Chap' 34' The neuronal membrane at rest

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Chap. 3-4. The neuronal membrane at rest The
action potential
Why the gradients?
2
Nernst equation (for equilibrirm potential) E
ion 2.303 RT/zF log iono/ioni 61.54 mV
log Ko/Ki 61.54 x log 1/20 61.54 x
(-1.3) -80 mV (inside) Goldman equation
(consider relative permeability and conc.
gradient) Vm 61.54 mV log PkKo
PnaNao PkKi PnaNai
61.54 mV log 40(5) 1(150) 350
40(100) 1(15) 4015 -65 mV
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Death by lethal injection Dr. Jack
Kevorkian Euthanasia, Physician-assisted
suicide Ko ----gtdepolarization of heart muscles
March 26, 1999 -- Kevorkian convicted of
second-degree murder in Youk's death and delivery
of a controlled substance. He faces up to life in
prison on the second-degree murder charge and up
to seven years on controlled substance charge.
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Na/K ATPase? Role?
De-occlusion
Na K
Occlusion
Three distinct and sequential steps in the
release of sodium ions by the Na/K ATPase. Nature
403 898 - 901 (2000)
Depletion of K ion and high speed voltage jump
to isolate de-occlusion----gtrelease of three Na
ions one at a time, in a strict order
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Box 3.4. Shaker flies and their defective
potassium channels.
Cloning of Na Ca channels by biochemical
methods Cloning of Glutamate receptors by
expression cloning (mRNA injection into frog
oocytes and current measurement) Shaking
flies in the presence of ether---gt Cloning of K
channel
Lily Y. Jan
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Na channel (single chain) vs. K channel (tetramer)
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Voltage sensing and opening
Gating current
Voltage sensor in the S4
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Fig. 3.18. A view of the K channel pore.
Rod MacKinnon Nobel Prize in Chemistry,
2003 Lasker Award (the Albert Lasker Basic
Medical Research Award), 1999
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Nature. 2003 May 1423(6935)42-8. Nature. 2003
May 1423(6935)33-41. Science. 2003 Apr
4300(5616)108-12. Nature. 2003 Mar
13422(6928)180-5. Cell. 2002 Dec
27111(7)957-65. Cell. 2002 Oct
18111(2)231-9. Nature. 2002 May
30417(6888)523-6. Nature. 2002 May
30417(6888)515-22. Nature. 2002 Mar
21416(6878)261-2. Nature. 2002 Jan
17415(6869)287-94. Nature. 2001 Nov
1414(6859)43-8. Nature. 2001 Nov
1414(6859)37-42. Nature. 2001 Jun
7411(6838)657-61. Science. 2000 Jul
7289(5476)123-7. Cell. 1999 Jun
2597(7)943-52. Cell. 1998 Nov 2595(5)649-55.
Science. 1998 Apr 3280(5360)69-77. Science.
1998 Apr 3280(5360)106-9.
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K channels
PSD-95
1
2
3
SH3
GK
PDZ (PSD-95/Dlg/ZO-1) domain
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The Structure of the Potassium Channel Molecular
Basis of K Conduction and Selectivity. Doyle and
MacKinnon. Science 1998 April 3 280 69-77.
Fig. 3. Views of the tetramer. Streptomyces
lividans
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Fig. 5. (right). Molecular surface of KcsA and
contour of the pore.
Inverted teepee, or cone
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Figure 7. Two mechanisms by which the K channel
stabilizes a cation in the middle of the
membrane. First, a large aqueous cavity
stabilizes an ion (green) in the otherwise
hydrophobic membrane interior. Second, oriented
helices point their partial negative charge
(carboxyl end, red) towards the cavity where a
cation is located.
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How does the K channel structure account for its
prodigious ion selective properties (over Na
ions)? Nearly complete dehydration of
ions Carbonyl oxygen may act like surrogate water
Molecular spring may keeps the diameter
constant This may prevent the filter from
accommodating a Na ion with its smaller radius.
Balance of energetic costs and gains!
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Figure 8. Detailed views of the K channel
selectivity filter.
H
H
O
K
O
O
Two K ions about 7.5 angstroms apart
Electrostatic repulsive forces
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How is the high conduction achieved? 1. Multiple
K binding sites 2. Two K ions at the given
time 3. More than one ion to overcome affinity
with repulsion
FEBS Lett. 2003 Nov 27555(1)62-5
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Dutzler and MacKinnon X-ray structure of a ClC
chloride channel at 3.0 A reveals the molecular
basis of anion selectivity. Nature. 2002 Jan
17415(6869)287-94.
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Figure 3 Structure of the StClC dimer.
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Figure 5 Structure of the StClC selectivity
filter.
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Figure 6 Ion conduction pathway
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Nature 417, 523 - 526 (30 May 2002)
Glycine hinge
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Nature. 2003 May 1423(6935)33-41
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A major problem in K channel crystal analysis
a particularly loose protein
structure Solutions (1) antibody molecules
(so-called Fab fragments) as a scaffold to aid
crystallization (2) Use a particularly rugged
channel protein (KvAP from the archaebacterium
Aeropyrum pernix)
A 'paddle' that extends out from the channel core
into the membrane's fluid interior
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A novel voltage-gating mechanism
Figure 8 Hypothesis for gating charge movements
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Nature. 2003 May 1423(6935)42-8.
Experimental tool 1 Fab
Fab (green) is added to the external or internal
side of a planar lipid membrane to determine
whether the epitope is exposed.
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Experimental tool 2 Biotin
Figure 2 a, Experimental strategy KvAP channels
with biotin tethered to a site-directed cysteine
can be 'grabbed' by avidin in solution to affect
channel function c, Representative traces showing
the effects of avidin on wild-type and mutant
biotinylated KvAP channels. Currents in the
absence (black traces) or presence of internal
(blue traces) or external (red traces) avidin.
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Figure 5 Positions within the membrane of the
voltage-sensor paddles during closed and opened
conformations, and a hypothesis for coupling to
pore opening.
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Figure 1 Voltage sensing in a potassium ion
channel. a, The control of ion flow through
voltage-gated channels is very sensitive to the
voltage across the cell membrane. By comparison,
an electronic device such as a transistor is much
less sensitive to applied voltage. b, The voltage
sensors in a bacterial potassium channel are
charged 'paddles' that move through the fluid
membrane interior. Four voltage sensors (two of
which are shown here) are linked mechanically to
the channel's 'gate'. Each voltage sensor has
four tethered positive charges (arginine amino
acids) the high sensitivity of channel gating
results from the transport of so many charges, 16
in all, most of the way across the membrane.
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Intracellular and extracellular recording
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Resting potential, rising phase, overshoot,
falling phase, undershoot
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Figure 4.2. The effect of injecting a positive
charge into a neuron.
VIR (Ohms law) Current injection--gtDepolarizati
on--gtAction potential
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AP All-or-none, threshold Current size--gt AP
frequency
Absolute refractory period Relative refractory
period
Why do we need the action potential?
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Reversal/equilibrium potential ENa or
EK Membrane potential (Vm) Goldman
equation Permeability Conc. gradient
___1___ 20
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Ion flows during Action Potential Gconductance1
/R I/V Hodgkin and Huxley Nobel Prize in
1963 Determined the nature of ion flows in the
AP of the giant axon of squid.
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IV curve used to show the characteristics of a
current of interest - Conductance - Identity of
ions
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H
H
Ion selectivity of Na channel
O
K
O
O
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The voltage-sensitive sodium channel is a
bell-shaped molecule with several cavities. Sato
et al. Nature 4091047 - 1051 (2001)
Cryo EM 19A
Top side
bottom
Figure 2 Surface representation of the sodium
channel protein.
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Figure 3 Domains and internal cavities of the
sodium channel protein and the sequence-based
structure prediction.
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The Patch-clamp method. Why important? Real-time
monitoring of individual channels
- Conductance? - Open time? - Probability of
opening?
Erwin Neher Gigaseal Nobel Prize in physiology
or medicine, 1991
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Population current
Voltage
Current
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Closed, open, inactivated
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Factors influencing conduction velocity 1)
Myelination 2) Diameter of axons
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(No Transcript)
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Local anesthesia Cocaine Lidocaine Use-dependen
t blockade
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Multiple Sclerosis (hardening) Shiverer,
CMT Caused by demyelination (an autoimmune
disease) Nearby cells to multiply until they
form thick, scar-like tissue --
the sclerosis or plaque. Problems in sensory and
motor function Difficulty walking, bladder and
bowel problems, reduced vision, fatigue,
sexual dysfunction and dizziness Capricious
remissions and relapses over many years Attack
axons in the brain, spinal cord and optic
nerve No cure..Interferon beta
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Saltatory nerve conduction (from the Latin
meaning to leap)
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Spike-initiation zone. How?
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Diverse electric behavior neurons
Steady frequency Aspinous stellate
cells Slowing of firing (adaptation) Spiny
pyramidal cells Why adaptation? Slowly
activating K channel Burst of spikes/action
potentials Some large pyramidal neurons