ClC-2 chloride channels contribute to HTC cell volume homeostasis.
ClC-2 chloride channels contribute to HTC cell volume homeostasis.
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ClC-2 氯离子通道有助于 HTC 细胞体积稳态。
DOI：
10.1152/ajpgi.2001.280.3.g344
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Fitz,JG
中科院分区：
文献类型：
--
作者：
Roman,RM;Smith,RL;Feranchak,AP;Clayton,GH;Doctor,RB;Fitz,JG
膜Cl−通道在细胞体积稳态和调节体积敏感性细胞转运和代谢中起重要作用。ClC-2通道cDNA的异源表达导致肿胀激活的Cl−电流的出现，这与细胞体积调节中的作用一致。由于异源模型中的通道特性可能被细胞背景修饰，我们评估了内源性ClC-2蛋白在细胞体积调节中是否具有重要的功能。如大鼠HTC肝癌细胞的全细胞膜片钳技术所示，当非ClC-2电流被DIDS（100 μM）阻断时，细胞体积增加刺激了内向整流Cl−电流。从HTC细胞中分离到与大鼠脑ClC-2同源的cDNA;在原代大鼠肝细胞和胆管细胞中证明了ClC-2 cDNA的相同序列。原位杂交、免疫细胞化学和Western blot检测ClC-2 mRNA和膜蛋白表达。细胞内递送抗体至ClC-2的基本调节结构域降低了HEK-293细胞中表达的ClC-2依赖性电流。在HTC细胞中，相同的抗体阻止了细胞体积增加或暴露于嘌呤能受体激动剂ATP引起的内源性Cl−电流的激活，并延迟了HTC细胞体积从肿胀中恢复。这些研究提供了进一步的证据，证明哺乳动物ClC-2通道蛋白具有功能，并表明在HTC细胞中，它们有助于膜氯离子渗透性和细胞体积稳态的生理变化。
Membrane Cl−channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl−currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl−currents when non-ClC-2 currents were blocked by DIDS (100 μM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl−currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl−permeability and cell volume homeostasis.