Ma T, Frigeri A, Tsai S T, Verbavatz J M, Verkman A S
Department of Medicine, University of California, San Francisco 94143-0532.
J Biol Chem. 1993 Oct 25;268(30):22756-64.
CHIP28 is a major water transporting protein in erythrocytes and plasma membranes in kidney proximal tubule and thin descending limb of Henle. Chinese hamster ovary cells were stably transfected with the coding sequence of cloned rat kidney CHIP28k using expression vectors containing cytomegalovirus or Rous sarcoma virus promoters. Clonal cell populations expressed a 1.3-kilobase mRNA on Northern blot probed by CHIP28k cDNA and a 28-kDa protein on immunoblot probed by a polyclonal CHIP28 antibody. The clone with greatest expression produced approximately 8 x 10(6) copies of CHIP28k protein/cell. Plasma membrane osmotic water permeability (Pf), measured by stopped-flow light scattering, was 0.004 cm/s in control (vector-transfected) cells (10 degrees C) and 0.014 cm/s in the CHIP28k-transfected cells. Pf in CHIP28k-transfected cells had an activation energy of 4.9 kcal/mol and was reversibly inhibited by HgCl2. CHIP28k expression did not affect the transport of protons and the small polar non-electrolytes urea and formamide. CHIP28k immunoreactivity and function was then determined in subcellular fractions. Pf in 6-carboxyfluorescein-labeled endocytic vesicles, measured by a stopped-flow fluorescence quenching assay, was 0.002 cm/s (control cells) and 0.011 cm/s (CHIP28k-transfected cells); Pf in transfected cells was inhibited by HgCl2. Immunoblotting of fractionated endoplasmic reticulum, Golgi, and plasma membranes revealed high densities of CHIP28k (approximately 5000 monomers/microns 2 in plasma membrane) with different glycosylation patterns; functional water transport activity was present only in Golgi and plasma membrane vesicles. Antibody detection of CHIP28k by confocal fluorescence microscopy and immunogold electron microscopy revealed localization to plasma membrane and intracellular vesicles. These studies establish a stably transfected somatic cell line that strongly expresses functional CHIP28k water channels. As in the original proximal tubule cells, the expressed CHIP28k protein is a selective water channel that is functional in endocytic vesicles and the cell plasma membrane.
CHIP28是红细胞以及肾近端小管和髓袢细降支的质膜中的一种主要水转运蛋白。使用含有巨细胞病毒或劳斯肉瘤病毒启动子的表达载体，将克隆的大鼠肾脏CHIP28k的编码序列稳定转染到中国仓鼠卵巢细胞中。在通过CHIP28k cDNA进行Northern杂交检测时，克隆细胞群体表达一种1.3千碱基的mRNA，在用多克隆CHIP28抗体进行免疫印迹检测时表达一种28 kDa的蛋白质。表达量最高的克隆产生约8×10⁶个CHIP28k蛋白拷贝/细胞。通过停流光散射测量的质膜渗透水通透性（Pf），在对照（载体转染）细胞中（10℃）为0.004 cm/s，在CHIP28k转染细胞中为0.014 cm/s。CHIP28k转染细胞中的Pf具有4.9千卡/摩尔的活化能，并被HgCl₂可逆抑制。CHIP28k的表达不影响质子以及小的极性非电解质尿素和甲酰胺的转运。然后在亚细胞组分中测定CHIP28k的免疫反应性和功能。通过停流荧光猝灭测定法测量，6-羧基荧光素标记的内吞小泡中的Pf，在对照细胞中为0.002 cm/s，在CHIP28k转染细胞中为0.011 cm/s；转染细胞中的Pf被HgCl₂抑制。对内质网、高尔基体和质膜分级分离后的免疫印迹显示CHIP28k具有高密度（质膜中约5000个单体/微米²）且糖基化模式不同；功能性水转运活性仅存在于高尔基体和质膜小泡中。通过共聚焦荧光显微镜和免疫金电子显微镜对CHIP28k进行抗体检测，显示其定位于质膜和细胞内小泡。这些研究建立了一个稳定转染的体细胞系，该体细胞系强烈表达功能性CHIP28k水通道。与原始近端小管细胞一样，所表达的CHIP28k蛋白是一种选择性水通道，在内吞小泡和细胞质膜中具有功能。
