Linking cellular metabolism to mitochondrial outer membrane potential

The voltage-dependent anion channel (VDAC) of the mitochondrial outer membrane (MOM) responds to transmembrane voltage through intrinsic gating and voltage-dependent interactions with cytosolic proteins, such as α-synuclein and β-tubulin. Despite this characteristic voltage sensitivity found in vitro, the existence of a substantial MOM potential (ΔΨ_MOM) in vivo remains controversial. Using pH sensors targeted to the cytosol and the intermembrane space (IMS), we measured the difference in proton concentration across the MOM and calculated a ΔΨ_MOM of ~33 mV, positive from the IMS side in HeLa cells; in contrast, HEK-293 cells lacked substantial ΔΨ_MOM. Hexokinase 2 (HK2) is known to be overexpressed in cancer cells. Consistent with the previously proposed role of VDAC- HK complexation in ΔΨ_MOM generation, we observed lower HK2 expression in HEK-293 cells than in HeLa cells. In addition, by studying pH changes in the IMS and cytosol in response to changes in glucose and glucose-6-phosphate concentrations in HeLa cells, we establish a relationship between ΔΨ_MOM and metabolic activity in cancer cells. Thus, our results demonstrate the metabolism-dependent generation of ΔΨ_MOM and provide strong evidence that VDAC regulation by voltage, observed in in vitro studies, is highly relevant to cell physiology.