Transcript Lecture-22

The final in-class slide (but not the
posted slides) has a 1-point
extra credit question.
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Bi 1
“Drugs and the Brain”
Lecture 22 Revised 5/18/06
Monday, May 15, 2006
1. Long-QT syndrome;
2. Epilepsy
3. Huntington’s Disease
1
Action potentials and the electrocardiogram
Na+ channels conduct
K+ channels conduct
Action Potential
measured with
intracellular
electrode
~ 100 mV
~ 1 sec
R
Electrocardiogram
measured
on the skin
~ 100 mV
T
P
QS
2
Monday, May 15. 2006 8:52 AM
Kaiser Sunset Facility
Cardiology Lab, Treadmill facility
Part of Bi1 lecturer
Bi1 lecturer’s
baseline EKG
3
An approximate explanation for the electrocardiogram, slide 1
The left ventricle pumps against the greatest resistance
therefore it has thickest walls;
therefore its currents are the largest;
therefore it contributes most of the ECG.
4
like Lecture 6
An approximate explanation for the electrocardiogram, slide 2
The capacitive currents are largest
dV
I C
  (V  Ei ) g i ;
dt
i
i  Na, K , Cl
An extracellular
Vext  IRext
electrode pair
Rext
records IR drops
proportional to the
(absolute value) of
extracellular
Na+
K+
Cl-
Na+
K+
Cl-
the 1st derivative of
G
membrane potential.
C
G
C
E
E
cytosol
5
An approximate explanation for the electrocardiogram, slide 3
Vext  IRext
Rext
chest
Only a small fraction of the current
flows across the resistance between
chest and a limb.
This produces a V ~
times
smaller than the transmembrane
potential.
103
The ECG records this signal
Vext  IRext
Rext
C
extracellular
Na+K C
+ lG
E
intracellular
Na+K C
+ l-
C
G
E
leg
6
Action potentials and the electrocardiogram
Na+ channels conduct
K+ channels conduct
Action Potential
measured with
intracellular
electrode
~ 100 mV
~ 1 sec
R
Electrocardiogram
measured
on the skin
~ 100 mV
T
P
QS
7
Two classes of V-dependent channel explain cardiac electrophysiology
in long-QT Syndrome. ~ 8 genes (complementation groups)
a heart-specific Na channel fails to inactivate completely
Action Potential
Electrocardiogram
R
Normal heart rhythm
T
P
QS
Arrhythmia
Q-T
Or, one of several heart-specific K channels fails to activate
8
A cardiac K channel is also the target for drug-induced arrhythmias
Primary subunit
Auxiliary subunit
P
KvLQT2
hERG
Human ether-a-go-go
related gene
KCNE2
(MiRP1)
Seldane® blocks hERG and was pulled from the market;
Allegra® does not
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Epilepsies: Repeated Seizures
Seizure: Massive derangement of brain function caused by excessive and
synchronized function in a group of neurons.
A seizure can range from a “focal” 3-sec loss of consciousness, barely
noticeable (like a “space out”) . . .
to a “generalized” event that causes a person to tense for several sec before a
several sec jerking of his entire body.
Prevalence: ~ 5% of the general population experiences one or more seizures.
The repeated seizures termed epilepsy occur in ~0.5% of the population.
Causes: brain injury (included a traumatic blow to the head), chronic illness, and
inherited vulnerabilities .
Genetics: ~ 50% of epilepsies involve an inherited vulnerability.
10
Epilepsies caused by Bi 1 Molecules
Genetics: ~ 50% of human epilepsies involve an inherited vulnerability.
Many knockout mice have seizures. Most of these genes are not associated
with human epilepsies.
Nestler Table 21-3 lists ion channel defects that produce some inherited
epilepsies (also discussed in Problem set 7).
KNCQ, a family of K channels (loss of function).
SCN, a Na channel (gain of function).
CHRN, nicotinic acetylcholine receptors (gain or loss, still uncertain).
Problem Set 6, Q1; see next slides.
In general, the causal links are less well understood than for long-QT
syndrome.
11
An exemplar inherited epilepsy:
Autosomal dominant nocturnal frontal lobe epilepsy.
First described as a disease, 1994.
The first epilepsy gene mapped and sequenced (1995).
Seizures arise during phase 2 sleep (rather than “rapid eye-movement sleep”;
Sometimes confused with nightmares.
Some patients display abnormal brain waves (as in Nestler Figures 21-5, 21-6).
Controlled by carbamazepine, not by valproate
12
from Lecture 3:
Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005)
~ 2200
amino acids
in 5 chains
(“subunits”),
Binding
region
MW
~ 2.5 x 106
Membrane
region
Colored by
secondary
structure
Colored by
subunit
(chain)
Cytosolic
region
http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9
13
from Lecture 3:
How the binding of agonist (acetylcholine or nicotine)
might open the channel: June 2003 view
Ligand-binding
domain
M1
M2
M3
M4
14
ADNFLE and slow-channel myasthenic syndrome
Ligand-binding domain
a4
b2
IC loop
M3
M4
Autosomal Dominant Nocturnal Frontal Lobe Epilepsy
2'
6'
9' 10'
14'
18'
22'
L
I T L C I S V L L S L T V F L L L I T X X X
T L C I S V L L A L T V F L L L I S K I V
Slow-Channel Myasthenic Syndrome:
Muscle
a1
b
e
d
M2
V
Brain
M1
M
M
C
T
Abnormally long channel duration
T
G
T
S
L
L
V
V
S
S
S
A
I
I
I
I
S
F
N
S
V
A
V
V
L
L
L
L
L
L
L
L
S
T
A
A
L
L
Q
Q
T
T
T
S
V
V
V
V
F
F
F
F
L
L
L
L
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F
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V
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L
Aligned Sequences of Mouse Muscle AChR M2 Domains
I
L
I
I
V
A
A
S
15
from Lecture 8:
Procaine Blocks Na+ Channels from inside the cell
inside
“Trapped” or
“Use-Dependent”
Blocker
Functioning
channel
procaine-H+
procaine-H+
procaine
16
from Lecture 8:
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
based on Lecture 3:
Some drugs compete with
nicotine or acetylcholine
Carbamazepine,
an antiepileptic drug,
binds in the pore
~ 40 Angstroms
(4 nm)
transmembrane
domain
Nicotinic acetylcholine receptor
18
from Lecture 21
Cystic Fibrosis
Huntington’s Disease
1. Clinical description
1. Clinical description
2. Genetics
2. Genetics
3. Gene structure
3. Gene structure
4. CFTR as a protein
4. Huntingtin as a protein
5. Physiology of CFTR
5. Physiology of huntingtin
6. What’s wrong with F508?
6. What’s wrong with the HD protein?
7. The cholera connection
8. Selective advantage of CF?
9. Therapeutic approaches:
Incremental approaches
Gene therapy
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1. Clinical description
Onset at 30-40 yr.
Neurons in the striatum and cerebral cortex die,
leading to movement disorders (“chorea”), dementia, and eventually death.
Woody Guthrie 1912-1967
Mother died of Huntington’s chorea; Woody began suffering in ~ 1945
He had 8 children.
20
from several previous lectures
Again, we highlight neurons that make dopamine;
here, note their postsynaptic targets in the striatum
“striped”
GABA-producing
“medium spiny” neurons
die in HD
Nestler Figure 8-6
21
like Lecture 21
2. Genetics
Huntington’s is a rare autosomal dominant disease (1 in 104 - 105 persons).
heterozygous
mutant parent
“carrier”
HD
WT huntingtin
HD phenotype
homozygous
WT parent
WT huntingtin
WT huntingtin
normal phenotype
Dominance:
50% of offspring have HD
heterozygous
“carrier”
heterozygous
“carrier”
homozygous
WT
homozygous
WT
HD
HD
WT
WT
WT
WT
WT
WT
HD phenotype
HD phenotype
normal phenotype
normal phenotype
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3. Gene structure
(from Lecture 20)
First localized to 4p16.3 (~ 2.2 Mb) in
1983.
Gene product identified in 1993.
Personal decision:
does a person at risk for HD submit to
the decisive test based on DNA
sequencing?
Mutation
5’ (N-terminus)
3’ (C-terminus)
210 kb in length
67 exons, 3144 amino acids = 9432 nt coding region (~ 4% of the gene)
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4. Huntingtin as a protein
A CAG repeat, encoding glutamine,
is amplified. When (CAG)n grows
beyond n = 42, the disease occurs.
As n increases, age of onset
decreases.
Eight other human
neurodegenerative diseases are
caused by expanded triplet
repeats.
A baffling aspect of these diseases:
the proteins are expressed widely
in brain and other tissues,
yet each is toxic in a different,
highly specific group of neurons
and produces a distinct pathology.
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5. Physiology of huntingtin
We don’t know the normal function of huntingtin.
“Knockout mice” for huntingtin die early in embryonic development,
before the nervous system develops.
6. What’s wrong with the mutant huntingtin?
Mice expressing mutant huntingtin exhibit a progressive neurologic phenotype with
many of the features of HD, including
-choreiform movements,
-involuntary stereotypic movements,
-tremor, and epileptic seizures,
-nonmovement disorder components.
Evidently the mutant huntingtin has a destructive effect
that is not provoked by wild type huntingtin;
thus HD is produced by a “gain-of-function” mutation.
25
Improper protein aggregates in HD
An N-terminal fragment of huntingtin containing the polyglutamine stretch
accumulates as aggregates in cells.
The aggregates often appear in the nucleus.
aggregate
When this fragment is expressed in mice,
aggregate
Nucleus
or even in yeast,
the fragment aggregates as well.
It is not known whether the fragment is itself toxic, or whether the nuclear
localization is important for toxicity.
Huntingtin interacts with several other proteins in the cell.
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Drosophila provides insights, as usual
wild type
fly
Seymour Benzer found recently that
fly
expressing
polyglutamine
repeats
glutamine repeats
plus “chaperone”
proteins
polyglutamine repeats also distort the
development of fruit fly eyes.
The polyglutamine repeat has been
low-power
electron
microscope
tagged with GFP, and the proteins
clearly aggregate
Normal development can be “rescued”
with “chaperone” proteins, which help to
light
microscope
fold or eliminate misfolded proteins.
But the aggregates remain, suggesting
that the aggregates themselves are not
GFP
toxic.
27
from lecture 21
Cl-
out
in
N
R-domain
CFTR-F508
C
polyglutamine forms
b-sheets
Misfolded mutant proteins: a postulated common theme in inherited disease
28
like Lecture 11
(FRET) detects polyglutamine aggregates
blue photon
yellow photon
< 10 nm
virtual
cyan photon
Cyan Fluorescent Protein (CFP)
Yellow Fluorescent Protein (YFP)
29
No interaction, no FRET
fused to
CFP
fused to
YFP
30
Aggregation leads to FRET
fused to
CFP
fused to
YFP
31
A type of fluorescence microscopy: fluorescence recovery after photobleaching
(Ataxin is another triple repeat protein)
1. Use a laser to bleach all the GFP-tagged protein within the rectangle
2. Watch unbleached mobile GFP-tagged “short” ataxin (above) diffuse into the
square from other regions of the cell
But “long” ataxin in aggregates (below) is immobile for many minutes
PNAS
(2002),
99, 9310
32
from Lecture 18
Controlled proteolysis takes place in the proteasome
Mutant huntingtin may
escape proteolysis in
proteasomes because
(1) there are no
proteasomes in the nucleus
(2) mutant huntingtin may
be in a complex that cannot
be degraded
modified from Little Alberts 1st edition Fig 7-32
33
Intracellular inclusions in some neurodegenerative diseases
Alzheimer’s Disease
Parkinson’s Disease
Huntington’s Disease
We don’t know whether these aggregates are part of the disease process,
Or simply relatively harmless epiphenomema.
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Bi 1
“Drugs and the Brain”
End of Lecture 22
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