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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

The final in-class slide (but not the posted slides) has a 1-point extra credit question.

You may not communicate this question to another student; no collaboration.

2

Action potentials and the electrocardiogram

Electrocardiogrammeasured on the skin

Action Potentialmeasured with

intracellularelectrode

P

S

R

T

Q

K+ channels conductNa+ channels conduct

~ 100 V

~ 100 mV

~ 1 sec

3

Monday, May 15. 2006 8:52 AM Kaiser Sunset Facility

Cardiology Lab, Treadmill facility

Part of Bi1 lecturer

Bi1 lecturer’sbaseline EKG

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An approximate explanation for the electrocardiogram, slide 1

The left ventricle pumps against the greatest resistancetherefore it has thickest walls; therefore its currents are the largest; therefore it contributes most of the ECG.

5

An approximate explanation for the electrocardiogram, slide 2like Lecture 6

CE

G

Na+ K+ Cl-extracellular

cytosol

CE

G

Na+ K+ Cl-

ClKNai

gEVdt

dVCI

iii

,,

;)(

extRextext IRV

The capacitive currents are largest

An extracellular

electrode pair

records IR drops

proportional to the

(absolute value) of

the 1st derivative of

membrane potential.

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chest

leg

extRextext IRV

Only a small fraction of the current flows across the resistance between chest and a limb.

This produces a V ~ 103 times smaller than the transmembrane potential.

The ECG records this signal

An approximate explanation for the electrocardiogram, slide 3

CE

G

Na+K+

Cl-

extracellular

intracellular

CE

G

Na+K+

Cl-

extRextext IRV

7

Action potentials and the electrocardiogram

Electrocardiogrammeasured on the skin

Action Potentialmeasured with

intracellularelectrode

P

S

R

T

Q

K+ channels conductNa+ channels conduct

~ 100 V

~ 100 mV

~ 1 sec

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ElectrocardiogramAction Potential

Two classes of V-dependent channel explain cardiac electrophysiology in long-QT Syndrome. ~ 8 genes (complementation groups)

Q-T

P

S

R

T

Q

a heart-specific Na channel fails to inactivate completely

Or, one of several heart-specific K channels fails to activate

Normal heart rhythm

Arrhythmia

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Primary subunit Auxiliary subunit

KvLQT2

hERG

Human ether-a-go-go related gene

KCNE2

(MiRP1)

A cardiac K channel is also the target for drug-induced arrhythmias

P

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.

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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.

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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

An exemplar inherited epilepsy:Autosomal dominant nocturnal frontal lobe epilepsy.

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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:

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How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 view

M2

M1

M3

M4

Ligand-bindingdomain

from Lecture 3:

15

ADNFLE and slow-channel myasthenic syndrome

Aligned Sequences of Mouse Muscle AChR M2 Domains

M2M1 M3 M4

2' 6' 10' 14' 18'9'

Autosomal Dominant Nocturnal Frontal Lobe Epilepsy

I T C I V L L S L T V F L L L I TL S LL

V

MMCT

TGTS

SSSA

IIII

SFNS

VAVV

LL

L

L

LL

STAA

LLQQ

TT

S

V

VV

FF FF

LLLL

LLFL

VL

L

ILII

VAAS

LLVV

Slow-Channel Myasthenic Syndrome:Abnormally long channel duration

LL

TV

L

X X X

T C I V L L A L T V F L L L I SL S K I V

22'

Ligand-binding domain IC loop

Muscle

Brain

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inside

Procaine Blocks Na+ Channels from inside the cell

procaineprocaine-H+

procaine-H+

Functioningchannel

“Trapped” or“Use-Dependent”

Blocker

from Lecture 8:

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Local anestheticsDental surgery (procaine = Novocain®)Sunburn medications

Antiarrhythmics (heart) “use-dependent blocker”example: (procainamide)

Antiepileptics / anticonvulsants (brain) “use-dependent blocker” (phenytoin = Dilantin® )

Na+ channel blockers in medicine

from Lecture 8:

18Nicotinic acetylcholine receptor

Carbamazepine, an antiepileptic drug,

binds in the pore

Some drugs compete with nicotine or acetylcholine

~ 40 Angstroms(4 nm)

transmembranedomain

based on Lecture 3:

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Cystic Fibrosis

1. Clinical description

2. Genetics

3. Gene structure

4. CFTR as a protein

5. Physiology of CFTR

6. What’s wrong with F508?

7. The cholera connection

8. Selective advantage of CF?

9. Therapeutic approaches:Incremental approachesGene therapy

Huntington’s Disease

1. Clinical description

2. Genetics

3. Gene structure

4. Huntingtin as a protein

5. Physiology of huntingtin

6. What’s wrong with the HD protein?

from Lecture 21

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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 ~ 1945He had 8 children.

1. Clinical description

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Again, we highlight neurons that make dopamine; here, note their postsynaptic targets in the striatum

Nestler Figure 8-6

from several previous lectures

“striped” GABA-producing“medium spiny” neurons

die in HD

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Huntington’s is a rare autosomal dominant disease (1 in 104 - 105 persons).

heterozygousmutant parent

“carrier”

HD

WT huntingtin

HD phenotype

homozygousWT parent

normal phenotype

like Lecture 21

WT huntingtin

WT huntingtin

HD

WT

HD phenotype

heterozygous“carrier”

HD

WT

HD phenotype

heterozygous“carrier”

WT

normal phenotype

homozygousWT

WT

normal phenotype

homozygousWT

WT WT

Dominance:50% of offspring have HD

2. Genetics

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First localized to 4p16.3 (~ 2.2 Mb) in 1983.

Gene product identified in 1993.

(from Lecture 20)

210 kb in length

67 exons, 3144 amino acids = 9432 nt coding region (~ 4% of the gene)

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)

3. Gene structure

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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.

4. Huntingtin as a protein

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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.

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?

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An N-terminal fragment of huntingtin containing the polyglutamine stretch

accumulates as aggregates in cells.

The aggregates often appear in the nucleus.

When this fragment is expressed in mice,

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.

Improper protein aggregates in HD

Nucleus

aggregate

aggregate

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Seymour Benzer found recently that

polyglutamine repeats also distort the

development of fruit fly eyes.

The polyglutamine repeat has been

tagged with GFP, and the proteins

clearly aggregate

Normal development can be “rescued”

with “chaperone” proteins, which help to

fold or eliminate misfolded proteins.

But the aggregates remain, suggesting

that the aggregates themselves are not

toxic.

Drosophila provides insights, as usual

wild typefly

fly expressing

polyglutamine repeats

glutamine repeats plus “chaperone” proteins

GFP

low-powerelectron microscope

lightmicroscope

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CFTR-F508

N

CR-domain

Cl- out

in

from lecture 21

Misfolded mutant proteins: a postulated common theme in inherited disease

polyglutamine forms-sheets

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< 10 nm

(FRET) detects polyglutamine aggregates

Cyan Fluorescent Protein (CFP) Yellow Fluorescent Protein (YFP)

blue photon

virtualcyan photon

yellow photon

like Lecture 11

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No interaction, no FRET

fused toYFP

fused toCFP

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Aggregation leads to FRET

fused toYFP

fused toCFP

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A type of fluorescence microscopy: fluorescence recovery after photobleaching (Ataxin is another triple repeat protein)

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

1. Use a laser to bleach all the GFP-tagged protein within the rectangle

33modified from Little Alberts 1st edition Fig 7-32

Controlled proteolysis takes place in the proteasome

from Lecture 18

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

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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