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 (ΔΨMOM) 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 ΔΨMOM of ~33 mV, positive from the IMS side in HeLa cells; in contrast, HEK-293 cells lacked substantial ΔΨMOM. Hexokinase 2 (HK2) is known to be overexpressed in cancer cells. Consistent with the previously proposed role of VDAC- HK complexation in ΔΨMOM 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 ΔΨMOM and metabolic activity in cancer cells. Thus, our results demonstrate the metabolism-dependent generation of ΔΨMOM and provide strong evidence that VDAC regulation by voltage, observed in in vitro studies, is highly relevant to cell physiology.