Hypotonic activation of short ClC3 isoform is modulated by direct interaction between its cytosolic C-terminal tail and subcortical actin filaments
Hypotonic activation of short ClC3 isoform is modulated by direct interaction between its cytosolic C-terminal tail and subcortical actin filaments
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DOI：
10.1074/jbc.m700379200
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发表时间：
2007-06-08
影响因子：
4.8
通讯作者：
Yamboliev, Ilia A.
中科院分区：
文献类型：
--
作者：
McCloskey, Diana T.;Doherty, Lynda;Yamboliev, Ilia A.
ClC3短异构体（sClC3）在某些细胞类型中起着体积敏感的向外整流阴离子通道（VSOAC）的作用。在以前的研究中，我们已经表明sClC3的低渗活化与细胞肿胀介导的肌动蛋白细胞骨架重塑有关。在本研究中，我们验证了sClC3的胞质尾部直接与肌动蛋白结合的假设，这种结合调节了通道的低渗激活。体外共沉淀实验表明，谷胱甘肽s -转移酶融合的scc3细胞质C端（gst - scc3 - ct）与丝状肌动蛋白（F-actin）有很强的结合，但与球状单体肌动蛋白（G-actin）没有结合。gst融合的N端（GST-sClC3-NT）对g和F-actin均表现出较低的结合亲和力。用逐渐截断的GST-sClC3-CT共沉淀实验表明，F-actin结合区位于scc3的690和760氨基酸之间。两种合成的定位细胞质sClC3-CT基本簇的肽（CTP2，异亮氨酸716到亮氨酸734；CTP3，脯氨酸688到脯氨酸709）在体外阻止了GST-sClC3-CT与F-actin的结合。在NIH/3T3细胞中透析这两种肽（但不包括合成肽CTP1（异亮氨酸737到谷氨酰胺748）），最大电流密度分别降低60%和38%。基于这些结果，我们得出结论，通过与皮质下肌动蛋白丝的直接相互作用，sClC3有助于NIH/3T3细胞低渗应激诱导的VSOACs。
Short ClC3 isoform (sClC3) functions as a volume-sensitive outwardly rectifying anion channel (VSOAC) in some cell types. In previous studies, we have shown that the hypotonic activation of sClC3 is linked to cell swelling-mediated remodeling of the actin cytoskeleton. In the present study, we have tested the hypothesis that the cytosolic tails of sClC3 bind to actin directly and that binding modulates the hypotonic activation of the channel. Co-sedimentation assays in vitro demonstrated a strong binding between the glutathione S-transferase-fused cytosolic C terminus of sClC3 (GST-sClC3-CT) to filamentous actin (F-actin) but not to globular monomeric actin (G-actin). The GST-fused N terminus (GST-sClC3-NT) exhibited low binding affinity to both G-and F-actin. Co-sedimentation experiments with progressively truncated GST-sClC3-CT indicated that the F-actin binding region is located between amino acids 690 and 760 of sClC3. Two synthetic peptides mapping basic clusters of the cytosolic sClC3-CT (CTP2, isoleucine 716 to leucine 734; and CTP3, proline 688 to proline 709) prevented binding of GST-sClC3-CT to F-actin in vitro. Dialysis into NIH/3T3 cells of these two peptides (but not of synthetic peptide CTP1 (isoleucine 737 to glutamine 748)) reduced the maximal current density by 60 and 38%, respectively. Based on these results, we have concluded that, by direct interaction with subcortical actin filaments, sClC3 contributes to the hypotonic stress-induced VSOACs in NIH/3T3 cells.