Molecular identification of ATP sensitive Mitochondrial Potassium Channel (mitoKATP)

Mitochondria ATP-sensitive potassium channels (mitoKATP) were first described in 1991 by direct patch clamp of isolated mitoplasts (i.e. mitochondria lacking the outer membrane). Since then, a growing amount of evidences showed that they are implicated in the control of a variety of mitochondrial functions. Most importantly, pharmacological modulation of the mitoKATP can efficiently protect the heart from ischemia/reperfusion injury. However, despite its huge therapeutic potential, the molecular identity of the mitoKATP is still unknown. Here we identify a protein complex that is sufficient to reconstitute a functional K+ permeant channel with the same pharmacological profile of the mitoKATP. In particular, we demonstrate that the mitoKATP is a heterooligomer composed by two different components. One is a previously uncharacterized protein of unknown function that we now name “mitoK”. The other is a protein belonging to the ABC superfamily here named “mitoSUR”. It possesses a nucleotide-binding site (a standard Walker motif) and provides ATP sensitivity to the mitoK pore-forming subunit through direct protein interaction. In detail, we demonstrated that mitoK is a mitochondrial protein located in the inner mitochondrial membrane (IMM), with two transmembrane domains and both its N- and C-termini exposed to the organelle matrix. Its overexpression induces a decrease in mitochondrial Ca2+ transients evoked by agonist stimulation, a drastic reduction of mitochondria membrane potential, fragmentation of the mitochondrial network and a derangement of the IMM ultrastructure, with the total collapse of the cristae. However, all these effect can be rescued by the concomitant overexpression of mitoSUR, but not by the expression of the mitoSURK513A mutant that lacks ATP binding. Most importantly, in vitro reconstitution of purified mitoK and mitoSUR in a planar lipid bilayer form a channel i) selective for monovalent cations, ii) blocked by tetraethyl ammonium (TEA, a general inhibitor of K+ channels), iii) sensitive to ATP, iv) activated by diazoxide and v) inhibited by 5-HD. Finally, we focused our efforts to understand the physiological role of mitoKATP. According to the available literature, mitoKATP channels are believed to be protective in ischemia/reperfusion injury. However, the broad conservation profile among all vertebrates suggests that the mitoKATP should also have a primary housekeeping function. In order to analyze the genuine physiological role of the mitoKATP, we generated Hela cells knockout for mitoK by using the Crispr/Cas9 technology. Two different guides targeting different gene regions were validated and several Hela mitoKKO clones were obtained. Overall, ablation of mitoK leads to no gross defects in mitochondria morphology, although an impairment of cristae structure becomes evident through electron microscopy. Mitochondrial membrane potential is overall intact and expression of respiratory chain complexes is unaffected. However, we noticed that Hela mitoKKO cells undergo to asynchronous, rapid and transient depolarizations of single mitochondria, a phenome known as “flickering” of mitochondria membrane potential. Therefore, oxygen consumption rate (OCR) is greatly impaired in Hela mitoKKO cells when compared to their wild type counterparts, in both basal, leaky (oligomycin induced) and maximal (i.e. FCCP induced) respiration. As already reported, mitochondrial K+ homeostasis is fundamental to regulate mitochondrial matrix volume: while excessive accumulation leads to organelle swelling, a decrease in K+ uptake is predicted to cause matrix contraction, with obvious consequences on the performance of the oxidative phosphorylation. Overall, our data indicates that mitoKATP is a central regulator of mitochondrial function that modulates the efficiency of energy production according to the ATP availability through the regulation of matrix volume.