Molecular cloning and expression of a chloride channel-associated protein pICln in human young red blood cells: association with actin.
Molecular cloning and expression of a chloride channel-associated protein pICln in human young red blood cells: association with actin.
复制标题
人类年轻红细胞中氯离子通道相关蛋白 pICln 的分子克隆和表达：与肌动蛋白的关联。
DOI：
10.1042/bj3270609
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Nagel,RL
中科院分区：
文献类型：
--
作者：
Schwartz,RS;Rybicki,AC;Nagel,RL
我们报道了从人网织红细胞中克隆和测序编码氯离子通道相关蛋白pICln的基因。人网织红细胞pICln(HrpICln)编码一个与人无色素睫状体上皮细胞pICln相同的蛋白质(预测分子质量为26293 Da)。利用全长HRpIClncDNA(约1.2kb)为探查人淋巴细胞中期染色体扩散，应用荧光原位杂交技术将人ICln基因定位于11q13。抗重组人高密度脂蛋白的多克隆抗体检测到条带约为1条。43 kDa和大约正常(AA)和镰刀状(SS)红细胞(RBC)鬼膜均为37 kDa。在SS幽灵和网织红细胞含量为9.8%的自身免疫性溶血性贫血患者的幽灵中，HRpICln的数量比AA幽灵高，提示HRpICln的表达或膜组装是年龄依赖性的。激光扫描共聚焦荧光显微镜可将HRpIC免疫定位于红细胞膜。富含网织红细胞的AA红细胞密度分离部分中HRpICln染色强度的增加与HRpICln膜含量与细胞年龄的依赖一致。用酵母双杂交系统证实，HRpICln与β-肌动蛋白在体内形成稳定的复合体。低离子强度的鬼膜提取会导致血影蛋白-肌动蛋白细胞骨架的提取，也会导致HRpICln的提取，这与这些蛋白在体内与红细胞结合的可能性是一致的。本文的结果证实了人红细胞中存在氯离子通道相关蛋白pICln，并提出了该蛋白在幼年红细胞的氯离子转运和容量调节中发挥作用的可能性。此外，RBC pICln与肌动蛋白的关联为测试RBC离子通道和细胞骨架之间的相互作用提供了一个模型。
We report the cloning and sequencing from human reticulocytes of cDNA coding for the Cl-channel-associated protein, pICln. Human reticulocyte pICln(HRpICln) cDNA encodes a protein (predicted molecular mass 26293 Da) identical with human non-pigmented ciliary epithelial cell pICln. By using full-length HRpIClncDNA (approx. 1.2 kb) to probe human lymphocyte metaphase-chromosome spreads, the location of the human IClngene was mapped to 11q13 by fluorescencein situhybridization analysis. Polyclonal antibodies to recombinant HRpIClndetected bands at approx. 43 kDa and approx. 37 kDa in both normal (AA) and sickle (SS) red blood cell (RBC) ghost membranes. In SS ghosts, and in ghosts from a patient with autoimmune haemolytic anaemia with 9.8% reticulocytes, the amount of HRpIClnwas increased compared with AA ghosts, suggesting that the expression or membrane assembly of HRpIClnis cell age-dependent. Laser scanning confocal fluorescent microscopy immunolocalized HRpIClnlargely to the RBC membrane. The increased staining intensity of HRpIClnin a reticulocyte-enriched AA RBC density-separated fraction is consistent with a dependence of HRpIClnmembrane content on cell age. HRpIClnand β-actin form stable complexesin vivo, demonstrated with the yeast two-hybrid system. Low-ionic-strength extraction of ghost membranes, which results in the extraction of the spectrin-actin cytoskeleton, also results in the extraction of HRpICln, consistent with the possibility for the association of these proteins in RBCsin vivo. The results presented here establish the presence of the Cl-channel-associated protein, pICln, in human RBCs, and raises the possibility that this protein has a role in RBC Cl-transport and volume regulation in young RBCs. Moreover the association of RBC pIClnwith actin offers a model in which to test interactions between RBC ion channels and the cytoskeleton.