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Bi 1 Lecture 8
Tuesday, April 11, 2006
The Central Dogma of Drugs and the Brain,
Part 1:
Drugs open and block ion channels
1
from Lecture 2
Atomic-scale Structures
H3CH 2C
N
morphine
procaine
nicotine
CH 3
N
N
CH 2CH 3
H2C
botulinum
toxin
HO
O
CH 2
N CH
3
O
C O
HO
morphine
NH 2
(Download to your computer;
Then open with Swiss-prot pdb viewer)
http://www.its.caltech.edu/~lester/Bi-1/morphine.pdb
http://www.its.caltech.edu/~lester/Bi-1/procaine.pdb
http://www.its.caltech.edu/~lester/Bi-1/nicotine.pdb
2
Drug interactions at the nicotinic acetylcholine receptor
Some drugs compete with nicotine
Some drugs bind on the axis
~ 100 Å
(10 nm)
3
Functioning channel
Drug-blocked channel
4
Model or
scheme
normal function
State 1
State 2
k21
open
closed
units: M-1s-1
units: s-1
simple block
k21
closed
all molecules
begin here at
t= 0
open
k23 = k+[Drug]
drug
blocked
5
current
time constant
= 1/k21
none
time constant
= 1/(k21+ k23)
Functioning channel
Drug-blocked channel
6
n =1
0
Counts/bin
open time histogram
counts/bin
Daniel Kegel ‘86
statistical analysis;
bit-mapped
graphics displays
http://www.kegel.com/
Christmas ‘83
“May I borrow your notebook computer over
vacation, please? I’m installing a radio link.”
time constant =
1
k 21
Time
time
Jan 1, 1984
7
Some single-molecule recordings suggest . . . .
5 pA
Acetylcholine only
20 ms
Acetylcholine + blocking drug (QX-222)
8
. . . . the foot-in-the-door scheme
current
time
9
Model or
scheme
normal function
simple block
State 1
all molecules
begin here at
t= 0
State 2
k21
open
closed
k21
open
closed
k23 = k+[Drug]
drug
blocked
k23 = k+[Drug]
k21
foot-in-the-door
closed
drug
blocked
open
k32
Not allowed
10
n =1
time constant
= 1/k21
0
time constant
= 1/(k21+ k23)
+
etc
11
Analyzing the foot-in-the-door model of drug block
k23 = k+[Drug]
k21
foot-in-the-door
closed
drug
blocked
open
k32
Not allowed
time constant
= 1/(k21 + k23)= 1/(k21 + k+[Drug]) P
time constant
P
= 1/k32
 ndt  1 / k
21
(P)
12
Macroscopic predictions of blocking schemes
http://www.its.caltech.edu/~
lester/Bi-1/normal
function.mws
1
channels open
0.8
normal closing
simple block
0.6
foot-in-the-door block:
two exponentials
http://www.its.caltech.edu/~l
ester/Bi-1/simpleblock.mws
0.4
0.2
0
0
1
2
3
4
5
http://www.its.caltech.edu/
~lester/Bi-1/Foot-in-doorspecific.mws
time, ms
Maple worksheets describing macroscopic
time course for the current according to
several models. You must have Maple
installed on your computer.
http://www.its.caltech.edu/
~lester/Bi-1/Foot-in-doorgeneral.mws
13
A use-dependent blocker
stimuli
impulses fail
impulses
(voltage)
channel
population
(currents)
threshold
pronounced block
at brief intervals
little block
at long intervals
14
inside
Functioning channel
“Trapped” or
“Use-Dependent”
Blocker
15
Procaine Blocks Na+ Channels from inside the cell
inside
Functioning
channel
“Trapped” or
“Use-Dependent”
Blocker
procaine-H+
procaine-H+
procaine
16
Na+ channel blockers in medicine
Local anesthetics
Dental surgery (procaine = Novocain®)
Sunburn medications
Antiarrhythmics (heart)
“use-dependent blocker”
example: (procainamide)
Antiepileptics / anticonvulsants (brain)
“use-dependent blocker”
(phenytoin = Dilantin® )
17
from Lecture #2
H3CH 2C
+ CH CH
2
3
HN
procaine
H2C
Charged amine: may bind
to charged groups on the
protein
Ester: hydrolyzed to
terminate drug action
CH 2
O
C O
Aromatic: may bind
to nonpolar groups
on the protein
NH 2
How do we determine the detailed binding site for a blocking drug?
18
from Lecture 7
Site-Directed Mutagenesis on Ion Channels
DNA
Mutate the desired codon(s)
Latin, ‘in glass”
RNA polymerase promoter
in vitro RNA synthesis
measure
Express by injecting into
immature frog eggs
measure 19
Site-directed mutagenesis alternating with single-channel measurements of block
DNA
Mutate the desired codon(s)
Latin, ‘in glass”
RNA polymerase promoter
in vitro RNA synthesis
Express by injecting into
immature frog eggs
measure
20
Result:
structural interpretations of drug binding
21
from Lecture 5:
Cells have evolved elaborate processes for pumping out intracellular
Na+ and Ca2+.
These gradients can be used in two ways:
1. The gradients are used for uphill “exchange” to control the
concentrations of other small molecules.
2. Transient, local increases in intracellular Ca2+ and Na+
concentrations can now be used for signaling inside cells!
Two examples of Ca2+ channel blockers that regulate intracellular signals . . .
22
Baldomero “Toto” Olivera Ph D ‘71
(Distinguished alumnus award, Commencement 2002)
23
Geographer’s cone
Magician’s cone
Leopard cone
Cloth-of-gold cone
24
A conotoxin:
25 amino acids
held together by disulfide bonds
individual conotoxins specifically block individual ion channels
This conotoxin blocks Ca2+ channels.
Slightly modified, it is now the drug, ziconotide.
It suppresses transmission at pain synapses in the spinal cord.
(Swiss-prot viewer must be
installed on your computer)
http://www.its.caltech.edu/~lester/Bi-1/conotoxin-annotated.pdb
25
A second example:
Channel block in memory and learning.
D. O. Hebb (1949),
The Organization of Behavior: A Neuropsychological Theory
"When an axon of cell A is near enough to excite B and repeatedly or
persistently takes part in firing it, some growth process or metabolic
change takes place in one or both cells such that A’s efficiency, as one of
the cells firing B, is increased"
26
from Lecture 4
Hebb’s Idea, in 2006 terms
Postsynaptic
neuron
Presynaptic
neuron
Excitatory Inhibitory
terminal terminal
presynaptic
terminal
axon
dendrites
cell
body
nucleus
presynaptic
terminal
When these two neurons fire simultaneous impulses
via this synapse,
this synapse is strengthened.
Memory depends on a “coincidence detector”
postsynaptic
dendrite
synaptic
cleft
Nestler Figure 2-2
(rotated)
27
A Glutamate Receptor
glutamatebinding
site
natural transmitter
“agonist”
glutamate
synthetic agonist
NMDA
glutamate
28
The NMDA receptor is blocked by Mg2+
in a voltage-dependent manner
Mg2+
glutamate
outside
Functioning channel
inside
-30 mV or more positive
Mg2+-blocked channel
-60 mV or more negative
29
from Lecture 5
Time required to exchange waters of hydration
Na+ , K+
1 ns
(~ 109/s)
Ca2+
5 ns
(2 x 108/s)
Mg2+
10 ms
(105/s)
Conclusion:
Na+ , K+, and Ca2+ can
flow through single
channels at rates > 1000fold greater than Mg2+
Mg2+ is suitable for
transporters, but not for
channels.
30
The NMDA receptor conducts only when
1. The membrane potential is more positive than -30 mV
2. Glutamate is present
(intracellular concentrations of glutamate and Mg2+ are nearly irrelevant)
Action potential
plus
glutamate
functioning channel
Mg
-30 mV
Na+, Ca2+
outside
inside
A molecular coincidence detector leading to Na+ and Ca2+ influx,
with many intracellular effects (lectures 12, 14)
31
How ”tight” is the gigaohm seal?
Strong suction
3. Mechanically tight
The patch breaks,
but the seal remains intact,
allowing electrical contact with the cytosol.
This leads to . . .
1 mm
Little Alberts 12-22A
© Garland
32
The whole-cell patch clamp:
We usually measure macroscopic currents
Feynman’s Idea:
record small currents from a single
channel in the patch
A
The whole-cell clamp:
record larger currents from all the
other channels in the cell
A
33
High-Throughout Patch Clamping, 2006:
Device Fabrication applied to Drug Discovery
Conventional patch configurations
require a micromanipulator and microscope
Planar patch configurations require only suction
and can run in parallel on multiple wells
Glasslike material
http://www.axon.com/downloads/Xpress_Animations/PatchXpress.mpg
1. Channels are expressed in small “immortalized” mammalian cells, not frog eggs
2. The experiments use wild type human channels, not mutated channels
3. Gigaseal -> rupture -> Whole-cell clamp
(note the capacitive currents)
4. Voltage-gated channels, then ligand-gated channels
5. Desensitization
6. Ignore the advertisement at the end
34
End of Lecture 8
35