Kv7.4 channels regulate potassium permeability in neuronal mitochondria

Mitochondrial K⁺ permeability regulates neuronal apoptosis, energy metabolism, autophagy, and protection against ischemia-reperfusion injury. Kv7.4 channels have been recently shown to regulate K⁺ permeability in cardiac mitochondria and exert cardioprotective effects. Here, the possible expression and functional role of Kv7.4 channels in regulating membrane potential, radical oxygen species (ROS) production, and Ca²⁺ uptake in neuronal mitochondria was investigated in both clonal (F11 cells) and native brain neurons. In coupled mitochondria isolated from F11 cells, K⁺-dependent changes of mitochondrial membrane potential (ΔΨ) were unaffected by the selective mitoBK_Ca channel blocker iberiotoxin and only partially inhibited by the mitoK_ATP blockers glyburide or ATP. Interestingly, K⁺-dependent ΔΨ decrease was significantly reduced by the Kv7 blocker XE991 and enhanced by the Kv7 activator retigabine. Among Kv7s, western blot experiments showed the expression of only Kv7.4 subunits in F11 mitochondrial fractions; immunocytochemistry experiments showed a strong overlap between the Kv7.4 fluorescent signal and that of the mitochondrial marker Mitotracker. Silencing of Kv7.4 expression significantly suppressed retigabine-dependent decrease in ΔΨ in intact F11 cells. Expression of Kv7.4 subunits was also detected by western blot in isolated mitochondria from total mouse brain and by immunofluorescence in mouse primary cortical neurons. Pharmacological experiments revealed a relevant functional role for Kv7.4 channels in regulating membrane potential and Ca²⁺ uptake in isolated neuronal mitochondria, as well as ΔΨ and ROS production in intact cortical neurons. In conclusion, these findings provide the first experimental evidence for the expression of Kv7.4 channels and their contribution in regulating K⁺ permeability of neuronal mitochondria.