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“Top Ten Reasons for 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”. *. Hille, 1992. Doyle et al ., 1998. Selectivity filter. Selectivity filter. Out. K. K. +. +. +. +. +. +. In. Gate. Gate.

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“Top Ten Reasons for why the Selectivity Filter is the Gate”

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

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

  3. S4 S4 O C O C Macroscopic currents vs. Single channel behavior Voltage sensor Gate Resting Active Closed Open I msec, sec < 10 sec

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

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

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

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

  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.”

  9. The Selectivity Filter ... 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 ...

  10. Conclusion from subconductance analysis: is the Gate 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.” is the Gate

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

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

  13. Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 10. 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)

  14. Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 10. 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).

  15. Top Ten Reasons for Why the Selectivity Filter is the Gate Reason # 10. 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).

  16. 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.

  17. 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 KscA 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).

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

  19. 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: 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;

  20. 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: 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.

  21. 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: 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.

  22. 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.

  23. 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).

  24. 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).

  25. 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)

  26. 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.

  27. 1.0 G G max drk1-LS drk1-S 0.5 0.0 -40 0 40 80 120 E (mV) m Drk1-S: triple mutation in S4  threshold +80 mV Drk1-LS: additional mutation V76L (selectivity filter)

  28. 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).

  29. 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)

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

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

  32. D G Y G V T T L drk1

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

  34. 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).

  35. 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 KscA (Meuser et al., 1999, FEBS Lett. 462: 447).

  36. Open state stability depends on direction of K flux

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

  38. 0.2 pA 3 msec 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.

  39. 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 helices: energetically expensive

  40. 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 helices: energetically expensive. Affinity-switching allows selectivity filter to gate the channel efficiently.

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

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

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

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

  45. M.C. Simulation Results for 1-site Model 100% Normalized K flux 10% 1% 0.001 0.010 0.100 1.000 Probability of being in low affinity state

  46. K selectivity and flux as a function of P_low for 2-site model 10000 10000 Without ion-ion repulsion With ion-ion repulsion 1000 1000 100 100 K/Na flux ratio 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

  47. The gate ?

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