““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.”
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 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
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 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).
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 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.
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 9.Reason # 9.
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).
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 9.Reason # 9.
Permeant ions bind with high affinity in the pore.Permeant ions bind with high affinity in the pore.
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 8.Reason # 8.
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;
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 8.Reason # 8.
Permeant ions bind with high affinity in the pore.Permeant ions bind with high affinity in the pore.
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.
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 8.Reason # 8.
Permeant ions bind with high affinity in the pore.Permeant ions bind with high affinity in the pore.
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.
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 8.Reason # 8.
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.
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 7.Reason # 7.
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).
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 7.Reason # 7.
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).
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 7.Reason # 7.
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 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)
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 7.Reason # 7.
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.
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 6.Reason # 6.
-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).
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 5.Reason # 5.
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)
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 5.Reason # 5.
Mutations in the selectivity filter affect single Mutations in the selectivity filter affect single channel gating.channel gating.
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 4.Reason # 4.
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).
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 3.Reason # 3.
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).
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 2.Reason # 2.
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.
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 1.Reason # 1.
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.
Top Ten Reasons for Top Ten Reasons for
Why the Selectivity Filter is the GateWhy the Selectivity Filter is the Gate
Reason # 1.Reason # 1.
0.2 pA
3 msec
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, 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
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 Affinity-switching allows selectivity filter to gate the channel efficientlychannel efficiently..
Top Ten Reasons for
Why the Selectivity Filter is the Gate
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 ?