area under the curve while reducing the peak
plasma DNP concentrations with a sustained-release coating increased the ratio of toxic to effective dose 25-fold over liver-targeted DNP and
1250-fold over unaltered DNP. These data support
the potential utility of mitochondrial protonophores and other mitochondrial uncoupling agents
for the treatment of the related epidemics of
NASH, metabolic syndrome, and T2D.
REFERENCES AND NOTES
1. V. Ratziu, S. Bellentani, H. Cortez-Pinto, C. Day, G. Marchesini,
J. Hepatol. 53, 372–384 (2010).
2. V. T. Samuel et al., J. Biol. Chem. 279, 32345–32353
3. R. J. Perry et al., Cell Metab. 18, 740–748 (2013).
4. R. J. Perry et al., Nat. Med. 20, 759–763 (2014).
5. I. A. Leclercq et al., J. Clin. Invest. 105, 1067–1075 (2000).
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39, 1286–1296 (2004).
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We thank J. Dong, C. Soroka, J. P. Camporez, M. Jurczak,
J. Stack, M. Kahn, C. Borders, Y. Kosover, A. Nasiri, G. Butrico,
M. Batsu, and W. Zhu for their invaluable technical assistance;
C. Frassetto and A. Barkley for their work to formulate the CRMP;
B. Ehrlich for assistance with the thermal algesia studies;
M. Kashgarian for expert analysis of renal histology; and A. Ray
and C. Tay for toxicology advice. Yale University has applied for a
patent (provisional patent application 61/919, 003) related to the
use of CRMP and similar protonophores for the treatment of
metabolic diseases, including NAFLD/NASH and T2D. This research
was funded by grants from the United States National Institutes
of Health (R01 DK-40936, R24 DK-085638, U24 DK-059635, T32
DK-101019, P30 DK-45735, P30 DK-34989, and UL1 TR-000142) and
the Novo Nordisk Foundation Center for Basic Metabolic Research,
University of Copenhagen, Copenhagen, Denmark.
Materials and Methods
Figs. S1 to S13
11 October 2014; accepted 5 February 2015
Published online 26 February 2015;
K2P channel gating mechanisms
revealed by structures of TREK-2 and
a complex with Prozac
Yin Yao Dong,1 Ashley C. W. Pike,1 Alexandra Mackenzie,1,2 Conor McClenaghan,2,3
Prafulla Aryal,2,3,4 Liang Dong,1† Andrew Quigley,1 Mariana Grieben,1
Solenne Goubin,1‡ Shubhashish Mukhopadhyay,1 Gian Filippo Ruda,1,5
Michael V. Clausen,2 Lishuang Cao,6 Paul E. Brennan,1,5 Nicola A. Burgess-Brown,1
Mark S. P. Sansom,3,4 Stephen J. Tucker,2,3§ Elisabeth P. Carpenter1,3§
TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by
multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the
mechanisms that control channel gating are unclear. Here we present crystal structures
of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations
and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a
state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane
fenestrations found in only one of these two conformations. Channel activation by
arachidonic acid and mechanical stretch involves conversion between these states through
movement of the pore-lining helices. These results provide an explanation for TREK
channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects
of the serotonin reuptake inhibitor Prozac.
Two-pore domain potassium (K2P) channels contribute to the background leak potas- sium currents in nearly all cells and exhibit versatile, polymodal patterns of regulation. This functional diversity contributes to regu-
lation of the resting membrane potential in many
excitable and nonexcitable tissues. K2P channels
represent important clinical targets for the treat-
ment of cardiovascular disease and several neuro-
logical disorders, including pain and depression (1).
The archetypal polymodal K2P channels TREK-1
and TREK-2 are regulated by physical factors
such as mechanical stretch, voltage, and temperature; by natural ligands including polyunsaturated fatty acids such as arachidonic acid (AA);
and by intra- and extracellular pH (pHint and
pHext) (1–3). Their activity can also be modulated
by diverse pharmacological agents such as volatile anesthetics, neuroprotective drugs, and antidepressants such as fluoxetine (Prozac) (1–6). Such
diverse regulation allows these channels to couple cellular electrical activity to a variety of signaling pathways; consequently, they represent
important pharmacological targets (6). In particular, TREK channels are inhibited in vitro by
fluoxetine and its active metabolite norfluoxetine
at physiologically relevant concentrations (4, 5, 7).
This selective serotonin reuptake inhibitor is
used in the treatment of a range of depressive
and anxiety disorders. In addition to its principal
effect of directly inhibiting serotonin transporters,
fluoxetine also inhibits several G protein–coupled
receptors and ion channels (4, 8). TREK-1 knockout mice appear resistant to depression, suggesting that TREK channel inhibition by fluoxetine
may contribute to its antidepressant effects (5, 8).
Inhibition of TREK channels in the cardiovascular system may also contribute to some of the
drug’s known side effects (9). Norfluoxetine is a
state-dependent blocker of TREK channels (4)
and is used here as a tool compound to probe the
structural basis of TREK channel inhibition.
The molecular and structural mechanisms
that allow K2P channels to sense such diverse
stimuli are poorly understood. Structures of two
members of the K2P channel family (TRAAK and
TWIK-1) reveal that they share many basic structural features with classical tetrameric K+ channels
but assemble as dimers with a pseudotetrameric
pore (10–12). Also, they do not appear to gate via
constriction of the cytoplasmic entrance to the
pore. Instead, this lower part of the conduction
pathway remains open even when the channel is
closed, and gating occurs primarily within the
selectivity filter (2, 13, 14). However, the mechanisms that relay regulatory stimuli to the pore,
and how drugs modulate this process, remain
To understand the mechanisms of polymodal
K2P channel gating and inhibition by drugs, we
solved the crystal structure of human TREK-2 in
two conformations at 3.4 and 3.9 Å resolution
(15) (figs. S1 and S2 and table S1). The truncated
protein used for crystallization retains many functional properties exhibited by wild-type TREK-2,
including activation by stretch and AA and inhibition by norfluoxetine (fig. S3). The two TREK-2
structures show the classic K2P channel fold
(10–12), with four transmembrane helices (M1
to M4), two pore-forming regions per chain (P1
and P2), and an extracellular cap domain (Fig. 1,
A and B). TREK-2 exhibits the domain swap seen
in TRAAK (10) (fig. S4).
Differences between the two conformations
are centered around the lower sections of the
M2, M3, and M4 helices (Fig. 1, C and D; fig.
S5; and movie S1). In the 3.9 Å structure, these
1256 13 MARCH 2015 • VOL 347 ISSUE 6227 sciencemag.org SCIENCE
1Structural Genomics Consortium, University of Oxford, Oxford
OX3 7DQ, UK. 2Clarendon Laboratory, Department of Physics,
University of Oxford, Oxford OX1 3PU, UK. 3OXION Initiative in
Ion Channels and Disease, University of Oxford, Oxford OX1
3PN, UK. 4Department of Biochemistry, University of Oxford,
Oxford OX1 3QU, UK. 5Target Discovery Institute, Nuffield
Department of Medicine, University of Oxford, Oxford OX3 7FZ,
UK. 6Pfizer Neusentis, Granta Park, Cambridge CB21 6GS, UK.
*These authors contributed equally to this work. †Present address:
Department of Clinical Neurosciences, Cambridge Institute for
Medical Research, University of Cambridge, Cambridge CB2 0XY,
UK. ‡Present address: Solenne Goubin, School of Veterinary
Medicine and Science, University of Nottingham, Sutton Bonington
Campus, Sutton Bonington, Leicestershire LE12 5RD, UK.
§Corresponding author. E-mail: email@example.com
(E.P.C.); firstname.lastname@example.org (S.J. T.)