Discovery of a novel response of ion channels to membrane potential, using molecular dynamics. More on this work here: https://doi.org/10.3389/fmolb.2020.00162

1. Molecular Domino Effect
Bignucolo Olivier, 16.11.2020
Faculty of Biology and Medicine
Department of Biomedical Sciences

2. Voltage-gated potassium channels 
THE most diverse group in the ion channel family
Functions in eukaryotic cells
- neural signaling
- cardiac rhythm
- potential drug target in cancer (Huang and Jan, JCB 2014)
e.g. restoration of the membrane potential after an action potential
Why voltage-gated K+ channels (Kv)?

3. Example heart:
ventricular action potential and ionic currents are tightly regulated by Kv channels
Schwartz et. al (2020)

4. General architecture of voltage-gated
ion channels
Voltage-Sensor Domain  VSD

5. Potassium channels: four subunits
arrange to form a tetramer
Voltage-Sensor Domain  VSD
4 VSDs
1 pore

6. Potassium channels: the S4 helix
harbours a conserved pattern involving basic residues
4 to 6 ARG/LYS

7. Schematic response of the channel to the
membrane potential fluctuations(Vm)

8. Crystal structures of voltage-sensor domains
in the active state
Bignucolo and Bernèche (2020)
S3
S4
S1
S2
Kv1.2 Kv1.2/2.1

9. “The” consensus describing the VSD response
to membrane polarization
Vargas et. al (2012)
Membrane depolarized
Negative charges
Positive charges

10. “The” consensus describing the VSD response
to membrane polarization
Vargas et. al (2012)
Membrane hyperpolarized

11. “The” consensus describing the VSD response
to membrane polarization
Vargas et. al (2012)

12. The consensus model is applicable to some channels
Bignucolo and Bernèche (2020)
S3
S4
S1
S2
Kv1.2 Kv1.2/2.1

13. Bignucolo and Bernèche (2020)
S3
S4
S1
S2
Kv1.2 Kv1.2/2.1 KvAP
The consensus model is difficult to apply to other
channels like e.g. KvAP

14. Bignucolo and Bernèche (2020)
S3
S4
S1
S2
Kv1.2 Kv1.2/2.1 KvAP
A sliding of S4 would bring hydrophobic residues in
contact with hydrophilic species

15. What is the response of this family of channels to
membrane hyperpolarization?
1- Presentation of the double bilayer simulation system
2- A novel response of the Voltage-Sensor Domain to membrane hyperpolarization
3- Apply the model to the full-length structure

16. What is the response of this family of channels to
membrane hyperpolarization?
1- The double bilayer simulation system
2- A novel response of the Voltage-Sensor Domain to membrane hyperpolarization
3- Apply the model on the full-length structure

17. Usual bilayer: ion concentration gradient not feasible
‘Upper side’
‘Lower side’
“Usual” system:
periodic boundary conditions:
 one solvent compartment
no ion differential
 no membrane potential

18. 1) duplicate the box
2) antiparallel duplication
Upper side

19. Upper side
Upper
side
1) copy the system
2) antiparallel duplication

20. Result: a biologically relevant system with
“inside” and “outside” compartments
Extracellular cell
compartment
Extracellular cell
compartment
Intracellular cell
compartment

21. One can generate a membrane potential (Vm)
Simply move a few ions “manually”
Vm = Vextracellular - Vintracellular
Black line : Vminit
Red line: Vmend

22. What is the response of this family of channels to
membrane hyperpolarization?
1- Presentation of the double bilayer simulation system
2- A novel response of the Voltage-Sensor Domain to
membrane hyperpolarization
3- Apply the model to the full-length structure

23. Constructed 65 such double bilayer systems and used
the electrostatic potential values for quality check
Deviation ≡ Vmend – Vminit
What is expected
deviation ~ 0  stable system

24. Four outliers  charge transport !!
In four simulations
electric charge transport

25. The charge translocation depends on the initial Vm
R = 0.82
p < 0.001
Bignucolo and Bernèche (2020)

26. What happened?
Observe the Arg133—Asp62 salt bridge
Bignucolo and Bernèche (2020)

27. What happened in the four trajectories?
1) Rupture of the R133—D62 salt bridge
2) breakage of S4
Bignucolo and Bernèche (2020)
S4 Nter
S4 Cter
Gly134
Vm ~ 0
S4 straight
salt bridge formed
Vm << 0
S4 broken at G134
rupture of the salt bridge

28. What happened in the four trajectories?
1) Rupture of the R133—D62 salt bridge
2) breakage of S4
Bignucolo and Bernèche (2020)
S4 Nter
S4 Cter
Gly134
Vm ~ 0
S4 straight
salt bridge formed
Vm << 0
S4 broken at G134
rupture of the salt bridge

29. What happened in the four trajectories?
1) Rupture of the R133—D62 salt bridge
2) Breakage of S4
Bignucolo and Bernèche (2020)
S4 Nter
S4 Cter
Gly134
Vm ~ 0
S4 straight
salt bridge formed
Vm << 0
S4 broken at G134
rupture of the salt bridge

30. What happened in the four trajectories?
1) Rupture of the R133—D62 salt bridge
2) Breakage of S4
3) This specific sequence: only prokaryotes and archaea
Bignucolo and Bernèche (2020)
S4 Nter
S4 Cter
Gly134
Vm ~ 0
S4 straight
salt bridge formed
Vm << 0
S4 broken at G134
rupture of the salt bridge

31. Co-occurrence of the salt bridge rupture and
the S4 breakage ? upon hyperpolarization
S4 bending angle time
evolution of trajectories in
which the distance between
Asp62 and Arg133 remained
stable at ∼ 2Å (nVSD = 126).
S4 bending angle time
evolution of the four
trajectories harboring a charge
translocation
S1
S2
D62
R133
S3
S4
Salt bridge distance of the four trajectories
harboring a charge translocation
S4 bending angle of the four
trajectories harboring a charge
translocation

32. Normalization to the time of the salt bridge
rupture  what happens to S4?
S4 bending angle time
evolution of trajectories in
which the distance between
Asp62 and Arg133 remained
stable at ∼ 2Å (nVSD = 126).
S4 bending angle time
evolution of the four
trajectories harboring a charge
translocation
S1
S2
D62
R133
S3
S4
Salt bridge distance of the four trajectories
harboring a charge translocation
S4 bending angle of the four
trajectories harboring a charge
translocation

33. The S4 breakage and the rupture of the salt
bridge occur simultaneously
S4 bending angle time
evolution of trajectories in
which the distance between
Asp62 and Arg133 remained
stable at ∼ 2Å (nVSD = 126).
S4 bending angle time
evolution of the four
trajectories harboring a charge
translocation
S1
S2
D62
R133
S3
S4
The time points of the salt
bridge rupture and initiation
of S4 bending superpose
exactly

34. Co-occurrence of the salt bridge rupture and
the S4 breakage upon hyperpolarization
Bignucolo and Bernèche (2020)
S4 bending angle time
evolution of trajectories in
which the distance between
Asp62 and Arg133 remained
stable at ∼ 2Å (nVSD = 122).
S1
S2
D62
R133
S3
S4 C-Ter

35. Co-occurrence of the salt bridge rupture and
the S4 breakage upon hyperpolarization
Bignucolo and Bernèche (2020)

36. Co-occurrence of the salt bridge rupture and
the S4 breakage upon hyperpolarization
Bignucolo and Bernèche (2020)

37. Co-occurrence of the salt bridge rupture and
the S4 breakage upon hyperpolarization
Displaced ions to
generate a positive
membrane potential

38. Restoration of the salt bridge and the S4
breakage upon depolarization
Restoration of the
salt bridge

39. The bending of S4 follows a similar trend
Restoration of the
salt bridge

40. What is the response of this family of channels to
membrane hyperpolarization?
1- The double bilayer simulation system
2- A novel response of the Voltage-Sensor Domain to membrane hyperpolarization
3- Apply the model to the full-length structure
Tao and MacKinnon (2019)

41. Dec. 2019: Full-length structure of KvAP
Structure published by Tao and MacKinnon (2019) inserted in a lipid bilayer
Brown spheres:
Phosphorous atoms
Each monomer a different colour:
blue, red, orange, grey

42. Details of the active state (at Vm = 0)
S1
S2
S3 S4 S5 pore helix and S6
Selectivity filter
Voltage-sensor Pore domain Voltage-sensor

43. G134 Ca
Pull force to induce the known bending of S4
Apply the model on the cryo-EM structure
1) Pull force  S4 bending after ~ 23 ns
2) 17 double bilayer systems, exposed to various Vm values

44. Apply the model on the cryo-EM structure
1) Pull force  S4 bending after ~ 23 ns
2) 17 double bilayer systems, exposed to various Vm values

45. Define the orientation of the S4 C-ter

46. Define the orientation of the S4 C-ter
in respect to the bilayer normal

47. Simple definition of S4 broken and S4 straight
> 60°
 broken
< 40°
 straight

48. Apply the model on the cryo-EM structure
1) Pull force  S4 bending after ~ 23 ns
2) 17 double bilayer systems, 136 VSDs exposed to various Vm
Vm << 0 --> S4 Broken, n = 19
Vm ~ 0 --> S4 Straight, n = 33
Discard the intermediate cases
for statistical analysis

49. How does the breakage of S4 affects the pore domain?
 we observe the S5 helix orientation
Bignucolo and Bernèche (2020)
S5
S4

50. How does the breakage of S4 affects the pore domain?
 Effect of the pull force on S4 C-ter
S5
S4

51. Confirm the model once again: hyperpolarization  S4 breakage
S5
S4

52. S4 C-ter along the lipid head groups  S5 reorientation ~ 20°
S5
S4

53. Vm ~ 0  restoration of S4 initial orientation
Bignucolo and Bernèche (2020)
S5
S4

54. Vm ~ 0  S4 straight again and S5 back to ~ 10°
Bignucolo and Bernèche (2020)
S5
S4

55. Successive conformational changes:
1) S4 breakage  2) S4 C-ter pushes S5 N-Ter
3) S5 C-ter pushes on the top of the pore helix
Bignucolo and Bernèche (2020)
S5
S4

56. Bignucolo and Bernèche (2020)
Successive conformational changes:
S4 breakage  S4 C-ter pushes S5 N-Ter

57. S4 breakage 
The pore helix N-ter approaches the selectivity filter

58. S4 breakage 
The pore helix N-ter approaches the selectivity filter

59. Molecular Domino Effect
S4 breakage  S4 C-ter pushes S5 N-Ter
S5 C-ter is constrained  pushes pore helix
 pore helix likely to restraint selectivity filter ≡ deactivation

60. Molecular Domino Effect
S4 breakage  S4 C-ter pushes S5 N-Ter
S5 C-ter is constrained  pushes pore helix
 pore helix likely to restraint selectivity filter ≡ deactivation

61. Molecular Domino Effect
S4 breakage  S4 C-ter pushes S5 N-Ter
S5 C-ter is constrained  pushes pore helix
 pore helix likely to restraint selectivity filter ≡ deactivation
Bignucolo and Bernèche (2020)

62. Relevance:
- First complete description of the interactions from S4 to the pore helix
in a Kv channel
- The “consensus” model should be extended to take this response into
account
- Other points, not shown here
- sequence unique to prokaryotes
- solution to the avidin accessibility experiment paradox
Bignucolo and Bernèche (2020)

63. Thanks!
This meeting:
Molecular Modelling Group
Vincent Zoete, Olivier Michielin
Ute Roehrig
and Team
The paper:
Simon Bernèche, Swiss Institute of Bioinformatics
Annaïse Jauch, University of Basel
Niklaus Johner, University of Basel
Stephan Kellenberger, University of Lausanne
and the reviewers of the journal

65. What is really new?
Shenkarev et. al (2010) NMR investigation detects a loss of helicity near Gly134
 Hints about a possible breakage of S4
No mention of the salt bridge
No membrane potential
Butterwick and Mackinnon (2010) The NMR structure of the VSD == 20 conformations
 Three conformations harboured a bended S4 helix
Not commented in the article
Freites and Tobias (2015) Very long MD simulation with membrane potential
 Detection of the salt-bridge rupture response to Vm
No mention of the S4 breakage

66. This is new:
The two conformational changes occur simultaneously and constitute
together the response of the VSD to the hyperpolarization
Shenkarev et. al (2010) NMR investigation detects a loss of helicity near Gly134
 Hints about a possible breakage of S4
No mention of the salt bridge
No membrane potential
Butterwick and Mackinnon (2010) The NMR structure of the VSD == 20 conformations
 Three conformations harboured a bended S4 helix
Not commented in the article
Freites and Tobias (2015) Very long MD simulation with membrane potential
 Detection of the salt-bridge rupture response to Vm
No mention of the S4 breakage

67. Sequence unique to prokaryotes

68. Decorrelation for good statistics

69. RMSD for one of the reviewers

70. Solution to the avidin accessibility experiment paradox