Novel Mechanism of Mitochondrial Respiration Control through Competition between Hexokinase-2 and Tubulin for VDAC Binding

Abstract

The voltage dependent anion channel (VDAC) is involved in regulation of metabolite flux across the mitochondrial outer membrane (MOM). Hexokinse II (HK2) is known to bind the MOM where it phosphorylates glucose into glucose-6-phosphate (G6P). High expression of HXK2 is a common phenotype of many cancers, where its concentration can be 200 times of that in noncancerous cells, and is implicated in the Warburg effect. It is believed that VDAC serves as a HXK2 binding site in the MOM. The 15 amino acid N-terminal sequence of HXK2 is responsible for mitochondrial binding and, when conjugated to TAT (TAT-HK2), binds to mitochondria with higher affinity than native HXK2, causing HXK2 detachment. We have previously found that dimeric tubulin reversibly binds and partially blocks VDAC inhibiting metabolite flux across the MOM. Now we show that this binding can be attenuated by TAT-HXK2 peptide as well as by full length HXK2. We have found that TAT-HXK2 and recombinant full length HXK2 inhibit tubulin blockage of VDAC reconstituted into planar lipid bilayers without altering characteristic channel properties such as single channel conductance and selectivity. Binding of HXK2 to VDAC is verified by the generation of high-frequency excess current noise without channel closure. HXK2 bound to VDAC prevents subsequent tubulin binding, but only when added before tubulin, and inhibits tubulin-induced VDAC blockage in a dose dependent manner. Moreover, G6P, which is known to cause HXK2 detachment from the MOM, fully reverses the inhibition of tubulin-VDAC binding. This suggests that HXK2 detachment from VDAC (and hence the MOM) is caused by a HXK2 conformational change upon G6P binding. Thus we propose a novel mechanism of mitochondrial respiration control in cancer cells through the competition between HXK2 and tubulin for VDAC binding.