Nature - USA (2020-10-15)

Nature | Vol 586 | 15 October 2020 | 459

chain was most important for sealing the cavity with K245. Indeed, a
smaller side chain in N243A impairs pHint activation (by around 70%),
whereas a larger side chain in N243R has no significant effect despite
its positive charge (Fig. 3g). W244A results in a more subtle loss of

pHint activation (by around 47%), which is probably due to increased

flexibility of TM4 upon loss of anchoring interactions between W244

and TM3 (Fig. 3b, c Extended Data Fig. 6d, e). Whereas K245 is able to

rotate from a hydrophobic pocket at high pH towards the conduction

TASK2 pH 6.5 (closed channel)

ab c

TASK2 pH 8.5 (open channel)

Intracellular

gate

Selectivity

lter

gate

TM4 TM4

TM2

TM3 TM3

TM2 TM2

TM3 TM3

TM2

TM4 TM4

50

40

30

20

10

012345

Radius of conduction pathway (Å)

Distance along

conduction axis (Å)

Intracellular

gate

S0 Selectivity lter gate

S1

S2

S4

S3

TASK2 closed

TASK2 open

Fig. 2 | Comparison of open and closed TASK2 structures captured at high
and low pH. a, b, Structures of TASK2 determined at pH 8.5 (a) and pH 6.5 (b).
The surface of the conduction pathway through the channel is shown in grey.
c, The radius of the channel interior as a function of distance along the

conduction pathway. The plot is drawn at the same scale as the surfaces in a

and b. The positions of the K+ coordination sites S0–S4, the extracellular

selectivity filter gate and the intracellular gate at the membrane–cytoplasm

interface are indicated.

W244

V246

K245

N243

TM4

TASK2 closed

+

~90º

TM4

W244

V246

K245

N243

Overlay

W244

N243

K245

N243

W244

K245

C

C

a

d

c

g

TASK2 closed

TASK2 open

W244

V246

N243 K245

TM4

TASK2 open

ef

90º

K245

N243

W244

W244

K245+ N243

+

C

C

b

TM4 TM4

TM2

TM3

C

TM3

TM2

TASK2 open TASK2 closed

Conduction axis Conduction axis

**** *** NS * ****

WT

K245AN243AN243RW244A

N243K/K245N

0

1

2

3

I(pH

int

9)/

I(pH

7)int

Fig. 3 | A TASK2 intracellular gate controlled by pHint. a, Overlay of closed
and open conformations of TASK2 viewed from the membrane plane,
highlighting conformational changes in the cytoplasmic end of TM4.
b, c, Surface representation of the region boxed in a viewed from the
cytoplasmic side for open (b) and closed (c) channels. The residues involved in
gating and the positive charge on K245 at low pH are indicated. d–f, View from
the membrane plane of one TM4 and intracellular gating residues from the
open conformation (d), closed conformation (e) and both structures overlaid
(f). In d and e, a dashed line is drawn at the centre of the K+ conduction axis for

reference. g, Normalized fold activation of current by alkaline pHint (pHint = 9.0/

pHint = 7.0 at 0 mV) for wild-type (WT) TASK2 (1.95 ± 0.11) and mutants K245A

(1.03 ± 0.04), N243A (1.32 ± 0.08), N243R (1.67 ± 0.16), W244A (1.53 ± 0.13) and

N243K/K245N (1.11 ± 0.07). Mean ± s.e.m. are reported and plotted for n = 7, 3, 9,

4, 5 and 6 cells from 4, 2, 5, 3, 2 and 2 independent transfections, respectively.

Differences were assessed with one-way analysis of variance (ANOVA) with

Dunnett correction for multiple comparisons. ****P < 0.0001 for K245A and

N243K/K245N. ***P = 0.0002 for N243A. *P = 0.04 for W244A. P = 0.34 (NS) for

N243R.