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“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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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”

*


Top ten reasons for why the selectivity filter is the gate mark l chapman

Hille, 1992

Doyle et al., 1998

Selectivity filter

Selectivity filter

Out

K

K

+

+

+

+

+

+

In

Gate

Gate


Top ten reasons for why the selectivity filter is the gate mark l chapman

S4

S4

O

O

C

C

Voltage sensor

Gate

Resting

Active

Closed

Open

I

msec, sec

< 10 msec


Closed o pen transition the gate moves

Closed  Open transition: the gate moves

open

0.2 pA

3 msec

closed


Sublevels are visited during open c losed transitions

open

closed

Sublevels are visited during open-closed transitions

open

closed

1 pA

10 msec

open

closed


Subunit composition and closed open transition

Subunit composition and closedopen transition

open

H3

H2a

H2b

0.2 pA

H1

3 msec

closed


Drk1 l at threshold 40 mv sublevel visits abundant during early openings

drk1-L at threshold (–40 mV):sublevel visits abundant during early openings


Conclusion from subconductance analysis

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 analysis1

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


Conclusion from subconductance analysis2

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.


Top ten reasons for why the selectivity filter is the gate mark l chapman

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


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

Reason # 10.

The KcsA structure with 2 K ions in the selectivity filter represents the closed conformation.

Doyle et al, 1998


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

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)


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

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).


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

Reason # 9.

The selectivity filter has a different conformation in the open an closed state.


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

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).


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

Reason # 8.

Permeant ions bind with high affinity in the pore.


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

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;


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

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.


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

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.


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

Reason # 7.

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 mark l chapman

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).


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

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).


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

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)


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

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.


Top ten reasons for why the selectivity filter is the gate mark l chapman

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)


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

Reason # 5.

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 mark l chapman

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)


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

Reason # 4.

Mutations in the selectivity filter affect single channel gating.


Top ten reasons for why the selectivity filter is the gate mark l chapman

D378E

E

D

G

Y

G

V

0.5 pA

T

50 msec

T

drk1


Top ten reasons for why the selectivity filter is the gate mark l chapman

D

G

Y

G

V

T

T

L

drk1


Top ten reasons for why the selectivity filter is the gate mark l chapman

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)


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

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).


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

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).


Top ten reasons for why the selectivity filter is the gate mark l chapman

Open state stability depends on direction of K flux


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

Reason # 1.

The selectivity filter makes a better gate, because of energy considerations.


Top ten reasons for why the selectivity filter is the gate mark l chapman

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.


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

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


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

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.


Top ten reasons for why the selectivity filter is the gate mark l chapman

Monte Carlo simulation of affinity-switching selectivity filter

Na

K


Top ten reasons for why the selectivity filter is the gate mark l chapman

Monte Carlo simulation of affinity-switching selectivity filter

Na

K


Top ten reasons for why the selectivity filter is the gate mark l chapman

CLOSED

OPEN

K

K

Na

X

High-affinity state.

Low-affinity state.

High K selectivity.

No ion selectivity

No permeation.

Efficient Permeation.


Top ten reasons for why the selectivity filter is the gate mark l chapman

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


Top ten reasons for why the selectivity filter is the gate mark l chapman

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


Top ten reasons for why the selectivity filter is the gate mark l chapman

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


Top ten reasons for why the selectivity filter is the gate mark l chapman

The gate ?


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