Article
https://doi.org/10.1038/s41586-019-1498-3
Identification of an ATP-sensitive
potassium channel in mitochondria
Angela Paggio1,4, Vanessa checchetto2,4, Antonio campo^1 , roberta Menabò^3 , Giulia Di Marco^1 , Fabio Di lisa1,3, ildiko Szabo2,3,
rosario rizzuto^1 & Diego De Stefani^1
Mitochondria provide chemical energy for endoergonic reactions in the form of ATP, and their activity must meet cellular
energy requirements, but the mechanisms that link organelle performance to ATP levels are poorly understood. Here we
confirm the existence of a protein complex localized in mitochondria that mediates ATP-dependent potassium currents
(that is, mitoKAT P). We show that—similar to their plasma membrane counterparts—mitoKAT P channels are composed
of pore-forming and ATP-binding subunits, which we term MITOK and MITOSUR, respectively. In vitro reconstitution
of MITOK together with MITOSUR recapitulates the main properties of mitoKAT P. Overexpression of MITOK triggers
marked organelle swelling, whereas the genetic ablation of this subunit causes instability in the mitochondrial membrane
potential, widening of the intracristal space and decreased oxidative phosphorylation. In a mouse model, the loss of
MITOK suppresses the cardioprotection that is elicited by pharmacological preconditioning induced by diazoxide. Our
results indicate that mitoKAT P channels respond to the cellular energetic status by regulating organelle volume and
function, and thereby have a key role in mitochondrial physiology and potential effects on several pathological processes.ATP-sensitive potassium (KATP) channels act as sensors of cellular
metabolism. In the plasma membrane^1 , they couple cell excitability
with energy availability^2 ,^3. They have also been reported to be located
in intracellular membranes—for example, in mitochondria (that is,
mitoKATP)^4 ,^5 —but, in this context, their existence is a matter of debate^6.
MitoKATP mediates the electrophoretic uptake of potassium ions (K+),
which is driven by the negative mitochondrial membrane potential
(ΔΨm), and it is inhibited by physiological levels of ATP. MitoKATP was
first described in the early 1990s, through patch clamp of mitoplasts^4
or by partial purification techniques^5. MitoKATP has been character-
ized from a pharmacological point of view, and both openers (diazox-
ide) and inhibitors (sulfonylureas and 5-hydroxydecanoate (5-HD))
have been described—some of them with proposed specific action on
mitoKATP versus plasma membrane KATP channels^7. Drugs that tar-
get KATP channels are useful in the treatment of several pathologies.
Importantly, some of their uses are due to the modulation of plasma
membrane KATP^8 but others seem to depend on their effects on mito-
KATP. For example, pharmacological preconditioning with diazoxide
efficiently protects the heart from ischaemia–reperfusion injury^9 ,^10
even in the absence of cardiac plasma membrane KATP^11 ,^12. However,
the molecular identity and pharmacology of the mitoKATP channel
remain unknown^7 ,^13 ,^14.
MITOK is a cation channel
We screened a subset of mitochondrial proteins with unknown func-
tion and focused on a candidate protein that is encoded by the CCDC51
gene (NCBI code 79714; we hereafter name the CCDC51 gene MITOK),
the overexpression of which markedly impaired mitochondrial physiol-
ogy. The MITOK gene is conserved in vertebrates, in which it encodes a
unique 45-kDa protein with a predicted N-terminal mitochondrial tar-
geting sequence, one coiled-coil and two transmembrane domains. In
humans, the MITOK gene encodes for two isoforms: isoform 1, which
is full-length but has no predicted mitochondrial targeting sequence,
and isoform 2, which is a splice variant that lacks the first 109 amino
acids (34 kDa in size) and includes a supposed mitochondrial targeting
sequence (Fig. 1a). Analyses of RNA and protein levels using existing
datasets revealed that MITOK is expressed in all tissues in humans,
as is Ccdc51 (which we hereafter name Mitok) in mice^15 ,^16. First, we
experimentally validated the mitochondrial localization of MITOK in
humans and mice. Immunofluorescence showed a full co-localization
of MITOK with a mitochondrial marker in HeLa cells (Fig. 1b), and
subcellular fractionation of mouse liver revealed a progressive enrich-
ment of MITOK in mitochondria and mitoplasts, and the absence of
this protein on the outer membrane (Fig. 1c). Carbonate extraction
confirmed membrane insertion (Extended Data Fig. 1a), which indi-
cates that MITOK is in the inner mitochondrial membrane. To inves-
tigate the topology of MITOK, we used two antibodies—one against
the N-terminal half, and the other covering the C-terminal half, of
MITOK (Fig. 1d). Digestion of mouse mitoplasts using proteinase K
caused the loss of full-length MITOK and the appearance of a smaller
fragment that was recognized by the N-terminal antibody (Extended
Data Fig. 1b), which indicates that a portion of the protein is protected
inside the organelle. By contrast, no residual signal was detected with an
antibody against the region between the two transmembrane domains,
which indicates that this part is exposed to the intermembrane space.
MITOK is a two-pass protein of the inner mitochondrial membrane
with the N and C termini exposed towards the matrix. Next, we cloned
and tagged (with Flag, V5 and GFP) mouse MITOK and investigated
the effect of its overexpression. In terms of morphology, MITOK over-
expression causes organelle fragmentation (Extended Data Fig. 1c) and
swelling (Fig. 1e). At functional level, MITOK overexpression caused
a drop in ΔΨm (Extended Data Fig. 1d) and agonist-induced mito-
chondrial Ca^2 + uptake (an additional readout for changes in ΔΨm)
(Extended Data Fig. 1e). For the human isoforms, we designed both
specific (isoform 1 versus isoform 2) and non-specific (pan-isoforms)
primers for quantitative PCR. Both isoforms can be detected in HeLa
cells at the transcript level, although isoform 2 is expressed one tenth
the level of isoform 1 (Extended Data Fig. 1f). Despite this, only a(^1) Department of Biomedical Sciences, University of Padova, Padova, Italy. (^2) Department of Biology, University of Padova, Padova, Italy. (^3) CNR Institute of Neuroscience, Padova, Italy. (^4) These authors
contributed equally: Angela Paggio, Vanessa Checchetto. *e-mail: [email protected]; [email protected]
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