Mechanisms of mitochondrial Drp1 inducing excessive opening of mPTP channel through LRRK2-HK2 after hypoxia

Abstract:

Objective To explore the regulatory mechanisms of mitochondrial dynamin-related protein 1（Drp1） on excessive opening of mitochondrial permeability transition pore（mPTP） after hypoxia.Methods The opening of mPTP after hypoxia was observed by immunofluorescence in vascular smooth muscle cells（VSMCs）. Mitochondrial and cytoplasmic fractions were separated by ultracentrifugation. Co-IP and Western blot methods were used to detect the expression and distribution of Drp1，the binding of hexokinase-2（HK2） to voltage dependent anion channel（VDAC），and the binding of Drp1 to leucine-rich repeat kinase 2（LRRK2）. The potential binding proteins and binding sites of Drp1 were screened by protein molecular docking and protein chip. Mdivi-1，a Drp1 inhibitor，and mutation methods were used to explore the corresponding mechanisms.Results Mitochondrial translocation of Drp1 caused mPTP excessive opening in VSMCs after hypoxia（P<0.05）. Reducing mitochondrial Drp1 expression by Mdivi-1 could inhibit mPTP opening and reduce the release of cytochrome C（CytC）（P<0.05）. The mitochondrial separation of HK2 caused by the decrease of HK2-Thr473 phosphorylation after hypoxia leaded to the destruction of mPTP structure. After restoring HK2 activity by HK2 T473D mutation，the binding of HK2 to mitochondria and the opening of mPTP after hypoxia were significantly improved（P<0.05）. The results of Drp1 protein chip showed that Drp1 could bind to kinase LRRK2 and block its active site after hypoxia. After the combination of Drp1-LRRK2 was broken by the site mutation of Drp1 T595A，the activity of HK2 and the mitochondrial separation of HK2 were obviously improved（P<0.05），and the opening of mPTP channel was obviously reduced（P<0.05）.Conclusion Mitochondrial Drp1 may reduce HK2-Thr473 phosphorylation and cause HK2 mitochondrial separation by binding and blocking the active site of kinase LRRK2，which finally induces the excessive opening of mPTP after hypoxia.