Bi / CNS 150 Lecture 4
Monday, October 5, 2015
Voltage-gated channels (no action potentials today)
Henry Lester
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The Bi / NB / CNS 150 2015
Home Page
http://www.cns.caltech.edu/bi150/
Please note:
Henry Lester’s office hours
Read the book
If you drop the course,
or if you register late,
please email Jaron
(in addition to the Registrar’s cards).
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Ion Channels in the News
5 October 2015:
Discovered the avermectins
(including ivermectin)
“for therapies that have revolutionized
the treatment of some of the most
devastating parasitic diseases”
Ivermectin irreversibly activates a Cl channel
Found among many invertebrates
Next: PDB file 3RIF
Todays news exemplifies how contributions to neuroscience come from many fields
. . . In this cases, parasitology
When ivermectin binds to the invertebrate glutamate-activated Cl- channel “GluCl”,
the cell becomes “clamped” at the Cl- Nernst potential (~-70 mV).
This prevents the cell from firing action potentials.
The parasite cannot feed, and dies.
Today’s news also exemplifies how
electricity is the language of neurons . . .
and of many other excitable cells.
Vertebrate neurons,
engineered to express an optimized GluCl,
enabling the experimenter
to “silence” the neuron
by applying ivermectin
Frazier, Cohen, and Lester J Biol Chem 2013
Transfection:
Empty DNA
An older GluCl construct
Our optimized “silencing” construct
No IVM
20 nM
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From Lecture 1
In the “selectivity filter” of most K+ channels,
K+ ions lose their waters of hydration and are co-ordinated by backbone carbonyl groups
Ion selectivity filter
H2O
carbonyl
K+ ion
Gate
(Like Kandel Figure 5-15)
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Major Roles for Ion Channels
actually, DE
electrical
transmission in
axons:
electric field
open
closed
Future lectures:
chemical
transmission at
synapses:
[neurotransmitter]
closed
open
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The electric field across a biological membrane,
compared with other electric fields in the modern world
1.
A “high-voltage” transmission line
1 megavolt = 106 V.
The ceramic insulators have a length of ~ 1 m.
The field is ~ 106 V/m.
Dielectric breakdown fields (V/m)
Ceramic
8 x 107
Silicone Rubber
3 x 107
Polyvinyl chloride
7 x 106
2.
A biological membrane
The “resting potential” ~ the Nernst potential for K+, -60 mV.
The membrane thickness is ~ 3 nm = 30 Å.
The field is (6 x 10-2 V) / (3 x 10-9 m) = 2 x 107 V/m !!!
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From Lecture 1
open channel
=
conductor
=
Na+ channel
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Max Delbruck
Carver Mead
Richard Feynman
http://en.wikipedia.org/wiki
/Carver_Mead
1973
H. A. L
http://www.nytimes.com/2015/
09/27/technology/smallerfaster-cheaper-over-thefuture-of-computer-chips.html 9
Intracellular recording with sharp glass electrodes
extracellular
V
R=
104 W-cm2
C=
1 mF/cm2
E
intracellular
 = RC = 10 ms;
too large!
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A better way: record the current from channels directly?
A
Feynman’s idea
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A single voltage-gated Na+ channel
A
-20 mV
-80 mV
5 pA = 104 ions/ms
20 ms
Dynamic range
10 ms to 20 min : 108
2 pA to 100 nA
50,000 chans/cell
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Press release for 1991 Nobel Prize in Physiology or Medicine:
http://www.nobel.se/medicine/laureates/1991/press.html
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“Shaker”, a well-studied voltage-gated K+ channel
“Shaker”, a Drosophila mutant first studied in (the late) Seymour Benzer’s lab
by graduate students Lily & Yuh-Nung Jan (now at UCSF);
Gene isolated simultaneously by L & Y-N Jan lab
& by Mark Tanouye (Benzer postdoc, then Caltech prof, now at UC Berkeley).
Simulation of Shaker gating
Francisco Bezanilla's simulation program at the Univ. of Chicago.
http://nerve.bsd.uchicago.edu/model/rotmodel.html
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The Hodgkin-Huxley formulation of a neuron membrane
Today we emphasize H & H’s description of channel gating
(although they never mentioned channels, or measured a single channel)
Channel opening and closing rate constants are functions of voltage--not of time:
The conformational changes are “Markov processes”.
The rate constants depend instantaneously on the voltage--not on the
history of the voltage.
These same rate constants govern both the macroscopic (summed) behavior and
the single-molecule behavior.
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Demonstrating the Bezanilla model, #1
This channel is actually Shaker with inactivation removed (Shaker-IR).
Based on biochemistry, electrophys, site-directed mutagenesis, X-ray crystallography,
fluorescence.
Two of 4 subunits. Outside is always above (show membrane). Green arrows = K+.
C1 and C2 are closed states, A is “active” = open.
6 helices (S1-S6) + P region, total / subunit.
Structure corresponds roughly to slide 7,
The two green helices (S5, S6 + P) correspond to the entire Xtal structure on slide 4.
First use manual opening. Channel opens when all 4 subunits are “A”.
Note the charges in S4 (5/subunit, but measurements give ~ 13 total). Alpha-helix
with Lys, Arg every 3 rd residue.
Countercharges are in other helices.
Note the S4 charge movement, “shots”. Where is the field, precisely? Near the top.
Note the “hinge” in S6, usually a glycine.
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Demonstrating the Bezanilla model, #2
Read the explanation on the simulation.
Show plot. Manual. Then Voltage (start at default, 0 mV ““delayed rectifier”.
Although we simulate sequentially, the cell adds many channels in parallel.
Not an action potential; this is a “voltage jump” or “voltage clamp” experiment.
Describe shots (measure with fluorescence, very approximately).
I = current. Note three types of I.
Describe gating current (average = I(gate); its waveform does not equal the
I(average).
Show -30 mV (delayed openings,) -50 mV (no openings), 0 (default).
Note tail current.
Note I(gate).
There are many V-gated K channels, each with its own V-sens and kinetics.
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Reminder of Lecture 1: Atomic-scale structure of (bacterial) Na+ channels
shows that here, too, partial loss of water is important for permeation
(As in Kandel Figure 5-1, Na+ channels select with their side chains)
Views
from the
extracellular
solution
The entire water-like pathway
Views
from the
membrane
plane
PDB files
4EKW, 4DXW
Payandeh et al, Nature 2011;
Zhang et al, Nature 2012
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Voltage-gated Na+ channels, and Blockade by Tetrodotoxin
The pufferfish toxin,
from PubChem
Payandeh et al,
Nature 2011;
Zhang et al,
Nature 2012;
Animal Na+ channels have
an inactivation flap
PDB files: 4EKW, 4DXW
Inactivation: a property of all voltage-gated Na+ channels
and of
Some voltage-gated K+ channels
http://nerve.bsd.uchicago.edu/Na_chan.htm
Site home:
http://nerve.bsd.uchicago.edu/
This model is ~ 10 years older than the K+ channel simulation.
Na+ channel has only one subunit, but it has 4 internal repeats
(it’s a “pseudo-tetramer”).
The internal repeats resemble an individual K+ subunit. The “P” region differs,
governing the ion selectivity.
Orange balls are Na+.
Note that the single-channel current (balls inside cell) requires two events:
a) All 3 S4 must move up, in response to DV;
b) Open flap. When the flap closes, the channel “inactivates”.
The flap may be linked to the 4th S4 domain.
The synthesized macroscopic current shows a negative peak, then decays.
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Next lecture employs electrical circuits
Review your material from Phys 1b, practical
See also Appendix A in Kandel
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Henry Lester’s Office Hours
Monday, Wednesday, Friday
1:15 – 2
In / near the Red Door
End of Lecture 4
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