Site-specific phosphorylation of VDAC1 decreases its susceptibility to oligomerization.

Abstract

The voltage-dependent anion channel 1 (VDAC1) is the primary pathway for metabolite and ion flux across the outer mitochondrial membrane. Oligomerization of VDAC1 that results in the formation of a mega-pore is a critical step in the release of cytochrome c and mitochondrial DNA that triggers apoptosis and inflammasome activation, respectively. However, the underlying mechanism that leads to mega-pore formation is unresolved. Previously, we identified phosphorylation of VDAC1 at S35 to be associated with cardioprotection. Here we report a novel finding that S35 phosphorylation regulates VDAC1 oligomerization and, subsequently, mega-pore formation. In 90 min recordings of channel activity of recombinant VDAC1 in planar lipid bilayers, wild-type (WT) VDAC1 exhibited cumulative current increases due to multiple channel insertions. In 50% of the recordings, a dramatic increase in cumulative channel conductance was observed (>1.8 µS). This was accompanied by a progressive shift at baseline, suggestive of high local ionic accumulation at the bilayer surface. These distinct biophysical characteristics were attributed to mega-pore formation. The probability of similar cumulative large conductances with associated baseline shifts was reduced in the phosphomimetic S35E VDAC1 (20% vs 50% in WT), while it was increased in a non-phosphorylatable mutant S35A (90% vs 50% in WT). In VDAC1 knock-out H9c2 cells expressing WT or mutant (S35A/S35E) VDAC1, 400 µM H2O2 induced the formation of VDAC1 oligomers. However, compared to WT, the formation of trimers and higher order oligomers was significantly decreased in cells expressing the S35E mutant, while it was significantly increased in cells expressing the S35A mutant. These effects are consistent with the corresponding biophysical characteristics observed in the bilayer experiments. Our results suggest that phosphorylation of S35 decreases, while dephosphorylation increases, the susceptibility of VDAC1 to oligomerization and mega-pore formation.