1. Why the Selectivity Filter is the Gate”
“Top Ten Reasons for
Why the Selectivity Filter is the Gate”
Mark L. Chapman
Antonius M. J. VanDongen
(*) “Letterman” *
“Top ten reasons for: Why the selectivity filter is the gate” – ML Chapman & AMJ VanDongen

2. Hille, 1992 Doyle et al., 1998 Selectivity filter Out In Gate K +
X-ray structure confirms picture from Hille’s textbook

3. I Voltage sensor Gate S4 Resting Active Closed Open msec, sec
Macroscopic current kinetics reflects movement of the 4 voltage sensors.
Single channel open-close behavior reflects movement of the “gate”.
msec, sec C O
< 10 msec

4. Closed  Open transition: the gate moves
During the transition from “closed” to “open” , the channel gate moves (changes conformation).
0.2 pA
3 msec
closed

5. Sublevels are visited during open-closed transitions
1 pA
10 msec
In the drk1 (Kv2.1) K channel, short-lived subconductance levels (arrows) are visited during open-closed transitions.
open
closed

6. Subunit composition and closedopen transitionH3
H2a
H2b
The sublevels seen in the previous slide are proposed to result from heteromeric pore conformations, in which some but not all four subunits support permeation. This is the subunit-subconductance hypothesis.
0.2 pA H1
3 msec
closed

7. drk1-L at threshold (–40 mV): sublevel visits abundant during early openings
A model based on our subunit-subconductance hypothesis (see Chapman et al 1997) predicts that sublevels should be more abundant at activation threshold. This was indeed the case.

8. Conclusion from subconductance analysis.
From: Chapman et al., 1997, Biophys. J. 72: 708.
“Ions could be prevented from translocating in the ‘closed’ conformation because of an energy well that is too deep (i.e. a high-affinity binding site). A conformational change that reduces the depth of the well would enable the channel to support ion permeation. ... permeation and gating are coupled: the same structure that controls permeation is also responsible for opening and closing the channel.”
Conclusion from subconductance analysis: permeation and gating are strongly coupled.

9. Conclusion from subconductance analysis.
From: Chapman et al., 1997, Biophys. J. 72: 708.
“Ions could be prevented from translocating in the ‘closed’ conformation because of an energy well that is too deep (i.e. a high-affinity binding site). A conformational change that reduces the depth of the well would enable the channel to support ion permeation. ... permeation and gating are coupled: the same structure that controls permeation is also responsible for opening and closing the channel.”
The selectivity filter
The same structure the controls permeation (“the selectivity filter”) …..

10. Conclusion from subconductance analysis.
From: Chapman et al., 1997, Biophys. J. 72: 708.
“Ions could be prevented from translocating in the ‘closed’ conformation because of an energy well that is too deep (i.e. a high-affinity binding site). A conformational change that reduces the depth of the well would enable the channel to support ion permeation. ... permeation and gating are coupled: the same structure that controls permeation is also responsible for opening and closing the channel.”
The selectivity filter is the gate.
Is also responsible for opening and closing the channel: the selectivity filter is the gate

11. The selectivity filter is the gate Mechanism: Affinity switching.
C O
High affinity
Low affinity
Possible mechanisms by which the filter could gate the channel: Affinity Switching
Closed state: traps K ions
Open state: release bound ions
Selectivity filter alters conformation

12. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 10.
The KcsA structure with 2 K ions in the selectivity filter represents the closed conformation.
Doyle et al, 1998
Reason #10. The KcsA structure represents the closed conformation

13. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 10.
The KcsA structure with 2 K ions in the selectivity filter represents the closed conformation.
The structure was obtained at a pH where the channel is closed (Clapham 1999, Cell 97: )
#10. The structure was obtained at a pH where the channel is closed.

14. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 10.
The KcsA structure with 2 K ions in the selectivity filter represents the closed conformation.
The structure was obtained at a pH where the channel is closed (Clapham 1999, Cell 97: )
The electrophysiological properties of the open KcsA channel are incompatible with the published crystal structure (Meuser et al., 1999, FEBS Letters 462: ).
#10. The electrophysiological properties of the open KcsA channel are incompatible with the published structure.

15. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 9.
The selectivity filter has a different conformation in the open an closed state.
Reason #9. The selectivity filter has a different conformation in the open and closed state.

16. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 9.
The selectivity filter has a different conformation in the open an closed state.
In the open state, single KcsA channels:
are poorly ion selective
permeate partially hydrated K ions
have a wider diameter than seen in the crystal structure.
(Meuser et al., 1999, FEBS Letters 462: 447).
#9. In the open state, single KcsA channels:
-are poorly ion selective
- permeate partially hydrated K ions
- have a wider diameter than seen in the crystal structure

17. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 8.
Permeant ions bind with high affinity in the pore.
Reason #8. Permeant ions bind with high affinity in the pore.

18. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 8.
Permeant ions bind with high affinity in the pore.
This was first described for Ca2+ ions in Ca channels
Armstrong & Neyton, 1991, Ann. N.Y. Acad. Sci. 635:18-25;
Kuo & Hess, 1993, J. Physiol. 466: ;
Yang et al., 1993, Nature 366: ;
Ellinor et al., 1995, Neuron 15:
Polo-Parada, & Korn, 1997, J. Gen. Physiol. 109: ;
#8. This was first described for Ca ions in Ca channels

19. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 8.
Permeant ions bind with high affinity in the pore.
K ions also bind with high affinity in the K channel pore:
mM K concentrations block Na conductance
Kiss et al., 1998, J. Gen. Physiol. 111: ;
Immke & Korn, 2000, J. Gen. Physiol. 115:
#8. K ions also bind with high affinity in the K channel pore: uM K concentrations block Na conductance of K channel.

20. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 8.
Permeant ions bind with high affinity in the pore.
K ions also bind with high affinity in the K channel pore:
mM K concentrations block Na conductance
Kiss et al., 1998, J. Gen. Physiol. 111: ;
Immke & Korn, 2000, J. Gen. Physiol. 115:
Short closed times in single channel records result from K ions acting as pore blockers
Choe et al., J. Gen. Physiol. 112:
#8. Short closed times in single channel records result from K ions acting as pore blockers.

21. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 7.
An alternative is needed for the cytoplasmic constriction acting as a gate, since it is not universally found.
Reason #7. An alternative is needed for the cytoplasmic constriction acting as a gate, since it is not universally found.

22. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 7.
An alternative is needed for the cytoplasmic constriction acting as a gate, since it is not universally found.
Inward rectifying K channels have a wide internal entrance (Lu et al., 1999, PNAS 96: 9926).
#7. Inward rectifying K channels have a wide internal entrance.

23. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 7.
An alternative is needed for the cytoplasmic constriction acting as a gate, since it is not universally found.
Inward rectifying K channels have a wide internal entrance (Lu et al., 1999, PNAS 96: 9926).
Glutamate receptors, which have an inverted topology, have a wide external vestibule
(Kuner et al., 1996, Neuron 17: 343).
#7. Glutamate receptors, which have an inverted topology, have a wide external vestibule.

24. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 7.
An alternative is needed for the cytoplasmic constriction acting as a gate, since it is not universally found.
Inward rectifying K channels have a wide internal entrance (Lu et al., 1999, PNAS 96: 9926).
Glutamate receptors, which have an inverted topology, have a wide external vestibule
(Kuner et al., 1996, Neuron 17: 343).
In CNG1, the cytoplasmic constriction does not prevent K ions from entering the vestibule.
(Flynn and Zagotta, this meeting)
#7. In CNG1, the cytoplasmic construction does not prevent K ions from entering the vestibule.

25. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 6.
There is a strong coupling between sensor movement and the conformation of the selectivity filter.
The effect of mutations in S4 on activation properties depends critically on whether the selectivity filter contains a Val or Leu at position 76.
Reason #6. There is a strong coupling between voltage sensor movement and the conformation of the selectivity filter.

26. drk1-LS drk1-S Drk1-S: triple mutation in S4  threshold +80 mV
Drk1-LS: additional mutation V76L (selectivity filter)
-40 40 80
120 E m
(mV)
0.0
0.5
1.0 G
max
drk1-LS
drk1-S
Reason #6.
Drk1-S: triple mutations in S4: threshold shifted +80 mV
Drk1-LS: additional mutation V76L (in the filter): normal threshold.

27. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 5.
Open state stability is determined by the permeating ion species, linking gating to selectivity.
(Spruce et al., 1989, J. Physiol. 411: 597).
Reason #5, Open state stability is determined by the permeating ion species, linking gating to selectivity.

28. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 5.
Open state stability is determined by the permeating ion species, linking gating to selectivity.
Spruce et al., 1989, J. Physiol. 411: 597.
Open times are very different for K and Rb in KcsA.
Lisa Heginbotham (personal communication)
Eduardo Perozo et al. (this meeting)
#5. Open times are very different for K and Rb ions in KcsA.

29. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 4.
Mutations in the selectivity filter affect single channel gating.
Reason #4. Mutations in the selectivity filter affect single channel gating.

30. D378E E D G Y G V
0.5 pA T
50 msec T
drk1
#4. Mutation D378E in the GYGD signature sequence of drk1 strongly reduces mean open time and opening frequency.

31. D G Y G V T T L
drk1
#4. Mutation V374L in the filter strongly increases open state stability.

32. D  E: Destabilization open stateG Y G
V  L: Stabilization open state &
subconductances (drk1) V T T
#4. Mutations at 3 different positions in the filter alter open state stability and/or stabilize sublevels in both drk1 and Shaker. A T
T  S: Stabilization open state &
subconductances (Shaker)

33. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 3.
In the NMDA receptor, a conserved Asparagine residue critical for Ca permeability and Mg block, stabilizes subconductance levels.
(Schneggenburger & Ascher, 1997, Neuron 18: 167).
Reason #3. In the NMDA receptor, a conserved Arginine residue critical for Ca permeability and Mg block, stabilizes subconductance levels.

34. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 2.
The direction of the K flux determines:
the open state stability in drk1.
which (sub)conductance levels predominate in KcsA (Meuser et al., 1999, FEBS Lett. 462: 447).
Reason #2. The direction of the K flux determines:
- the open state stability in drk1
- which (sub)conductance levels predominate in KcsA.

35. Open state stability depends on direction of K flux

36. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 1.
The selectivity filter makes a better gate, because of energy considerations.
Reason #1. The selectivity filter makes a better gate, because of energy considerations.

37. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 1.
The selectivity filter makes a better gate, because of energy considerations.
Single channel gating:
Highly reversible.
C-O transition timescale: microseconds.
Closed-Open transition requires little free energy.
#1. Single channel gating:
- highly reversible
- C-O transition timescale = microseconds
- Closed-open transition requires little free energy
0.2 pA
3 msec

38. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 1.
The selectivity filter makes a better gate, because of energy considerations.
Single channel gating:
Highly reversible, timescale of microseconds.
Closed-Open transition requires little free energy.
Rotation of 4 S6 a-helices: energetically expensive
#1. Rotation of four S6 alpha-helices = energetically expensive.

39. Why the Selectivity Filter is the Gate
Top Ten Reasons for
Why the Selectivity Filter is the Gate
Reason # 1.
The selectivity filter makes a better gate, because of energy considerations.
Single channel gating:
Highly reversible, timescale of microseconds.
Closed-Open Transition requires little free energy.
Rotation of four S6 a-helices: energetically expensive.
Affinity-switching allows selectivity filter to gate the channel efficiently.
Affinity-switching allows selectivity filter to gate the channel efficiently.

40. Monte Carlo simulation of affinity-switching selectivity filterNa K
Monte Carlo simulation of affinity-switching selectivity filter.

41. Monte Carlo simulation of affinity-switching selectivity filterNa K
Equal opportunity version.

42. CLOSED OPEN K K Na X High-affinity state. Low-affinity state.
High K selectivity.
No ion selectivity
The affinity switching model.
No permeation.
Efficient Permeation.

43. M.C. Simulation Results for 1-site Model
1000
K selectivity
100
(K/Na flux ratio) 10
Results from MC simulation 1
0.001
0.010
0.100
1.000
Probability of being in low affinity state

44. M.C. Simulation Results for 1-site Model
100%
Normalized
K flux
10%
Result from MC simulations of 1-site model: 100-fold K selectivity with only 5-fold reduction of flux rate. 1%
0.001
0.010
0.100
1.000
Probability of being in low affinity state

45. K selectivity and flux as a function of P_low for 2-site model
10000
10000
With ion-ion repulsion
Without ion-ion repulsion
1000
1000
K/Na flux ratio
100
100
Results for 2-site model 10 10 1 1
0.01
0.1 1
0.01
0.1 1
Prob of being in low-affinity state
Prob of being in low-affinity state

46. The gate ?
The selectivity filter: is it the gate?