Several charged residues in the voltage-dependent anion channel, VDAC1, have been identified to be essential for hexokinase II (HK II) binding. Phosphorylation of VDAC1 at threonine 51 by GSK3β has also been reported to detach HK II from the channel. A serine residue S215, located on the cytoplasmic loop of VDAC1, is a potential phosphorylation target based on the consensus phosphorylation sequence for GSK3β. In the present study, we investigated the effects of a phosphomimetic S215E on the functional interaction between VDAC1 and HK II. Wild-type (WT) recombinant rat VDAC1 was obtained from OriGene. S215E was generated by site-directed mutagenesis. The WT-VDAC1 or the S215E-VDAC1 protein was reconstituted in planar lipid bilayers for electrophysiological measurements. The reconstitution of the proteins in the bilayers was confirmed by the characteristic voltage-dependence of VDAC1 in response to a voltage ramp protocol from −80 to +80 mV. Following functional confirmation, a solution change prevented further insertion of additional channel proteins into the bilayer. In the absence of HK II, S215E-VDAC1 maintained the characteristic voltage-dependence as WT-VDAC1, indicating that the mutation had no significant effects on channel gating. With the addition of HK II (60 kU/ml; human recombinant HK II, Genway), WT-VDAC1 conductance decreased to 55±5% (n=8) of its initial value. This inhibition was maintained throughout a 24-min recording period. In contrast, HK II had no significant effect on the S215E-VDAC1, where conductance remained unchanged (95±7% of its initial value; n=6). These findings provide strong evidence on the critical role of charged residues in the VDAC1-HK II interactions. Furthermore, our results implicate the role of phosphorylation in modulating this interaction. Molecular dynamics simulations are ongoing to further delineate the VDAC1-HK II interactions.