1. Mark L. Chapman Antonius M. J. VanDongen (*) “Letterman” * “Top Ten Reasons for why the Selectivity Filter is the Gate”

2. + + + + + + Selectivity filter Gate In Out Selectivity filter Gate K K Hille, 1992Doyle et al., 1998 Hille 1992 model & KcsA X-ray structure

3. RestingActive Voltage sensor S4 ClosedOpen Gate I CO msec, sec < 10  sec CO Macroscopic currents vs. Single channel behavior

4. 0.2 pA 3 msec closed open Closed-Open transition: the gate moves

5. Sublevels are visited during open-closed transitions 1 pA 10 msec open closed open closed

6. Subunit composition and Closed-Open transitions 0.2 pA 3 msec closed open H3 H2a H1 H2b

7. drk1-L at threshold: sublevel visits abundant during early openings

8. Conclusion from subconductance analysis: Chapman et al., 1997, Biophys. J. 72: 708. 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.”

9. Chapman et al., 1997, Biophys. J. 72: 708. 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... Conclusion from subconductance analysis:

10. Chapman et al., 1997, Biophys. J. 72: 708. 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: The Selectivity Filter … is the Gate

11. The selectivity filter is the gate C O High affinity Low affinity Closed state: traps K ions Open state: releases bound ions Selectivity filter alters conformation Gating Mechanism: Affinity Switching

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

13. The KscA 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: 547-550) Top Ten Reasons for Why the Selectivity Filter is the Gate Reason #10

14. The KscA 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: 547-550) The electrophysiological properties of the open KcsA channel are incompatible with the published crystal structure (Meuser et al., 1999, FEBS Letters 462: 447- 452). Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 10.

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

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

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

18. Permeant ions bind with high affinity in the pore. This was first described for Ca 2+ ions in Ca channels Armstrong & Neyton, 1991, Ann. N.Y. Acad. Sci. 635:18-25; Kuo & Hess, 1993, J. Physiol. 466: 657-682; Yang et al., 1993, Nature 366: 158-161; Ellinor et al., 1995, Neuron 15:1121-1132. Polo-Parada, & Korn, 1997, J. Gen. Physiol. 109:693-702; Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 8.

19. Permeant ions bind with high affinity in the pore. K ions also bind with high affinity in the K channel pore:  M K concentrations block Na conductance Kiss et al., 1998, J. Gen. Physiol. 111: 195-206; Immke & Korn, 2000, J. Gen. Physiol. 115: 509-518. Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 8.

20. Permeant ions bind with high affinity in the pore. K ions also bind with high affinity in the K channel pore:  M K concentrations block Na conductance Kiss et al., 1998, J. Gen. Physiol. 111: 195-206; Immke & Korn, 2000, J. Gen. Physiol. 115: 509-518. Short closed times in single channel records result from K ions acting as pore blockers Choe et al., 1998. J. Gen. Physiol. 112: 433-446. Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 8.

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

22. 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). Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 7.

23. 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). (Kuner et al., 1996, Neuron 17: 343). Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 7.

24. 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). (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) Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 7.

25. 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. Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 6.

26. -4004080120 E m (mV) 0.0 0.5 1.0 G G max drk1-LS drk1-S Drk1-S: triple mutation in S4  threshold +80 mV Drk1-LS: additional mutation V76L (selectivity filter)

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

28. 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) Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 5.

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

30. 0.5 pA 50 msec D378E drk1 E D G G Y V T T

31. D G G Y V T T L

32. D G G Y V T T A T D  E: Destabilization open state V  L: Stabilization open state & subconductances (drk1) T  S: Stabilization open state & subconductances (Shaker)

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

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

35. Open state stability depends on direction of K flux

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

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

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

39. 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  helices: energetically expensive. Affinity-switching allows selectivity filter to gate the channel efficiently. Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 1.

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

41. High-affinity state. High K selectivity. No permeation. Low-affinity state. No ion selectivity Efficient Permeation. CLOSED OPEN X KKNa

42. 0.0010.0100.1001.000 1 10 100 1000 Probability of being in low affinity state K selectivity (K/Na flux ratio) M.C. Simulation Results for 1-site Model

43. 1% 10% 100% 0.0010.0100.1001.000 Probability of being in low affinity state Normalized K flux M.C. Simulation Results for 1-site Model

44. K/Na flux ratio Prob of being in low-affinity state K selectivity and flux as a function of P_low for 2-site model 1 10 100 1000 10000 0.010.11 0.010.11 With ion-ion repulsion Prob of being in low-affinity state 1 10 100 1000 10000 Without ion-ion repulsion

45. The gate ?