Structural Mechanisms of Two Pore Domain Potassium (K2P) Channel Gating

Transcription

1 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

2 The voltage-gated-like (VGL) ion channel chanome (from Yu & Catterall 2004, Science STKE 253, 15)

3 15 mammalian K2P channels in 6 families 6 families of K2P channels N TALK TREK TASK NH 2 T c TWIK THIK PSD motif. A A COOH c TRESK K2P channels are dimers

4 K2P structure (TREK1) Based on KcsA Based on KvAP

5 K2P structure (TREK1) Based on KcsA Based on KvAP Based on TRAAK (Brohawn et al 2012)

6 K2P structure (TREK1)

7 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

8 Development of Resting Membrane Potential and IK SO in cerebellar granule neurons (CGNs) with time in culture Watkins & Mathie (1996) J Physiol 491:

9 K2P channel expression in the mouse cerebellum Aller et al (2005) J Neurosci 25:

10 IK SO is blocked by extracellular acidification Millar et al (2000) PNAS 97:

11 TASK-3 KO mice have a reduced IK SO and a compromised neuronal excitability Brickley et al (2007) J Neurosci 27:

12 TASK3 is selectively blocked by Zinc 2500 Zinc (100 M) 600 Zinc (100 M) Current (pa) Control Zinc (100 M) 1000 pa 100 ms Current (pa) pa Control Zinc (100 M) 100 ms Time (Seconds) Time (Seconds) TASK3 TASK1

13 E70 in the M1/P1 loop and H98 at the selectivity filter have a role in zinc regulation of TASK3 channels E70 N H98 NH 2 T c PSD motif. A A COOH c

14 E70 & H98 contribute to TASK-3 zinc sensitivity TASK-3 E70K_H98A Current (pa) Zinc (100 M) zinc control % Inhibition Time (s) -20 TASK-3 TASK-3 H98A TASK-3 E70K TASK-3 E70K_H98A Clarke et al (2008) J Biol Chem 283:

15 K2P structure Based on KcsA Based on KvAP Based on TRAAK (Brohawn et al 2012)

16 Muscarinic ACh receptor activation inhibits IKSO mv -60 mv I (pa) control 10 M muscarine Time (ms) Mathie (2007) J Physiol 578:

17 How does M 3 receptor activation inhibit TASK channels? Mathie (2007) J Physiol 578:

18 Does G q inhibit TASK-3 directly? TASK Agonist GPCR 1500 G q Current (pa) 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:

19 A region of 6 amino acids of TASK channels is critical for transducing their regulation by G q -coupled receptors TASK-3 Current (pa) TASK3 Muscarine (0.1 M) VLRFLT Time (s) Current (pa) 800 Muscarine (0.1 M) TASK3 VLRFLT-DEL Time (s) Veale et al (2007) BJP 152:

20 K2P channels have gate(s) which may control their activity Selectivity filter Gate? Bundle Crossing Gate?

21 Does A237 in TASK3 regulate the bundle crossing gate? from Ashmole et al (2009) J Physiol

22 A237T in TASK3 occludes modulation by muscarine Current (pa) Muscarine (0.1 M) Time (Seconds) Current (pa) Time (ms) muscarine (0.1 M) Emma Veale unpublished data

23 Do different regulators of TASK3 act through different gates? zinc? G q?

24 The Pore Structure and Gating Mechanism of K2P Channels NH 2 COOH Dr Stephen J. Tucker

25 Long chain QAs can block K2P channels from intracellular side TREK µm TEA 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

26 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: /emboj This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License.

27 Use high-affinity pore blocker to probe gating mechanism Gate? Blocker Gate? State-dependent accessibility of QA ion to its blocker site?

28 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)

29 Mutation of W275 blunts regulation of TREK1 Bagriantsev et al 2011 & 2012 EMBO J

30 Gating of TREK1: FILTER & GATE Selectivity Filter acts as Primary Gating Mechanism

31 K2P channel gating Closed? Open? Open/closed?

32 Enhancement of TREK channels by Fenamates Takahira et al (2005) Pflugers Arch 451:

33 Enhancement of TREK1 by BL-1249 (1 M) 2500 Current (pa) BL-1249 (1 M) Time (s) 1000 pa 200 ms % Change TREK1 0

34 Enhancement of TREK1 by BL-1249 (1 M) 2500 Current (pa) BL-1249 (1 M) pa Time (s) Effect of BL-1249 (1 M) on TREK1_Y284A 200 ms % Change TREK1 Y284A 1000 Current (pa) BL-1249 (1 M) 1000 pa 200 ms Time (s)

35 Important residues that influence fenamate action in TREK1 TMs 1 and 2 TMs 3 and 4 (rotated 90 o )

36 M1 M42

37 Enhancement of TREK1_1-41Del by BL-1249

38 Enhancement of TREK1_1-41Del by FFA

39 Enhancement of TREK1_1-41Del_Y284A by FFA

40 Fenamates AND point mutations which alter TREK1 gating at the selectivity filter overcome N terminus truncation

41

42 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

43 K2P channel - colleagues University of Kent Emma Veale Ehab Al Moubarak Mickael El Hachmane Kate Rees Mustafa Hassan Fraser Eldin Vadim Sumbayev Gurprit Lall Imperial and UCL Chris Watkins Julie Millar Catherine Clarke David Boyd Louise Kennard 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