Nature | Vol 586 | 15 October 2020 | 457Article
Structural basis for pH gating of the
two-pore domain K
+
channel TASK2Baobin Li1,2,3,5, Robert A. Rietmeijer1,2,3,4,5 & Stephen G. Brohawn1,2,3 ✉TASK2 (also known as KCNK5) channels generate pH-gated leak-type K+ currents to
control cellular electrical excitability^1 –^3. TASK2 is involved in the regulation of
breathing by chemosensory neurons of the retrotrapezoid nucleus in the brainstem^4 –^6
and pH homeostasis by kidney proximal tubule cells^7 ,^8. These roles depend on channel
activation by intracellular and extracellular alkalization^3 ,^8 ,^9 , but the mechanistic basis
for TASK2 gating by pH is unknown. Here we present cryo-electron microscopy
structures of Mus musculus TASK2 in lipid nanodiscs in open and closed
conformations. We identify two gates, distinct from previously observed K+ channel
gates, controlled by stimuli on either side of the membrane. Intracellular gating
involves lysine protonation on inner helices and the formation of a protein seal
between the cytoplasm and the channel. Extracellular gating involves arginine
protonation on the channel surface and correlated conformational changes that
displace the K+-selectivity filter to render it nonconductive. These results explain how
internal and external protons control intracellular and selectivity filter gates to
modulate TASK2 activity.TASK2 (TWIK-related acid-sensitive K+ channel 2) is a pH-gated member
of the two-pore domain K+ (K2P) channel family^1 ,^2. TASK2 is expressed
widely, including in neurons, immune cells, chondrocytes and epithelial
cells of organs including the kidney, and has been implicated in chem-
osensation, volume regulation and ionic homeostasis^3 ,^4 ,^7 ,^10 –^13. Upregula-
tion of TASK2 is associated with breast cancer proliferation, whereas
its loss of function underlies Balkan endemic nephropathy^14 ,^15. Physi-
ological roles of TASK2 have been related to its modulation by changes
in intracellular and extracellular pH (pHint and pHext)^3 ,^5 ,^8. In neurons of
the retrotrapezoid nucleus, inhibition of TASK2 by extracellular and/or
intracellular protons (as a proxy for blood [CO 2 ]^9 ) depolarizes the cell,
increases spike frequency and leads to increased respiration^4 –^6. In the
kidney proximal tubule, activation of TASK2 by extracellular alkaliza-
tion that results from electrogenic bicarbonate secretion hyperpolar-
izes the cell to support further bicarbonate efflux^7 ,^8.
TASK2 is predominantly closed at pH 6.5 and is activated by intracel-
lular and extracellular alkalization with a midpoint pH of 8.0–8.5^1 ,^3 ,^16.
Mutational analyses have identified proton sensors on either side of the
membrane: the extracellular residue R224 and the intracellular residue
K245^9 ,^17. Gating by pHext, but not pHint, is voltage- and [K+]ext-dependent,
which is consistent with selectivity filter (or C-type) gating by external,
but not internal, stimuli^1 ,^9. Still, the structural and mechanistic basis
for gating of TASK2 is unknown and expected to be distinct from other
K2P channels for which structures have been determined in detergent
micelles^18 –^23.
Removing the predicted unstructured C-terminal region of Mus
musculus TASK2 (resulting in TASK21–335) increased the expression
and biochemical stability of the purified channel for structural stud-
ies (Extended Data Fig. 1, Supplementary Figs. 1, 2). This construct
retained the hallmark features of TASK2 when expressed in cells;
activation by extracellular alkalization, intracellular alkalization and
intracellular phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ;
which requires an intact membrane proximal C-terminal region) was
indistinguishable from that of full-length TASK2 and comparable to
previous reports^1 ,^9 ,^16 ,^17 ,^24 (Fig. 1a–c, Extended Data Fig. 2a–e). Thus,
TASK21–335 was used for structural studies.
To determine the basis for channel gating in a lipid membrane-like
environment, we reconstituted TASK2 into nanodiscs made from
the scaffold protein MSP1D1^25 and the lipids DOPE, POPC and POPS
(Extended Data Fig. 1), and determined its structure at pH 8.5 and pH
6.5. Unmasked reconstructions show well-defined density for TASK2
and the surrounding nanodisc (Fig. 1d). Cryo-electron microscopy
(cryo-EM) maps were improved by partial signal subtraction of nanodisc
density followed by further classification and refinement (Extended
Data Figs. 3–5). The channel is twofold symmetric at high pH and asym-
metric at low pH. Although asymmetric and C 2 symmetric reconstruc-
tions of TASK2 at pH 8.5 were essentially indistinguishable, enforcing
C 2 symmetry in reconstructions of TASK2 at pH 6.5 resulted in a loss
of density features, especially around the intracellular halves of trans-
membrane helices.
Amino acids 6–260 from each chain (58 kDa total) correspond-
ing to the transmembrane and extracellular regions of the channel
were de novo modelled into the final reconstructions at pH 8.5 and
pH 6.5 (both at approximately 3.5 Å resolution) (Fig. 1d, e, Extended
Data Fig. 5, Extended Data Table 1). Like other K2P channels, TASK2 is
a domain-swapped homodimer, with each protomer chain contain-
ing four transmembrane-spanning helices (TM1–TM4), two reentrant
pore helices (PH1 and PH2), two selectivity filters (SF1 and SF2) andhttps://doi.org/10.1038/s41586-020-2770-2
Received: 16 April 2020
Accepted: 6 July 2020
Published online: 30 September 2020
Check for updates(^1) Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA. (^2) Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, USA.
(^3) California Institute for Quantitative Biology (QB3), University of California Berkeley, Berkeley, CA, USA. (^4) Biophysics Graduate Group, University of California Berkeley, Berkeley, CA, USA.
(^5) These authors contributed equally: Baobin Li, Robert A. Rietmeijer. ✉e-mail: [email protected]