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“Top Ten Reasons for Why the Selectivity Filter is the Gate” Mark L. Chapman

“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. S4. S4. O. O. C. C.

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“Top Ten Reasons for Why the Selectivity Filter is the Gate” Mark L. Chapman

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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

  3. S4 S4 O O C C Voltage sensor Gate Resting Active Closed Open I msec, sec < 10 msec

  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 transition open H3 H2a H2b 0.2 pA H1 3 msec closed

  7. drk1-L at threshold (–40 mV):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. 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

  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.

  11. C O High affinity Low affinity The selectivity filter is the gate 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 KcsA 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 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: 547-550)

  14. 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: 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 # 9. The selectivity filter has a different conformation in the open an closed state.

  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. • 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).

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

  18. 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;

  19. 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: 195-206; Immke & Korn, 2000, J. Gen. Physiol. 115: 509-518.

  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: mM 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.

  21. 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.

  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. Inward rectifying K channels have a wide internal entrance (Lu et al., 1999, PNAS 96: 9926).

  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). Glutamate receptors, which have an inverted topology, have a wide external vestibule (Kuner et al., 1996, Neuron 17: 343).

  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). In CNG1, the cytoplasmic constriction does not prevent K ions from entering the vestibule. (Flynn and Zagotta, this meeting)

  25. 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.

  26. 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)

  27. 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).

  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. Open times are very different for K and Rb in KcsA. Lisa Heginbotham (personal communication) Eduardo Perozo et al. (this meeting)

  29. Top Ten Reasons for Why the Selectivity Filter is the Gate 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

  31. D G Y G V T T L drk1

  32. 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)

  33. 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).

  34. 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).

  35. Open state stability depends on direction of K flux

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

  37. 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.

  38. 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

  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 four S6 a-helices: energetically expensive. • Affinity-switching allows selectivity filter to gate the channel efficiently.

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

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

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

  43. 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

  44. 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

  45. 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

  46. The gate ?

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