medway school of pharmacy
Structural Mechanisms of Two Pore Domain
Potassium (K2P) Channel Gating
Alistair Mathie
Medway School of Pharmacy
University of Kent
Royal Society Industry Fellow
The voltage-gated-like (VGL) ion channel “chanome”
(from Yu & Catterall 2004, Science STKE 253, 15)
15 mammalian K2P channels in 6 families
6 families of K2P channels
N
TALK
TREK
TASK
NH 2
T
c
THIK
PSD motif.
TWIK
TRESK
K2P channels are dimers
c
A
A
COOH
K2P structure (TREK1)
Based on KcsA
Based on KvAP
K2P structure (TREK1)
Based on KcsA
Based on KvAP
Based on TRAAK
(Brohawn et al 2012)
K2P structure (TREK1)
Themes
• Physiological Roles of K2P channels
• How do regulatory compounds act on K2P channels
to produce their effect?
• Do K2P channels have a single gate at the selectivity
filter?
• Regulation of TREK1 isoforms
Development of Resting Membrane Potential
and IKSO in cerebellar granule neurons (CGNs)
with time in culture
Watkins & Mathie (1996) J Physiol 491: 401-412
K2P channel expression in the mouse cerebellum
Aller et al (2005) J Neurosci 25: 11455-11467
IKSO is blocked by extracellular acidification
Millar et al (2000) PNAS 97: 3614-3618
TASK-3 KO mice have a reduced IKSO and a
compromised neuronal excitability
Brickley et al (2007) J Neurosci 27: 9329-9340
TASK3 is selectively blocked by Zinc
600
2500
Zinc (100 M)
Current (pA)
2000
1500
Control
1000
Zinc (100 M)
Current (pA)
Zinc (100 M)
400
Control
Zinc (100 M)
200
500 pA
500
1000 pA
100 ms
100 ms
0
0
100
200
Time (Seconds)
TASK3
300
400
0
0
100
Time (Seconds)
TASK1
200
300
E70 in the M1/P1 loop and H98 at the selectivity
filter have a role in zinc regulation of
TASK3 channels
E70
H98
N
NH 2
T
c
PSD motif.
c
A
A
COOH
E70 & H98 contribute to TASK-3 zinc sensitivity
TASK-3 E70K_H98A
100
Current (pA)
1600
1200
zinc
800
control
400
0
% Inhibition
80
Zinc (100 M)
60
40
20
0
0
100
200
Time (s)
300
-20
TASK-3
TASK-3
H98A
TASK-3
E70K
TASK-3
E70K_H98A
Clarke et al (2008) J Biol Chem 283: 16985-92
K2P structure
Based on KcsA
Based on KvAP
Based on TRAAK
(Brohawn et al 2012)
Muscarinic ACh receptor activation inhibits IKSO
-20 mV
-20
-30
-40
800
-50
-60 mV
-60
0
200
400
600
800
1000
1200
I (pA)
600
control
400
200
10 M muscarine
-20
0
-40
-60
0
-80
-100
0
200
500
400
600
1000 1500
Time (ms)
800
2000
1000
Mathie (2007) J Physiol 578: 377-385
How does M3 receptor activation inhibit TASK channels?
Mathie (2007) J Physiol 578: 377-385
Does Gq inhibit TASK-3 directly?
TASK
Agonist
GPCR
1500
Current (pA)
Gq
1000
500
+ Gq*
+ Gq*
+ Gq*RT
0
TASK-3
TASK-3(PKC-)
Constitutively active Gq (Gq*) inhibits TASK-3 current.
So too does a further mutated form of Gq (Gq*,R256A,T257A)
which does not activate PLC.
Veale et al (2007) Mol Pharmacol 71: 1666-1675
A region of 6 amino acids of TASK channels is critical
for transducing their regulation by Gq-coupled receptors
TASK3
Current (pA)
TASK-3
VLRFLT
Muscarine (0.1 M)
800
600
400
200
0
0
50
100
150
200
250
300
Time (s)
Current (pA)
TASK3VLRFLT-DEL
Muscarine (0.1 M)
800
600
400
200
0
0
50
100
150
200
250
Time (s)
Veale et al (2007) BJP 152: 778-786
K2P channels have gate(s) which may control
their activity
Selectivity filter
Gate?
Bundle Crossing
Gate?
Does A237 in TASK3 regulate the
bundle crossing gate?
from Ashmole et al (2009) J Physiol
A237T in TASK3 occludes modulation by muscarine
800
2500
Muscarine (0.1 M)
1500
Current (pA)
Current (pA)
2000
600
400
200
0
1000
500
0
muscarine (0.1 M)
-500
0
50
100
150
Time (Seconds)
200
250
-1000
0
400
800
1200
1600
Time (ms)
Emma Veale – unpublished data
Do different regulators of TASK3 act through
different gates?
zinc?
Gq?
The Pore Structure and Gating Mechanism of
K2P Channels
NH2
COOH
Dr Stephen J. Tucker
Long chain QAs can block K2P channels
from intracellular side
TREK-1
100 µM TEA+
100 µM TButA+
10 µM TPen+
10 µM THex+
Giant i/o excised patches - rTREK-1 expressed in Xenopus oocytes
Use TPenA and THexA as probes of pore structure
Piechotta et al (2011) EMBO Journal
The QA ion binding site in TREK-1.
Figure 4 from Paula L Piechotta et al.
The EMBO Journal online publication
05 August 2011 doi:10.1038/emboj.2011.268
© This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License.
Use high-affinity pore blocker to probe
gating mechanism
Gate?
Blocker
Gate?
State-dependent accessibility of QA ion to its blocker site?
State-dependent accessibility of QA to its blocker site?
Helix bundle crossing does not restrict access of QA to
blocker site when the pH gate is closed (at pH 8.0)
Mutation of W275 blunts regulation of TREK1
Bagriantsev et al 2011 & 2012 EMBO J
Gating of TREK1:
FILTER & GATE
Selectivity Filter acts as Primary Gating Mechanism
K2P channel gating
Closed?
Open?
Open/closed?
Enhancement of TREK channels by Fenamates
Takahira et al (2005) Pflugers Arch 451: 474-478
Enhancement of TREK1 by BL-1249 (1 M)
2500
1000 pA
200 ms
1500
1000
500
BL-1249 (1 M)
200
0
0
50
100
150
Time (s)
200
250
TREK1
300
% Change
Current (pA)
2000
150
100
50
0
Enhancement of TREK1 by BL-1249 (1 M)
2500
Current (pA)
2000
1000 pA
200 ms
1500
1000
500
BL-1249 (1 M)
200
0
50
100
150
200
250
TREK1
300
Time (s)
1200
Effect of BL-1249 (1 M) on TREK1_Y284A
% Change
0
150
100
50
0
Current (pA)
1000
800
600
BL-1249 (1 M)
1000 pA
400
200 ms
200
0
0
50
100
150
Time (s)
200
250
Y284A
Important residues that influence fenamate
action in TREK1
TMs 1 and 2
TMs 3 and 4 (rotated 90o)
M42
M1
Enhancement of TREK1_1-41Del by BL-1249
Enhancement of TREK1_1-41Del by FFA
Enhancement of TREK1_1-41Del_Y284A by FFA
Fenamates AND point mutations which alter TREK1
gating at the selectivity filter “overcome”
N terminus truncation
Themes
• Physiological Roles of K2P channels
• How do regulatory compounds act on K2P channels
to produce their effect?
• Do K2P channels have a single gate at the selectivity
filter?
• Regulation of TREK1 isoforms
K2P channel - colleagues
University of Kent
Imperial and UCL
Emma Veale
Ehab Al Moubarak
Mickael El Hachmane
Kate Rees
Mustafa Hassan
Fraser Eldin
Chris Watkins
Julie Millar
Catherine Clarke
David Boyd
Louise Kennard
Vadim Sumbayev
Gurprit Lall
Stefan Trapp
Stephen Brickley
Bill Wisden
Jamie Vandenberg (Sydney, Australia)
Stephen Tucker (Oxford, UK)
Eddy Stevens, Lishuang Cao, and Kiyo Omoto (Pfizer Neusentis)
Current Support: Royal Society, BBSRC, Pfizer Neusentis