活的阴离子通道。然而,最近在Madin-Darby犬肾细胞和内皮细胞中的研究表明,该蛋白主要定位于细胞质。因此,有人推测,活化涉及pICln从细胞质的可逆易位和插入质膜。我们使用几种不同的方法来测试这个假设。将C6胶质瘤细胞分级分离成含有质膜和细胞质的组分表明,回收的pICln的约90%局限于胞质溶胶。溶胀对质膜组分中蛋白质的相对含量没有影响。免疫荧光显微镜显示pIClnis主要定位于肿胀和非肿胀细胞的细胞质。类似地,用绿色荧光蛋白标记的pICln构建体转染细胞未能揭示蛋白质的任何膜定位。这些发现并不支持pIClnis是一个由肿胀诱导的膜插入激活的体积调节阴离子通道的假设。
pIClnis found ubiquitously in mammalian cells and is postulated to play a critical role in cell volume regulation. Mutagenesis studies led to the proposal that pIClnis a swelling-activated anion channel. However, recent studies in Madin-Darby canine kidney cells and endothelial cells have shown that the protein is localized primarily to the cytoplasm. It has therefore been postulated that activation involves reversible translocation of pIClnfrom the cytoplasm and insertion into the plasma membrane. We tested this hypothesis using several different approaches. Fractionation of C6glioma cells into plasma membrane- and cytoplasm-containing fractions demonstrated that ∼90% of the recovered pIClnwas confined to the cytosol. Swelling had no effect on the relative amount of protein present in the plasma membrane fraction. Immunofluorescence microscopy revealed that pIClnis localized primarily, if not exclusively, to the cytoplasm of swollen and nonswollen cells. Similarly, transfection of cells with a green fluorescent protein-labeled pIClnconstruct failed to reveal any membrane localization of the protein. These findings do not support the hypothesis that pIClnis a volume regulatory anion channel activated by swelling-induced membrane insertion.
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