how ion channels move to create action potentials
TRANSCRIPT
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How Ion Channels Move to Create Action Potentials
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Suggests model where 2 states that differ in energy by qV
Where q is about 13e, or 13e/4 per S1-S4 sub-unit; V= -80mV.
q is part of channel—gating current, not ionic current!
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How does gate spontaneously shut-off? How fast?
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Nerve Impulse propagate, not spread, because Na+ spontaneously shut-off.
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Structure of Pore-Domain(S5-S6) is known
(KvAP, Kv1.2… all yield the same structure)
¼ of aKV channel(1 -subunit)
aS1 S2 S3 S4 S5 P S6
S1S1
S4 S4
S2 S2S3 S3
+++
+++
S5 S5S6 S6
b Voltage-sensingdomains (S1-S4)
surround the pore-domain (S5-S6)
Pore figure adapted fromJiang, Y. et al. Nature 417, 523-6. (2002).
But how S4 (and S1-S3) move, remain controversial.
Explains ion selectivity (K+ > Na+) and rapid ion flux.
Excellent agreement between LRET and Crystallography
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S1-S4 Voltage Sensor Lies on the Outside of S5/S6
Crystal Structure of S1-S6 Ion ChannelNobel Prize for Rod MacKinnon, 2006
But…
Channel has been crystallized in only one state. There is no crystal structure of a channel in the open and closed state. Also, there were
some serious problems with some (all?) states.
Need alternative techniques…lower resolution but can tell about channel in a more realistic setting.
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3 Models for how S4 moves
c) Helix: Twist and Rotation
TranslationRotation plus small rotation
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What can fluorescence tell us about Shaker K+ channel
opening/closing?
Shaker Channel– Can measure both Open & Closed States.
Inject mRNA in oocytes; wait 2-5 days; protein in membrane.
(Use mRNA where ionic current is blocked, if necessary).
www.mpibp-frankfurt.mpg.de/schwarz/oocytes.html
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(review)
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How to measure? Where to put probes?
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Problems with oocytes
1. Tremendous amount of autofluorescence
2. Donor only and acceptor only plus desired donor-acceptor only
Answer: Luminescence Resoance Energy Transfer (LRET).
1. Donor has long lifetime– gets away from autofluorescence
2. Can isolate donor-acceptor complex.
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Luminescent ChelatesL
n L
umin
esce
nce
10
100
1000
0 1 2 3 4 5 6 7Time (msec)
Tb3+
1.54 msec
Eu3+
0.61 msec
0
1 104
2 104
3 104
4 104
5 104
6 104
500 550 600 650 700Wavelength (nm)
7F
o
7F
1
7F
2
7F
3
7F
4
Eu3+ Tb3+
7F
6
7F
5
7F
4 7F
3Ln
Lum
ines
cenc
e
O
O-
O
COO-
3CH
ON
CO2-
NHC
C
N
N
N
O
CO2-CO2
-
O
Tb 3+
in
out
Energy
transfer
Donor
Acceptor
Comparatively short lifetime.
Emits where donor is dark.
Long lifetime
Sharply spiked in .
Can see D-A even if incomplete labeling: D-only, A-only.
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Example of LRET
6
11
1 ( )
DA A
D DA A
o
IE
R I IR
Fluor-escein
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O
O -
O
COO -
3CH
ON
CO 2-
NHC
C
N
N
N
O
CO 2-
CO 2-
O
Tb 3+
Low [KCl]
Inside
Outside
High [KCl]-60 mV (resting, closed)
0 mV (active, open)
in
out
K+
K+
K+
gate
Energy
transfer
Shaker Potassium Channel
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S4: Geometry & data of Shaker channel
RSA
RSC
Donor-only = 1.6 msec
Sensitized Emission = 92 sec, 566 sec
RSC = 28 Å
RSA = 41 Å
Time
Fluo
resc
ence
Cha, Nature, 1999
Two exponential= two-distances
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b.
c.
Voltage dependent movement
LRET is tracking Gating charge movement
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Three neighboring residues, 351, 352, 352
Move in different directions
farther
Shaker voltage sensor twists, does not translate too much.
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How it all adds up: Shaker voltage sensor twists, does not translate too much.
S4 Resting S4 Activated
Cha, Nature, 1999
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Class evaluation
1. What was the most interesting thing you learned in class today?
2. What are you confused about?
3. Related to today’s subject, what would you like to know more about?
4. Any helpful comments.
Answer, and turn in at the end of class.