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Top Ten Reasons for Why the Selectivity Filter
is the Gate Mark L. Chapman Antonius M. J.
VanDongen () Letterman

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(No Transcript)
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Voltage sensor
Gate
Resting
Active
Closed
Open
I
msec, sec
lt 10 msec
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Closed ? Open transition the gate moves
open
0.2 pA
3 msec
closed
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Sublevels are visited during open-closed
transitions
open
closed
1 pA
10 msec
open
closed
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Subunit composition and closed?open transition
open
H3
H2a
H2b
0.2 pA
H1
3 msec
closed
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drk1-L at threshold (40 mV)sublevel visits
abundant during early openings
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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.
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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

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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.

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The selectivity filter is the gate
Mechanism Affinity switching.
Closed state traps K ions Open state release
bound ions Selectivity filter alters conformation
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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
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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)
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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).
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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.
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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).

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Top Ten Reasons for Why the Selectivity Filter
is the Gate
Reason 8.
Permeant ions bind with high affinity in the
pore.
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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. 63518-25 Kuo Hess, 1993,
J. Physiol. 466 657-682 Yang et al., 1993,
Nature 366 158-161 Ellinor et al., 1995,
Neuron 151121-1132. Polo-Parada, Korn, 1997,
J. Gen. Physiol. 109693-702
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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.
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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.
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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.
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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).
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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).
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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)
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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.
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Drk1-S triple mutation in S4 ? threshold 80
mV Drk1-LS additional mutation V76L (selectivity
filter)
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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).
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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)
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Top Ten Reasons for Why the Selectivity Filter
is the Gate
Reason 4.
Mutations in the selectivity filter affect single
channel gating.
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D378E
E
D
G
Y
G
V
0.5 pA
T
50 msec
T
drk1
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D
G
Y
G
V
T
T
L
drk1
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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)
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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).
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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).

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Open state stability depends on direction of K
flux
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Top Ten Reasons for Why the Selectivity Filter
is the Gate
Reason 1.
The selectivity filter makes a better gate,
because of energy considerations.
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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.

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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

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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.

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Monte Carlo simulation of affinity-switching
selectivity filter
Na
K
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Monte Carlo simulation of affinity-switching
selectivity filter
Na
K
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CLOSED
OPEN
K
K
Na
X
High-affinity state.
Low-affinity state.
High K selectivity.
No ion selectivity
No permeation.
Efficient Permeation.
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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
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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
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K selectivity and flux as a function of P_low for
2-site model
10000
10000
With ion-ion repulsion
Without 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
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The gate ?