Mechanisms of epithelial sodium channel (ENaC) regulation by cortactin: Involvement of dynamin
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We have recently shown that epithelial sodium channels (ENaCs) are regulated by the actin-binding protein cortactin via the Arp2/3 protein complex. It has been also demonstrated that a GTPase dynamin, which is known to regulate clathrin-mediated endocytosis, can as well initiate signaling cascades regulated by cortactin. This study was designed to investigate the involvement of dynamin into cortactin-mediated regulation of ENaC. Initially, a recently described inhibitor of dynamin, dynasore, was used. However, use of this inhibitor seemed to be inappropriate due to discovered side effects. Thus, treatment of mpkCCDc14 cells monolayers with dynasore (in concentrations of 10 and 100 μM) resulted in a decrease in ENaC-mediated transepithelial currents. Besides, dynasore caused reduced amiloride-sensitive currents in CHO cells transfected with ENaC subunits. Therefore, the data demonstrated that dynasore down regulates both native and overexpressed channel’s activity and use of this drug is not appropriate for studies of ENaC endocytosis. We hypothesize that this effect is most likely caused either by dynasore’s toxic actions upon the cells or by enhanced endocytosis of ENaC-activating proteins. In the following experiments plasmids encoding mutant forms of dynamin and cortactin were used. Dominant negative dynamin (K44A) transfected into CHO cells together with ENaC subunits significantly increased amiloride-sensitive current density compared to cells transfected with ENaC only (control); additional transfection of cortactin together with the K44A dynamin resulted in current density restitution back to the control level. Moreover, ENaC overexpression with the SH3 domain of cortactin, which is responsible for dynamin binding, caused a decrease of ENaC current. Thus, we have shown in this study that cortactin can mediate ENaC activity not only via the Arp2/3 complex, but also through the dynamin-mediated processes.
KeywordsENaC cortactin dynamin endocytosis dynasore sodium transport epithelial cells
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