Duan D, Cowley S, Horowitz B, Hume J R
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046, USA.
J Gen Physiol. 1999 Jan;113(1):57-70. doi: 10.1085/jgp.113.1.57.
In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.
在许多哺乳动物细胞中，ClC-3容积调节性氯离子通道在渗透扰动期间维持多种正常细胞功能。然而，细胞容积对该通道的调节分子机制尚不清楚。由于近期的多项研究表明蛋白磷酸化/去磷酸化参与容积调节性离子转运系统的控制，我们使用定点诱变和膜片钳技术研究了ClC-3通道的磷酸化与容积调节之间的关系。在天然心肌细胞以及在NIH/3T3细胞中过表达时，ClC-3通道可通过细胞肿胀或抑制内源性PKC而开放，但可被PKC激活、磷酸酶抑制或细胞内Ca2+升高所关闭。位点特异性突变研究表明，ClC-3通道蛋白细胞内氨基末端共有PKC磷酸化位点内的一个丝氨酸残基（丝氨酸51）是该通道重要的容积感受器。这些结果提供了直接的分子和药理学证据，表明通道的磷酸化/去磷酸化在重组ClC-3通道及其天然对应物ICl.vol的容积敏感性调节中起关键作用。
