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1
Bi 1 Lecture 7
Monday, April 10, 2006 The Central Dogma of Drugs and the Brain,
Part 1:Drugs open and block ion channels
N
CH3N
Nicotine
2
[neurotransmitter]
openclosedchemical transmission atsynapses:
electric field
openclosedelectrical transmission inaxons:
actually, E
Major Roles for Ion Channelsfrom Lecture 6
3
nicotine procaine
N
CH3N
O
CH2
H2C
NH2
C O
NH3CH2C CH2CH3
botulinum toxin
Atomic-scale Structures
(Swiss-prot pdb viewer must be installed on your computer)
N
O
HO
HOCH3
morphine
morphine
http://www.its.caltech.edu/~lester/Bi-1/nicotine.pdb
http://www.its.caltech.edu/~lester/Bi-1/procaine.pdb
http://www.its.caltech.edu/~lester/Bi-1/morphine.pdb
from Lecture 2
4
nicotinic acetylcholine
receptor
Most drug receptors are membrane proteins
Outside the cell
Inside the cell = cytosol
(view in ~1995)
natural ligand(agonist)
nicotine, another agonist
Membrane = lipid bilayer
~ 100 Å= 10 nm
from Lecture 3
5
Overall topology of the nicotinic acetylcholine receptor(view in ~2000)
outside the cell:
5 subunitseach subunit has 4 -helices
in the membrane (20 membrane helices total)
Little Alberts figure 12-42© Garland publishing
from Lecture 3
6
The acetylcholine binding protein (AChBP) from a snail, discovered in 2001, strongly resembles the binding region
(Swiss-prot viewer must be installed on your computer)
Color by chainShow 2 subunits,Chains,Ribbons
5 subunits
Little Alberts figure 12-42© Garland publishing
http://www.its.caltech.edu/~lester/Bi-1/AChBP+Carb-5mer.pdb
from Lecture 3
7
http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdb
The AChBP binding site occupied by an acetylcholine analog (2004)http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdb
from Lecture 3
8
Binding region
Membrane region
Cytosolicregion
Colored by secondary
structure
Colored by subunit(chain)
Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005)
http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9
~ 2200 amino acids in 5 chains
(“subunits”),
MW ~ 2.5 x 106
from Lecture 3
9
How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 view
M2
M1
M3
M4
Ligand-bindingdomain
from Lecture 3
10
~60o
closedclosed
-OH
openopen
-OH
-OH
-OH
-OH
-OH
5 kinked -helices rotate,
removing the oily side chain ( = hydrophobic) from the pore
and exposing side chains with -OH groups.
This would provide a water-like (HOH) environment for the permeating ions.
One hypothesis about the nature of nicotinic receptor gating
in the transmembrane domain
13
5 pA = 104 ions/ms
20 ms
a nicotinic acetylcholine receptor exposed to acetylcholine
A sensitive electronic ammeter
dynamic range:5 s to 5 min
1 part in 108
Implementing Feynman’s Idea
14
How ”tight” is the gigaohm seal?
1. Electrically tight
See next slide
Little Alberts 12-22A© Garland
1 m
15
How ”tight” is the gigaohm seal?
1. Electrically tight
R = l/A
R ~ 109
= resistivity = 22 -cm;
l = length = 10 m;
A = area = 10 m x t (thickness);
Therefore
t ~ 2 x 10-11 m, or less than 1 Å!
t
pipette wall
membrane
t
pipette wall
membrane
16
acetylcholine in the pipette opens channels in the pipette
2. Chemically tight
The seal compartmentalizes molecules.
Molecules outside the pipette do not mix with molecules inside the pipette
acetylcholine outside the pipette opens channels outside the pipette
How ”tight” is the gigaohm seal?
17
Alberts omitted all of the electronics!
A
sensitive electronic ammeter
Little Alberts 12-22D© Garland
18
Max Delbruck
Richard Feynman
H. A. L
Carver Mead
“If you want to measure small, noisy signals, I have a Senior who can help”
from Lecture 6
19
Fred Sigworth ‘74 and Apostol’s Clock
http://www.math.caltech.edu/classes/ma1c-An/index.html#text
Ma 1c-Analytic track, Spring 2006:
http://www.info.med.yale.edu/bbs/faculty/sig_fr.html
Fred Sigworth’s Web page at Yale
20
Statistical analysis of single-molecule eventschannel opens
now we synchronize artificially on the opening event
n =1
0
23
Concentration of acetylcholine at a
synapse
Number of open channels
ms
0
high closed open
State 1 State 2
k21
all molecules begin here at
t= 0
units: s-1
24
We wish to test a hypothesis that a particular side chain governs channel opening/closing rates
~60o
closedclosed
-OH
openopen
-OH
-OH
-OH
-OH
-OH
25
Site-Directed Mutagenesis: the General Idea
RNA
Gene (DNA)
measure
Hypothesis about an important side chain(s)
Mutate the desired codon(s)
“Express” theprotein with an altered side chain(s)
26
in vitro RNA synthesisRNA polymerase promoter
DNA
measure
Site-Directed Mutagenesis on Ion Channels
Express by injecting into immature frog eggs
Mutate the desired codon(s)
Latin, ‘in glass”
measure
27
The identified side chain governs channel opening/closing rates
n =1
0
n =1
0
leucine (wild type)
alanine
29
Measured “dose-response” relations verify that an identified side chain governs agonist-receptor interactions
wild type (tryptophan)
phenylalanine
The instrument (~ 90 MB!):
http://www.moleculardevices.com/product_literature/download_form.php?docnum=475&prodid=108&useid=25&familyid=&interestid=
30
The cation- site in a nicotinic acetylcholine receptor
We know that acetylcholine and nicotine bind
within a “box” of aromatic amino acids;
Electrophysiology agrees with crystallography!
~ 8 Angstroms
tryptophan
31
Unnatural amino-acids define acetylcholine binding within 0.5 Angstroms
Quantum-mechanical calculations of cation- energy
Mea
sure
d ac
etyl
chol
ine
bind
ing
ener
gy
Dougherty group, Caltech Chemistry