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

““Top Ten Reasons for Top Ten Reasons for

Why the Selectivity Filter is the Gate”Why the Selectivity Filter is the Gate”

Mark L. ChapmanMark L. Chapman

Antonius M. J. VanDongenAntonius M. J. VanDongen

(*) “Letterman”(*) “Letterman”

**

+

+

+

+

+

+

Selectivity filter

Gate

In

Out

Selectivity filter

Gate

K

K

Hille, 1992 Doyle et al., 1998

RestingResting ActiveActive

Voltage sensorVoltage sensor

S4

S4

ClosedClosed OpenOpen

GateGate

II CC

OO

msec, secmsec, sec

< 10 < 10 secsecCC

OO

Closed Closed Open transition: Open transition: the gate movesthe gate moves

0.2 pA3 msec

closed

open

Sublevels are visited during open-closed transitionsSublevels are visited during open-closed transitions

open

closed

1 pA10 msec

open

closed

open

closed

Subunit composition and closedSubunit composition and closedopen open transitiontransition

0.2 pA3 msec

closed

open

H3

H2a

H1

H2b

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

Conclusion from subconductance analysis.Conclusion from subconductance analysis.

From: Chapman Chapman et al.et al., 1997, Biophys. J. 72: 708., 1997, Biophys. J. 72: 708.

““Ions could be prevented from translocating in the Ions could be prevented from translocating in the ‘closed’ conformation because of an energy well that ‘closed’ conformation because of an energy well that is too deep (i.e. a high-affinity binding site). A is too deep (i.e. a high-affinity binding site). A conformational change that reduces the depth of the conformational change that reduces the depth of the well would enable the channel to support ion well would enable the channel to support ion permeation. ... permeation and gating are coupled: permeation. ... permeation and gating are coupled: the same structure that controls permeation is also the same structure that controls permeation is also responsible for opening and closing the channel.”responsible for opening and closing the channel.”



““Ions could be prevented from translocating in the Ions could be prevented from translocating in the ‘closed’ conformation because of an energy well that ‘closed’ conformation because of an energy well that is too deep (i.e. a high-affinity binding site). A is too deep (i.e. a high-affinity binding site). A conformational change that reduces the depth of the conformational change that reduces the depth of the well would enable the channel to support ion well would enable the channel to support ion permeation. ... permeation and gating are coupled: permeation. ... permeation and gating are coupled: the same structure that the same structure that controls permeationcontrols permeation is also is also responsible for opening and closing the channel.”responsible for opening and closing the channel.”

• The selectivity filterThe selectivity filter



““Ions could be prevented from translocating in the Ions could be prevented from translocating in the ‘closed’ conformation because of an energy well that ‘closed’ conformation because of an energy well that is too deep (i.e. a high-affinity binding site). A is too deep (i.e. a high-affinity binding site). A conformational change that reduces the depth of the conformational change that reduces the depth of the well would enable the channel to support ion well would enable the channel to support ion permeation. ... permeation and gating are coupled: permeation. ... permeation and gating are coupled: the same structure that controls permeation is also the same structure that controls permeation is also responsible for responsible for opening and closingopening and closing the channel.” the channel.”

• The selectivity filter is the gate.The selectivity filter is the gate.

Mechanism: Affinity switching.Mechanism: Affinity switching.

The selectivity filter is the gateThe selectivity filter is the gate

C OC O

High affinityHigh affinity Low affinityLow affinity

Closed state: traps K ionsClosed state: traps K ions

Open state: release bound ionsOpen state: release bound ions

Selectivity filter alters conformationSelectivity filter alters conformation

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

Top Ten Reasons for

Why the Selectivity Filter is the Gate

Reason # 10.

Doyle et al, 1998

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

The structure was obtained at a pH where the channel The structure was obtained at a pH where the channel is closed (Clapham 1999, Cell 97: 547-550)is closed (Clapham 1999, Cell 97: 547-550)

Top Ten Reasons for Top Ten Reasons for

Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate

Reason # 10.Reason # 10.

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

The structure was obtained at a pH where the channel The structure was obtained at a pH where the channel is closed (Clapham 1999, Cell 97: 547-550)is closed (Clapham 1999, Cell 97: 547-550)

The electrophysiological properties of the open KcsA The electrophysiological properties of the open KcsA channel are incompatible with the published crystal channel are incompatible with the published crystal structure (Meuser et al., 1999, FEBS Letters 462: structure (Meuser et al., 1999, FEBS Letters 462: 447-452). 447-452).




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




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

In the In the openopen state, single KcsA channels: state, single KcsA channels:

• are poorly ion selective are poorly ion selective

• permeate partially hydrated K ions permeate partially hydrated K ions

• have a wider diameter than seen in the crystal have a wider diameter than seen in the crystal structure. structure.

(Meuser (Meuser et al.et al., 1999, FEBS Letters 462: 447)., 1999, FEBS Letters 462: 447).




Permeant ions bind with high affinity in the pore.Permeant ions bind with high affinity in the pore.





This was first described for CaThis was first described for Ca2+2+ ions in Ca channels ions in Ca channels

Armstrong & Neyton, 1991, Ann. N.Y. Acad. Sci. 635:18-25; Armstrong & Neyton, 1991, Ann. N.Y. Acad. Sci. 635:18-25;

Kuo & Hess, 1993, J. Physiol. 466: 657-682; Kuo & Hess, 1993, J. Physiol. 466: 657-682;

Yang et al., 1993, Nature 366: 158-161; Yang et al., 1993, Nature 366: 158-161;

Ellinor et al., 1995, Neuron 15:1121-1132.Ellinor et al., 1995, Neuron 15:1121-1132.

Polo-Parada, & Korn, 1997, J. Gen. Physiol. 109:693-702; Polo-Parada, & Korn, 1997, J. Gen. Physiol. 109:693-702;





K ions also bind with high affinity in the K channel pore:K ions also bind with high affinity in the K channel pore:

M K concentrations block Na conductanceM K concentrations block Na conductance

Kiss Kiss et alet al., 1998, J. Gen. Physiol. 111: 195-206; ., 1998, J. Gen. Physiol. 111: 195-206;

Immke & Korn, 2000, J. Gen. Physiol. 115: 509-518.Immke & Korn, 2000, J. Gen. Physiol. 115: 509-518.





K ions also bind with high affinity in the K channel pore:K ions also bind with high affinity in the K channel pore:

M K concentrations block Na conductanceM K concentrations block Na conductance

Kiss Kiss et alet al., 1998, J. Gen. Physiol. 111: 195-206; ., 1998, J. Gen. Physiol. 111: 195-206;

Immke & Korn, 2000, J. Gen. Physiol. 115: 509-518.Immke & Korn, 2000, J. Gen. Physiol. 115: 509-518.

Short closed times in single channel records Short closed times in single channel records result from K ions acting as pore blockersresult from K ions acting as pore blockers

Choe et al., 1998. J. Gen. Physiol. 112: 433-446.Choe et al., 1998. J. Gen. Physiol. 112: 433-446.




An alternative is needed for the cytoplasmic constriction An alternative is needed for the cytoplasmic constriction acting as a gate, since it is not universally found.acting as a gate, since it is not universally found.





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






Glutamate receptors, which have an inverted Glutamate receptors, which have an inverted topology, have a wide external vestibuletopology, have a wide external vestibule

(Kuner et al., 1996, Neuron 17: 343).(Kuner et al., 1996, Neuron 17: 343).






Glutamate receptors, which have an inverted topology, Glutamate receptors, which have an inverted topology, have a wide external vestibulehave 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 In CNG1, the cytoplasmic constriction does not prevent K ions from entering the vestibule.prevent K ions from entering the vestibule.

(Flynn and Zagotta, this meeting)(Flynn and Zagotta, this meeting)




There is a strong coupling between sensor movement There is a strong coupling between sensor movement and the conformation of the selectivity filter. and the conformation of the selectivity filter.

The effect of mutations in S4 on activation properties The effect of mutations in S4 on activation properties depends critically on whether the selectivity filter depends critically on whether the selectivity filter contains a Val or Leu at position 76.contains a Val or Leu at position 76.




-40 0 40 80 120

Em (mV)

0.0

0.5

1.0

GGmax

drk1-LS drk1-S

Drk1-S: triple mutation in S4 Drk1-S: triple mutation in S4 threshold +80 mV threshold +80 mV

Drk1-LS: additional mutation V76L (selectivity filter)Drk1-LS: additional mutation V76L (selectivity filter)

Open state stability is determined by the Open state stability is determined by the permeating ion species, linking gating to permeating ion species, linking gating to selectivity. selectivity.

(Spruce et al., 1989, J. Physiol. 411: 597).(Spruce et al., 1989, J. Physiol. 411: 597).




Open state stability is determined by the Open state stability is determined by the permeating ion species, linking gating to permeating ion species, linking gating to selectivity. selectivity.

Spruce et al., 1989, J. Physiol. 411: 597.Spruce et al., 1989, J. Physiol. 411: 597.

Open times are very different for K and Rb in KcsA.Open times are very different for K and Rb in KcsA.

Lisa Heginbotham (personal communication)Lisa Heginbotham (personal communication)

Eduardo Perozo et al. (this meeting)Eduardo Perozo et al. (this meeting)




Mutations in the selectivity filter affect single Mutations in the selectivity filter affect single channel gating.channel gating.




0.5 pA

50 msec

D378E

drk1

0.5 pA

50 msec

E

D

G

G

Y

V

TT

drk1

0.5 pA

50 msec

V374L

1.0 pA

50 msec

D

G

G

Y

V

TT

L

D

G

G

Y

V

T

TA

T

D E: Destabilization open state

V L: Stabilization open state &

subconductances (drk1)

T S: Stabilization open state &

subconductances (Shaker)

In the NMDA receptor, a conserved Asparagine In the NMDA receptor, a conserved Asparagine residue critical for Ca permeability and Mg residue critical for Ca permeability and Mg block, stabilizes subconductance levels.block, stabilizes subconductance levels.

(Schneggenburger & Ascher, 1997, (Schneggenburger & Ascher, 1997, Neuron 18Neuron 18: 167).: 167).




drk1 P A S F W W A T I T M T T V G Y G D I Y P

Shak P D A F W W A V V T M T T V G Y G D M T P

KcsA P R A L W W S V E T A T T V G Y G D L Y P

GluR0 Q N G M W F A L V T L T T V G Y G D R S P

NR1 S S A M W F S W G V L L N S G I G E G A P

The direction of the K flux determines: The direction of the K flux determines:

• the open state stability in the open state stability in drk1drk1..

• which (sub)conductance levels predominate inwhich (sub)conductance levels predominate in KcsA KcsA (Meuser et al., 1999, FEBS Lett. 462: 447). (Meuser et al., 1999, FEBS Lett. 462: 447).




0

5

604020

D378EO

pen

Tim

e in

mse

c

0-20-40-60-80

Membrane Potential in mV

outward current

inward current

Open state stability depends on direction of K fluxOpen state stability depends on direction of K flux

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





Single channel gating: 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.




0.2 pA

3 msec


Single channel gating: 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 Rotation of 4 S6 helices: energetically expensivehelices: energetically expensive

Top Ten Reasons for


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 Affinity-switching allows selectivity filter to gate the channel efficientlychannel efficiently..

Top Ten Reasons for


Reason # 1.

Na

K

Monte Carlo simulation of affinity-switching selectivity filterMonte Carlo simulation of affinity-switching selectivity filter

Na

K

Monte Carlo simulation of affinity-switching selectivity filterMonte Carlo simulation of affinity-switching selectivity filter

High-affinity state.High-affinity state.

High K selectivity. High K selectivity. No permeation.No permeation.

Low-affinity state.Low-affinity state.

No ion selectivity No ion selectivity

Efficient Permeation.Efficient Permeation.

CLOSEDCLOSED OPENOPEN

X

K K Na

0.0010.001 0.0100.010 0.1000.100 1.0001.000

11

1010

100100

10001000

Probability of being in low affinity stateProbability of being in low affinity state

K selectivityK selectivity

(K/Na (K/Na fluxflux ratio) ratio)

M.C. Simulation Results for 1-site Model M.C. Simulation Results for 1-site Model

1%

10%

100%

0.001 0.010 0.100 1.000

Probability of being in low affinity state

Normalized

K flux

M.C. Simulation Results for 1-site Model M.C. Simulation Results for 1-site Model

K/N

a fl

ux

rati

oK

/Na

flu

x ra

tio

Prob of being in low-affinity stateProb of being in low-affinity state

K selectivity and flux as a function of P_low for 2-site modelK selectivity and flux as a function of P_low for 2-site model

1

10

100

1000

10000

0.010.01 0.10.1 11 0.01 0.1 1

With ion-ion repulsion

Prob of being in low-affinity stateProb of being in low-affinity state

1

10

100

1000

10000

Without ion-ion repulsion

The gate ?

Documents

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