VDAC1 is a key component of the mitochondrial permeability transition pore To initiate apoptosis and certain other forms of cell death, mitochondria become permeable such that cytochrome c and other pre-apoptotic molecules resident inside the mitochondria enter the cytosol and activate apoptotic cascades We have shown recently that VDAC1 interacts directly with never-in-mitosis A related kinase 1 (Nek1), and that Nek1 phosphorylates VDAC1 on Ser193 to prevent excessive cell death after injury How this phosphorylation regulates the activity of VDAC1, however, has not yet been reported Here, we use atomic force microscopy (AFM) and cytochrome c conductance studies to examine the configuration of VDAC1 before and after phosphorylation by Nek1 Wild-type VDAC1 assumes an open configuration, but closes and prevents cytochrome c efflux when phosphorylated by Nek1. A VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1 under identical conditions, remains open and constitutively allows cytochrome c efflux Conversely, a VDAC1-Ser193Glu mutant, which mimics constitutive phosphorylation by Nek1, remains closed by AFM and prevents cytochrome c leakage in the same liposome assays Our data provide a mechanism to explain how Nek1 regulates cell death by affecting the opening and closing of VDAC1. (C) 2010 Elsevier Inc All rights reserved