Regulation of connexin43 protein complexes by intracellular acidification
Regulation of connexin43 protein complexes by intracellular acidification
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DOI：
10.1161/01.res.0000113924.06926.11
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发表时间：
2004-02-06
影响因子：
20.1
通讯作者：
Spray, DC
中科院分区：
文献类型：
--
作者：
Duffy, HS;Ashton, AW;Spray, DC
缺血诱导的星形胶质细胞或心肌细胞酸化通过关闭间隙连接通道并随后内化间隙连接蛋白来减少细胞间通讯。为了确定这种耦合的变化是否可能归因于连接蛋白43（Cx43）和其他蛋白质之间的相互作用的改变，我们应用尼日利亚菌素/高K+的方法来改变培养的皮质星形胶质细胞的细胞内pH值（pHi）。细胞内酸化伴随着Cx43的内化，Cx43支架蛋白封闭带-1（ZO-1）保留在细胞表面，表明ZO-1和Cx43在低pH下解离。免疫共沉淀研究表明，减少ZO-1的结合，并增加结合的c-Src Cx43在低pH值。共振镜光谱用于定量c-Src的SH 3结构域和ZO-1的PDZ结构域与Cx43（Cx43 CT）的羧基末端结构域的结合。数据表明，c-Src/Cx43 CT相互作用是高度pH依赖性的，而ZO-1/Cx43 CT相互作用不是。此外，c-Src与Cx43 CT的结合阻止并逆转ZO-1/Cx43 CT结合。我们推测，在低pHi时c-Src对Cx43的亲和力增加有助于Cx43与ZO-1的分离，这可能有助于Cx43的内化。这些数据表明，长期酸化可能重塑蛋白质-蛋白质相互作用，涉及Cx43，从而提供了一个重要的保护机制，以限制缺血性损伤后的病变扩散。
Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K+ method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury.