Transcript Folie 1

Michael Berger (Center for Brain Research,
Medical University Vienna, Austria):
Ligand/Receptor Interaction
L*
http://cwx.prenhall.com/horton/medialib/media_portfolio/09.html
Wenn Du mit anderen ein Schiff bauen willst,
Antoine de Saint Exupery
Wenn Du mit anderen ein Schiff bauen willst,
beginne nicht, mit Ihnen Holz zu sammeln,
Antoine de Saint Exupery
Wenn Du mit anderen ein Schiff bauen willst,
beginne nicht, mit Ihnen Holz zu sammeln,
sondern wecke in Ihnen die Sehnsucht
nach dem großen weiten Meer.
Antoine de Saint Exupery
What is a receptor?
What is a receptor?
A physical target mediating the
physiological effect of a drug.
What is a receptor?
A physical target mediating the
physiological effect of a drug.
What is a ligand?
What is a receptor?
A physical target mediating the
physiological effect of a drug.
What is a ligand?
A substance that (strongly)
binds to a tissue.
What is a receptor?
A physical target mediating the
physiological effect of a drug.
What is a ligand?
A substance that (strongly)
binds to a tissue.
What is an agonist?
What is a receptor?
A physical target mediating the
physiological effect of a drug.
What is a ligand?
A substance that (strongly)
binds to a tissue.
What is an agonist?
A substance that causes an effect, an
active change in the target tissue.
What is a receptor?
A physical target mediating the
physiological effect of a drug.
What is a ligand?
A substance that (strongly)
binds to a tissue.
What is an agonist?
A substance that causes an effect, an
active change in the target tissue.
What is an antagonist?
What is a receptor?
A physical target mediating the
physiological effect of a drug.
What is a ligand?
A substance that (strongly)
binds to a tissue.
What is an agonist?
A substance that causes an effect, an
active change in the target tissue.
What is an antagonist?
A substance that blocks the
effect of an agonist
What is a receptor?
A physical target mediating the
physiological effect of a drug.
What is a ligand?
A substance that (strongly)
binds to a tissue.
What is an agonist?
A substance that causes an effect, an
active change in the target tissue.
What is an antagonist?
A substance that blocks the
effect of an agonist
What is a transmitter?
What is a receptor?
A physical target mediating the
physiological effect of a drug.
What is a ligand?
A substance that (strongly)
binds to a tissue.
What is an agonist?
A substance that causes an effect, an
active change in the target tissue.
What is an antagonist?
A substance that blocks the
effect of an agonist
What is a transmitter?
A natural agonist released by a cell
and acting on a neighboring cell.
Association:
B+L
BL
KA =
[BL]
[B] . [L]
Association:
B+L
BL
KA =
[BL]
[B] . [L]
KA: association equilibrium constant
Association:
B+L
BL
KA =
[BL]
[B] . [L]
KA: association equilibrium constant
Dissociation:
BL
B+L
KD =
[B] . [L]
[BL]
KD: dissociation equilibrium constant
Association:
B+L
BL
KA =
[BL]
[B] . [L]
KA: association equilibrium constant
dimension: (concentration)-1
Dissociation:
BL
B+L
KD =
[B] . [L]
[BL]
KD: dissociation equilibrium constant
Association:
B+L
BL
KA =
[BL]
[B] . [L]
KA: association equilibrium constant
dimension: (concentration)-1
Dissociation:
BL
B+L
KD =
[B] . [L]
[BL]
KD: dissociation equilibrium constant
dimension: concentration
Association:
B+L
BL
Dissociation:
BL
B+L
Association:
B+L
BL
Dissociation:
BL
B+L
Strong binding: equilibrium is
on right side
on left side
Association:
B+L
BL
Dissociation:
BL
B+L
Strong binding: equilibrium is
KA =
on right side
on left side
[BL]
[B] . [L]
[B] . [L]
>> 1
KD =
[BL]
<< 1
Association:
B+L
BL
Dissociation:
BL
B+L
Strong binding: equilibrium is
KA =
on right side
on left side
[BL]
[B] . [L]
[B] . [L]
ln KA positiv
>> 1
KD =
[BL]
ln KD negativ
<< 1
Association:
B+L
BL
Dissociation:
BL
B+L
Strong binding: equilibrium is
KA =
on right side
on left side
[BL]
[B] . [L]
>> 1
[B] . [L]
KD =
ln KA positiv
Van't Hoff:
<< 1
[BL]
ln KD negativ
ΔGo = - RT . ln KA = + RT . ln KD
ΔGo: change in free enthalpy (Gibbs energy)
R: universal gas constant, 1.987 cal/(Mol . °K) or 8.314 J/(Mol . °K)
T: absolute temperature
The Van‘t Hoff equation allows the calculation of the free
enthalpy change of a reaction from the reaction‘s equilibrium
constant:
ΔGo (20 °C)
KA
KD
kcal/Mol
kJ/Mol
107 M-1
10-7 M
-9.4
-39.2
108 M-1
10-8 M
-10.7
-44.8
109 M-1
10-9 M
-12.0
-50.3
Van't Hoff:
ΔGo = - RT . ln KA = + RT . ln KD
ΔGo: change in free enthalpy (Gibbs energy)
R: universal gas constant, 1.987 cal/(Mol . °K) or 8.314 J/(Mol . °K)
T: absolute temperature
Examples for the change in free enthalpy Go in
various reactions
ΔGo (kcal/Mol)
Glucose + 6 O2
H2 + ½ O2
ATP
6 CO2 + 6 H2O
H2O
ADP + Pi -7.3
-686
-46
Examples for the change in free enthalpy Go in
various reactions
ΔGo (kcal/Mol)
Glucose + 6 O2
H2 + ½ O2
ATP
6 CO2 + 6 H2O
H2O
ADP + Pi -7.3
-686
-46
In these reactions, Go is reduced (exergonic processes)
Examples for the change in free enthalpy Go in
various reactions
ΔGo (kcal/Mol)
Glucose + 6 O2
H2 + ½ O2
ATP
6 CO2 + 6 H2O
H2O
ADP + Pi -7.3
-686
-46
In these reactions, Go is reduced (exergonic processes)
Bond dissociation energies
HO-H
CH3CH2-H
CH3-CH3
HO· + ·H
CH3CH2· + ·H
CH3· + ·CH3
118
101
90
The free enthalpy change ΔGo of a
reaction is composed of 2 terms:
Gibbs & Helmholtz: ΔGo = ΔHo – T . ΔSo
The free enthalpy change ΔGo of a
reaction is composed of 2 terms:
Gibbs & Helmholtz: ΔGo = ΔHo – T . ΔSo
change in enthalpy
The free enthalpy change ΔGo of a
reaction is composed of 2 terms:
Gibbs & Helmholtz: ΔGo = ΔHo – T . ΔSo
change in enthalpy
change in entropy, multiplied
by absolute temperature
! attention
℮ ∫ ∑ mathematics
∂
∞ √ % attention !
The free enthalpy change ΔGo of a
reaction is composed of 2 terms:
Gibbs & Helmholtz: ΔGo = ΔHo – T . ΔSo
change in enthalpy
change in entropy, multiplied
by absolute temperature
ln KD = ΔGo / RT = ΔHo / RT - ΔSo / R
• KD measured at various temperatures
• ln KD plotted against 1/T
! attention
℮ ∫ ∑ mathematics
∂
∞ √ % attention !
! attention
℮ ∫ ∑ mathematics
∂
∞ √ % attention !
ΔHo < 0: exotherm
(reaction mixture warms)
lg KD
055 °C
ΔSo > 0
(order is decreased)
0 °C
-3 -6 -9 -
‚
0.001
‚
0.002
●●
●●
‚
‚
0.003
0.004
1/T
Most common case: The
warmer (the lower 1/T), the
weaker the affinity (the
less negative lg KD).
ln KD = ΔGo / RT = ΔHo / RT - ΔSo / R
• KD measured at various temperatures
• ln KD plotted against 1/T
lg KD = 0.434 . ΔHo/R . 1/T – 0.434 . ΔSo/R
! attention
℮ ∫ ∑ mathematics
∂
[0.434 = 1/ln10]
∞ √ % attention !
! attention
℮ ∫ ∑ mathematics
∂
∞ √ % attention !
ΔHo < 0: exotherm
(reaction mixture warms)
lg KD
055 °C
ΔSo > 0
(order is decreased)
0 °C
-3 -6 -9 -
‚
0.001
‚
0.002
●●
●●
‚
‚
0.003
0.004
1/T
Most common case: The
warmer (the lower 1/T), the
weaker the affinity (the
less negative lg KD).
ln KD = ΔGo / RT = ΔHo / RT - ΔSo / R
Intersection with ordinate
gives information about ΔSo.
• KD measured at various temperatures
• ln KD plotted against 1/T
lg KD = 0.434 . ΔHo/R . 1/T – 0.434 . ΔSo/R
! attention
℮ ∫ ∑ mathematics
∂
[0.434 = 1/ln10]
∞ √ % attention !
! attention
℮ ∫ ∑ mathematics
∂
∞ √ % attention !
ΔHo < 0: exotherm
(reaction mixture warms)
lg KD
055 °C
ΔSo > 0
(order is decreased)
0 °C
-3 -6 -9 -
‚
0.001
‚
0.002
●●
●●
‚
‚
0.003
0.004
1/T
Most common case: The
warmer (the lower 1/T), the
weaker the affinity (the
less negative lg KD).
ln KD = ΔGo / RT = ΔHo / RT - ΔSo / R
Intersection with ordinate
gives information about ΔSo.
Slope allows access to ΔHo.
• KD measured at various temperatures
• ln KD plotted against 1/T
lg KD = 0.434 . ΔHo/R . 1/T – 0.434 . ΔSo/R
! attention
℮ ∫ ∑ mathematics
∂
[0.434 = 1/ln10]
∞ √ % attention !
! attention
℮ ∫ ∑ mathematics
∂
∞ √ % attention !
ΔHo < 0: exotherm
(reaction mixture warms)
lg KD
055 °C
ΔSo > 0
(order is decreased)
0 °C
-3 -6 -9 -
‚
0.001
‚
0.002
●●
●●
‚
‚
0.003
0.004
1/T
lg KD
0-
ΔSo < 0
(order is increased)
If order is increased,
driving force is even
more sensitive to high
temperatures
-3 -6 -9 -
‚
0.001
‚
0.002
●●
●●
‚
‚
0.003
0.004
1/T
ln KD = ΔGo / RT = ΔHo / RT - ΔSo / R
• KD measured at various temperatures
• ln KD plotted against 1/T
lg KD = 0.434 . ΔHo/R . 1/T – 0.434 . ΔSo/R
! attention
℮ ∫ ∑ mathematics
∂
[0.434 = 1/ln10]
∞ √ % attention !
! attention
℮ ∫ ∑ mathematics
∂
∞ √ % attention !
ΔHo < 0: exotherm
(reaction mixture warms)
lg KD
055 °C
ΔSo > 0
(order is decreased)
0 °C
-3 -6 -9 -
‚
0.001
‚
0.002
●●
●●
‚
‚
0.003
0.004
1/T
lg KD
0-
ΔSo < 0
(order is increased)
If order is increased,
driving force is even
more sensitive to high
temperatures
-3 -6 -9 -
‚
0.001
‚
0.002
●●
●●
‚
‚
0.003
0.004
1/T
It may be difficult to obtain
solid data that allow to
decide, if ΔSo is > or < 0.
ln KD = ΔGo / RT = ΔHo / RT - ΔSo / R
• KD measured at various temperatures
• ln KD plotted against 1/T
lg KD = 0.434 . ΔHo/R . 1/T – 0.434 . ΔSo/R
! attention
℮ ∫ ∑ mathematics
∂
[0.434 = 1/ln10]
∞ √ % attention !
! attention
℮ ∫ ∑ mathematics
∂
∞ √ % attention !
ΔHo < 0: exotherm
(reaction mixture warms)
lg KD
ΔHo > 0: endotherm
(reaction mixture cools)
lg KD
0-
055 °C
ΔSo > 0
(order is decreased)
0 °C
-3 -6 -9 -
‚
0.001
‚
0.002
●●
●●
-3 -6 -
‚
‚
0.003
0.004
-9 -
1/T
‚
0.001
‚
0.002
●●
●●
‚
‚
0.003
0.004
1/T
lg KD
0-
ΔSo < 0
(order is increased)
-3 -6 -9 -
‚
0.001
‚
0.002
●●
●●
‚
‚
0.003
0.004
1/T
Endotherm binding is
driven by decrease in
order only; here, driving
force increases with
temperature.
ln KD = ΔGo / RT = ΔHo / RT - ΔSo / R
• KD measured at various temperatures
• ln KD plotted against 1/T
lg KD = 0.434 . ΔHo/R . 1/T – 0.434 . ΔSo/R
! attention
℮ ∫ ∑ mathematics
∂
[0.434 = 1/ln10]
∞ √ % attention !
Mechanisms contributing to
ligand/receptor interaction:
1.
2.
3.
4.
5.
Ionic interaction
Hydrogen bonds
Hydrophobic interaction
Cation/p interaction
Van der Waals interaction
ionic interaction
attraction between 2 charges depends on
e1 . e2
D . r2
r ...
D ...
distance
dielectric constant
ionic interaction
attraction between 2 charges depends on
e1 . e2
D . r2
r ...
D ...
distance
dielectric constant
vacuum ...
hexane ...
H2O ...
1.0
1.9
78
ionic interaction
attraction between 2 charges depends on
e1 . e2
D . r2
r ...
D ...
distance
dielectric constant
vacuum ...
hexane ...
H2O ...
1.0
1.9
78
In water, ionic
interaction is hindered
by shells of water
molecules surrounding
each ion.
hydrogen bonds
B+L
BL
Formation of a hydrogen
bond is highly exergonic,
yields 3-7 kcal/mol
hydrogen bonds
B+L
BL
Formation of a hydrogen
bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor:
BH2O + LH2O
BL + H2OH2O
hydrogen bonds
B+L
BL
Formation of a hydrogen
bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor:
BH2O + LH2O
1. Break this bond.
BL + H2OH2O
hydrogen bonds
B+L
BL
Formation of a hydrogen
bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor:
BH2O + LH2O
2. Break this bond.
1. Break this bond.
BL + H2OH2O
hydrogen bonds
B+L
BL
Formation of a hydrogen
bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor:
BH2O + LH2O
BL + H2OH2O
2. Break this bond.
1. Break this bond.
3. Form this bond.
hydrogen bonds
B+L
BL
Formation of a hydrogen
bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor:
BH2O + LH2O
BL + H2OH2O
2. Break this bond.
1. Break this bond.
4. Form this bond.
3. Form this bond.
hydrogen bonds
B+L
BL
Formation of a hydrogen
bond is highly exergonic,
yields 3-7 kcal/mol
However, enthalpy balance is poor:
BH2O + LH2O
BL + H2OH2O
2. Break this bond.
1. Break this bond.
4. Form this bond.
3. Form this bond.
Hydrogen bond formation mainly driven by increase in
entropy, since the water molecules “get more freedom“
(2 kcal per mol of water).
hydrophobic interaction
Molecules or parts
of molecules
(„residues“) without
charge, that cannot
form a hydrogen
bond, are called
hydrophobic. They
aggregate together
to reduce the
contact with water
to a minimum.
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
hydrophobic interaction
Molecules or parts
of molecules
(„residues“) without
charge, that cannot
form a hydrogen
bond, are called
hydrophobic. They
aggregate together
to reduce the
contact with water
to a minimum.
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
2
H
H
H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
O H
O H
O H
1
8
3
7
4
6
5
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
10
H
H
H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
O H
O H
O H
9
16
11
15
12
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
14
13
hydrophobic interaction
Molecules or parts
of molecules
(„residues“) without
charge, that cannot
form a hydrogen
bond, are called
hydrophobic. They
aggregate together
to reduce the
contact with water
to a minimum.
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
O H
O H
O H
H
H
H
O H
O H
O H
H
H
H
O H
O H
O H
3
4
2
5
1
6
O H
O H
O H
H
H
H
O H
O H
O H
12
7
H
H
H
11
O H
8
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
H
H
H
H
H
H
H
H
O H
O H
O H
O H
O H
O H
O H
O H
10
9
hydrophobic interaction
• This example is nice, but wrong.
• Hydrogene bonds are never left open.
• In contact with an inert partner, water
molecules are highly ordered.
• Reduction of contact area leads to reduced
order.
hydrophobic interaction
• This example is nice, but wrong.
• Hydrogene bonds are never left open.
• In contact with an inert partner, water
molecules are highly ordered.
• Reduction of contact area leads to reduced
order.
• Reduction of Go by hydrophobic interaction is
always due to the entropy term T . ΔS
Gibbs & Helmholtz: ΔGo = ΔHo – T . ΔSo
hydrophobic interaction
• This example is nice, but wrong.
• Hydrogene bonds are never left open.
• In contact with an inert partner, water
molecules are highly ordered.
• Reduction of contact area leads to reduced
order.
• Reduction of Go by hydrophobic interaction is
always due to the entropy term T . ΔS
• Empirical rule: Δ Go = -0.03 x area hidden from
water (in Ǻ2).
cation/p interaction
A molluscan
acetylcholine
(AcCh) binding
protein, with high
sequence homology
to the AcCh binding
site of the nicotinic
receptor, has been
crystallized. The
binding pocket is
surrounded by tyr
and trp residues
(Bejc et al. 2001,
Nature 411: 269)
Van der Waals interaction
Two atoms
„touching“ each
other with their
electron shells
redistribute their
charges, resulting
in attraction.
http://www.columbia.edu/cu/biology/courses/c2005/lectures/lec02_06.html
Van der Waals interaction
range 3-4 Ǻ, turns into
repulsion at shorter
distances
contribution to ΔGo 0.51.0 kcal/Mol (lower than
hydrogen bond)
A „good“ ligand
undergoes 5-10 van der
Waals contacts with his
receptor.
hydrogen bond
Van der Waals
interaction
Van der Waals interaction
The ensemble of
van der Waals
interactions is
responsible for the
key/lock nature of
ligand/receptor
interaction.
1998 Leif Saul
Example for the interaction of a
hypothetical ligand with its receptor:
kcal/mol
formation of a hydrogen bond ...
loss of hydrogen bond with H2O...
- 5.0
+ 5.0
Example for the interaction of a
hypothetical ligand with its receptor:
kcal/mol
formation of a hydrogen bond ...
loss of hydrogen bond with H2O...
preliminary balance:
- 5.0
+ 5.0
±0
Example for the interaction of a
hypothetical ligand with its receptor:
kcal/mol
formation of a hydrogen bond ...
loss of hydrogen bond with H2O ...
2 H2O set free …
Hydrophobic interaction …
8 van der Waals contacts …
- 5.0
+ 5.0
- 4.0
- 2.0
- 4.7
Example for the interaction of a
hypothetical ligand with its receptor:
kcal/mol
formation of a hydrogen bond ...
loss of hydrogen bond with H2O ...
2 H2O set free …
Hydrophobic interaction …
8 van der Waals contacts …
balance:
- 5.0
+ 5.0
- 4.0
- 2.0
- 4.7
-10.7
Example for the interaction of a
hypothetical ligand with its receptor:
kcal/mol
formation of a hydrogen bond ...
loss of hydrogen bond with H2O ...
2 H2O set free …
Hydrophobic interaction …
8 van der Waals contacts …
- 5.0
+ 5.0
- 4.0
- 2.0
- 4.7
balance:
-10.7
ΔGo (20 °C)
KA
KD
kcal/Mol
kJ/Mol
107 M-1
10-7 M
-9.4
-39.2
108 M-1
10-8 M
-10.7
-44.8
109 M-1
10-9 M
-12.0
-50.3
How many receptors do we expect in a
responsive tissue?
Which analytical tools will be necessary to
detect them?
How many receptors do we expect in a
responsive tissue?
•
•
Theoretical assumption: the tissue consists of cubes
10 µm x 10 µm x 10 µm
Then, 1 mg tissue would consist of 100 x 100 x 100 =
106 cells
How many receptors do we expect in a
responsive tissue?
Josef Loschmidt
(1821-1895)
•
•
•
•
•
Theoretical assumption: the tissue consists of cubes
10 µm x 10 µm x 10 µm
Then, 1 mg tissue would consist of 100 x 100 x 100 =
106 cells
If each cell bears 1 binding site, this would result in
106 binding sites / mg tissue
1 fMol = 6 x 1023-15 = 6 x 108 molecules
106 molecules = 1/600 fMol
How many receptors do we expect in a
responsive tissue?
•
•
•
The most common binding sites occur at densities of 10 to
several 100 fMol/mg tissue.
This is much more than 1/600 fMol/mg tissue.
Thus, receptor-bearing cells have not only 1, but several
thousands of binding sites.
Freeze-fracture
analysis of AMPA
receptors labelled
with immuno gold
antibodies (5 nm)
at the postsynaptic
site on cerebellar
Purkinje cells
(climbing fiber
input). Tanaka et al
(2005) J Neurosci
25:799
Which analytical tools will be necessary to
detect them?
Labelling: Replacement of one or more protons by
tritium (3H; molecule practically unchanged)
Marie & Pierre Curie
Which analytical tools will be necessary to
detect them?
Radioactivity measured in
• Curie (Ci, mCi, µCi)
(the radioactivity
of 1 g radium)
Marie & Pierre Curie
Which analytical tools will be necessary to
detect them?
Radioactivity measured in
• Curie (Ci, mCi, µCi)
• Becquerel (Bq, decays / s)
• dpm (decays / min)
1 Bq = 60 dpm
Henry Becquerel
Which analytical tools will be necessary to
detect them?
Radioactivity measured in
• Curie (Ci, mCi, µCi)
• Becquerel (Bq, decays / s)
• dpm (decays / min)
1 µCi = 2 220 000 dpm
1 nCi = 2 220 dpm
1 pCi = 2.22 dpm
Henry Becquerel
Which analytical tools will be necessary to
detect them?
Comparison of 3H with other nuclides
(1 radioactive atom / molecule)
t½
14C
5 730 y
3H
12.4 y
35S
87 d
131I
8d
1
10
100
The shorter the half-life, the hotter the radioligand.
1000
fMol
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
A rule of thumb is
a principle with
broad application
that is not intended
to be strictly
accurate or reliable
for every situation.
(Wikipedia)
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
• 1 fMol = 10-15 x 6 . 1023 = 6 . 108 molecules
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
• 1 fMol = 10-15 x 6 . 1023 = 6 . 108 molecules
• t½ = 12.3 y = 4 500 d = 108 000 h = 6.48 . 106 min
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
• 1 fMol = 10-15 x 6 . 1023 = 6 . 108 molecules
• t½ = 12.3 y = 4 500 d = 108 000 h = 6.48 . 106 min
General idea: Since we know that half of the radioactive nuclei
will decay in 6.48 million minutes, we might obtain the number
of nuclei decaying in 1 minute simply by dividing half of the
number of nuclei by 6.48 millions.
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
• 1 fMol = 10-15 x 6 . 1023 = 6 . 108 molecules
• t½ = 12.3 y = 4 500 d = 108 000 h = 6.48 . 106 min
• dpm = 6 . 108 . 0.5 / 6.48 . 106 = 46
General idea: Since we know that half of the radioactive nuclei
will decay in 6.48 million minutes, we might obtain the number
of nuclei decaying in 1 minute simply by dividing half of the
number of nuclei by 6.48 millions.
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
• 1 fMol = 10-15 x 6 . 1023 = 6 . 108 molecules
• t½ = 12.3 y = 4 500 d = 108 000 h = 6.48 . 106 min
• dpm = 6 . 108 . 0.5 / 6.48 . 106 = 46
% original activity
100
80
t1/2 = 12.3 years
60
40
20
0
linear decay
0
5
10
15
20 years
0.5 would be correct, if the decay rate would be the
same for the whole decay period.
Which analytical tools will be necessary to
detect them?
How many dpm can be expected from 1 fMol 3H?
• 1 fMol = 10-15 x 6 . 1023 = 6 . 108 molecules
• t½ = 12.3 y = 4 500 d = 108 000 h = 6.48 . 106 min
• dpm = 6 . 108 . ln2 / 6.48 . 106 = 64
% original activity
100
80
t1/2 = 12.3 years
60
40 exponential decay
20
0
linear decay
0
5
10
15
20 years
0.5 would be correct, if the decay rate would be the
same for the whole decay period. However,
radioactive decay follows an exponential law;
therefore, 0.5 must be replaced by ln2 = 0.69.
Why the natural logarithm of 2?
! attention
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∞ √ % attention !
Which analytical tools will be necessary to
detect them?
A … number of radioactive nuclei
k … decay constant
dA/dt = -k . A
∫(1/A)dA = -k . ∫dt
ln(A/Ao) = -k . Δt
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Which analytical tools will be necessary to
detect them?
A … number of radioactive nuclei
k … decay constant
dA/dt = -k . A
∫(1/A)dA = -k . ∫dt
ln(A/Ao) = -k . Δt
A = Ao . e-k.Δt
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! attention
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Which analytical tools will be necessary to
detect them?
A … number of radioactive nuclei
k … decay constant
dA/dt = -k . A
∫(1/A)dA = -k . ∫dt
ln(A/Ao) = -k . Δt
A = Ao . e-k.Δt
k is related to t½:
! attention
ln(½) = -k . t½
k = ln2 / t½
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! attention
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Which analytical tools will be necessary to
detect them?
A … number of radioactive nuclei
k … decay constant
dA/dt = -k . A
∫(1/A)dA = -k . ∫dt
ln(A/Ao) = -k . Δt
A = Ao . e-k.Δt
k is related to t½:
ln(½) = -k . t½
k = ln2 / t½
for 1 min (Δt = 1):
-ΔA = k . A . 1
= ln2 / t½ . A
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Which analytical tools will be necessary to
detect them?
Therefore, it can be expected that 6 . 108 tritium nuclei
(1 fMol) will emit
6 . 108 . ln2 / 6.48 . 106 = 64 electrons / min.
A molecule labeled with one single 3H has a specific
radioactivity (short: specific activity) of 64 dpm / fMol.
Which analytical tools will be necessary to
detect them?
Therefore, it can be expected that 6 . 108 tritium nuclei
(1 fMol) will emit
6 . 108 . ln2 / 6.48 . 106 = 64 electrons / min.
A molecule labeled with one single 3H has a specific
radioactivity (short: specific activity) of 64 dpm / fMol.
Remember:
1 µCi = 2 220 000 dpm
1 nCi = 2 220 dpm
1 pCi = 2.22 dpm
64 dpm / fMol = 28.8 pCi / fMol = 28.8 Ci / mMol
Which analytical tools will be necessary to
detect them?
64 dpm / fMol = 28.8 pCi / fMol = 28.8 Ci / mMol
Which analytical tools will be necessary to
detect them?
64 dpm / fMol = 28.8 pCi / fMol = 28.8 Ci / mMol
Which analytical tools will be necessary to
detect them?
Comparison of 3H with other nuclides
(1 radioactive atom / molecule)
t½
1
10
100
1000
14C
5 730 y
0.14
1.4
14
140
3H
12.4 y
64
640
6.4*103
64*103
35S
87 d
3.3*103
33*103
330*103
3.3*106
131I
8d
36*103
360*103
3.6*106
36*106
dpm / mg tissue
fMol
Which analytical tools will be necessary to
detect them?
Comparison of 3H with other nuclides
(1 radioactive atom / molecule)
t½
1
10
100
1000
14C
5 730 y
0.14
1.4
14
140
3H
12.4 y
64
640
6.4*103
64*103
35S
87 d
3.3*103
33*103
330*103
3.3*106
131I
8d
36*103
360*103
3.6*106
36*106
dpm / mg tissue
most common experimental condition
fMol
Properties of 3H
• can replace 1H present in every organic
molecule
• does not change the properties of the
labeled molecule (no isotope effect)
• t½ 12.4 y
• b decay (emits electrons)
• radiation reaches in air 6 mm, in liquid
and tissue 6 µm
• relatively safe to work with (no shielding
required)
• the only risk is incorporation of > 1 mCi
• only reliable method of counting:
Properties of 3H
• can replace 1H present in every organic
molecule
• does not change the properties of the
labeled molecule (no isotope effect)
• t½ 12.4 y
• b decay (emits electrons)
• radiation reaches in air 6 mm, in liquid
and tissue 6 µm
• relatively safe to work with (no shielding
required)
• the only risk is incorporation of > 1 mCi
• only reliable method of counting: liquid
scintillation
Analytical techniques
equilibrium dialysis
non-equilibrium techniques for
receptors in solution
•
•
•
•
gel filtration
charcoal adsorption
precipitation
adsorption to glass fiber filters
non.-equilibrium techniques for
particulate receptors
• centrifugation
• filtration
• slice autoradiography
B + L*
BL*
Analytical techniques
B + L*
BL*
equilibrium dialysis
non-equilibrium techniques for
receptors in solution
•
•
•
•
gel filtration
charcoal adsorption
precipitation
adsorption to glass fiber filters
non.-equilibrium techniques for
particulate receptors
• centrifugation
• filtration
• slice autoradiography
B
BL*
L*
L*
Analytical techniques
equilibrium dialysis
non-equilibrium techniques for
receptors in solution
•
•
•
•
gel filtration
charcoal adsorption
precipitation
adsorption to glass fiber filters
non.-equilibrium techniques for
particulate receptors
• centrifugation
• filtration
• slice autoradiography
B + L*
BL*
Analytical techniques
equilibrium dialysis
non-equilibrium techniques for
receptors in solution
•
•
•
•
gel filtration
charcoal adsorption
precipitation
adsorption to glass fiber filters
non.-equilibrium techniques for
particulate receptors
• centrifugation
• filtration
• slice autoradiography
B + L*
BL*
Analytical techniques
B + L*
BL*
equilibrium dialysis
non-equilibrium techniques for
receptors in solution
•
•
•
•
gel filtration
charcoal adsorption
precipitation
adsorption to glass fiber filters
non.-equilibrium techniques for
particulate receptors
• centrifugation
• filtration
• slice autoradiography
polyethylene glycol
n = 6 000 – 8 000
Analytical techniques
equilibrium dialysis
non-equilibrium techniques for
receptors in solution
•
•
•
•
gel filtration
charcoal adsorption
precipitation
adsorption to glass fiber filters
non.-equilibrium techniques for
particulate receptors
• centrifugation
• filtration
• slice autoradiography
B + L*
BL*
Analytical techniques
equilibrium dialysis
non-equilibrium techniques for
receptors in solution
•
•
•
•
gel filtration
charcoal adsorption
precipitation
adsorption to glass fiber filters
non.-equilibrium techniques for
particulate receptors
• centrifugation
• filtration
• slice autoradiography
B + L*
BL*
Analytical techniques
equilibrium dialysis
non-equilibrium techniques for
receptors in solution
•
•
•
•
gel filtration
charcoal adsorption
precipitation
adsorption to glass fiber filters
non.-equilibrium techniques for
particulate receptors
• centrifugation
• filtration
• slice autoradiography
B + L*
BL*
Analytical techniques
B + L*
BL*
equilibrium dialysis
non-equilibrium techniques for
receptors in solution
•
•
•
•
gel filtration
charcoal adsorption
precipitation
adsorption to glass fiber filters
non.-equilibrium techniques for
particulate receptors
• centrifugation
• filtration
• slice autoradiography
L*
L*
L*
L*
L*
L*
L*
coating
glass
L*
L*
L*
L*
L*
Saturation & non-specific binding
Saturability: a radioligand can only be displaced if the
target density is low.
Other examples for saturability:
Langmuir isotherme (monomolecular layer on a surface),
enzyme reaction rate
(Michaelis-Menten).
http://www.steve.gb.com/science/membranes.html
Saturation & non-specific binding
T o S a v e t h is t e m p la t e ,C h o o s e
F ile :T e m p la t e :T e m p la t e S a v e .
BL (fMol)
4000
3000
no analysis
possible
2000
favorable
conditions
1000
0
0
500
1000
1500
L (nM)
2000
Saturation & non-specific binding
T o S a v e t h is t e m p la t e ,C h o o s e
F ile :T e m p la t e :T e m p la t e S a v e .
BL (fMol)
4000
3000
no analysis
possible
2000
favorable
conditions
1000
0
0
At low nM concentrations,
most of the radioligand L
is bound to saturable
high affinity sites.
500
1000
1500
L (nM)
2000
Saturation & non-specific binding
T o S a v e t h is t e m p la t e ,C h o o s e
F ile :T e m p la t e :T e m p la t e S a v e .
BL (fMol)
4000
3000
no analysis
possible
2000
favorable
conditions
1000
0
0
At low nM concentrations,
most of the radioligand L
is bound to saturable
high affinity sites.
500
1000
1500
L (nM)
2000
At high concentrations,
the linearly rising
non-specific binding
will dominate, and
specific binding
can no longer
be detected.
Saturation & non-specific binding
BL
B+L
KD =
[B] . [L]
[BL]
KD: dissociation equilibrium constant
BL
With increasing [L] more
binding sites are
occupied (BL) and free
sites (B) are lost. The
sum
150
100
50
BL + B = BM
0
0
10
20
L
30
remains constant.
Saturation & non-specific binding
KD =
[BL]
replace [B] by BM – [BL]:
BL
150
100
50
0
[B] . [L]
0
10
20
L
30
Saturation & non-specific binding
KD =
[B] . [L]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . [L]
[BL]
solve for [BL]:
50
0
[BL]
0
10
20
L
30
Saturation & non-specific binding
KD =
[B] . [L]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . [L]
[BL]
solve for [BL]:
50
0
[BL]
KD . [BL] = BM . [L] – [BL] . [L]
0
10
20
L
30
Saturation & non-specific binding
KD =
[B] . [L]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . [L]
[BL]
solve for [BL]:
50
0
[BL]
KD . [BL] = BM . [L] – [BL] . [L]
0
10
20
L
30
[BL] . ([L] + KD) = BM . [L]
Saturation & non-specific binding
KD =
[B] . [L]
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
(BM - [BL]) . [L]
100
solve for [BL]:
50
0
[BL]
KD . [BL] = BM . [L] – [BL] . [L]
0
10
20
L
30
[BL] . ([L] + KD) = BM . [L]
[L]
Langmuir isotherm
[BL] = BM .
[L] + KD
Saturation & non-specific binding
KD =
[B] . [L]
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
(BM - [BL]) . [L]
100
solve for [BL]:
50
0
[BL]
KD . [BL] = BM . [L] – [BL] . [L]
0
10
20
L
30
[BL] . ([L] + KD) = BM . [L]
[L]
Irving Langmuir
1881-1957
Nobel price 1932
Langmuir isotherm
[BL] = BM .
[L] + KD
Saturation & non-specific binding
KD =
[B] . [L]
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
(BM - [BL]) . [L]
100
[BL]
solve for [BL]:
50
0
wrong!
KD . [BL] = BM . [L] – [BL] . [L]
0
10
20
L
30
[BL] . ([L] + KD) = BM . [L]
[L]
Irving Langmuir
1881-1957
Nobel price 1932
Langmuir isotherm
[BL] = BM .
[L] + KD
Saturation & non-specific binding
KD =
[B] . [L]
replace [B] by BM – [BL]:
BL
150
KD =
100
50
0
[BL]
0
10
20
L
30
(BM - [BL]) . (Lo – [BL])
[BL]
correct
! attention
℮ ∫ ∑ mathematics
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∞ √ % attention !
Saturation & non-specific binding
[B] . [L]
KD =
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . (Lo – [BL])
[BL]
solve for [BL]:
50
KD . [BL] = BM . Lo – BM . [BL] – [BL] . Lo + [BL]2
0
0
10
20
30
L
! attention
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∞ √ % attention !
! attention
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∂
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Saturation & non-specific binding
[B] . [L]
KD =
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . (Lo – [BL])
[BL]
solve for [BL]:
50
KD . [BL] = BM . Lo – BM . [BL] – [BL] . Lo + [BL]2
0
0
10
20
L
! attention
30
[BL]2 – [BL] . (BM + Lo + KD) + BM . Lo = 0
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Saturation & non-specific binding
Sweet memories…
[B] . [L]
KD =
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . (Lo – [BL])
[BL]
solve for [BL]:
50
KD . [BL] = BM . Lo – BM . [BL] – [BL] . Lo + [BL]2
0
0
10
20
L
! attention
30
[BL]2 – [BL] . (BM + Lo + KD) + BM . Lo = 0
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∞ √ % attention !
! attention
℮ ∫ ∑ mathematics
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∞ √ % attention !
Saturation & non-specific binding
Sweet memories…
[B] . [L]
KD =
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . (Lo – [BL])
[BL]
solve for [BL]:
50
KD . [BL] = BM . Lo – BM . [BL] – [BL] . Lo + [BL]2
0
0
10
20
L
30
[BL]2 – [BL] . (BM + Lo + KD) + BM . Lo = 0
[BL] = ½ . {BM + Lo + KD - [(BM + Lo + KD)2 – 4 . BM . Lo]½}
! attention
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! attention
℮ ∫ ∑ mathematics
∂
∞ √ % attention !
Saturation & non-specific binding
Sweet memories…
[B] . [L]
KD =
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . (Lo – [BL])
[BL]
solve for [BL]:
50
KD . [BL] = BM . Lo – BM . [BL] – [BL] . Lo + [BL]2
0
0
10
20
L
30
[BL]2 – [BL] . (BM + Lo + KD) + BM . Lo = 0
[BL] = ½ . {BM + Lo + KD - [(BM + Lo + KD)2 – 4 . BM . Lo]½}
In this case, quantities Lo and KD are not entered as concentrations, but
as moles in the respective volume chosen, in the same units as B M.
! attention
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∞ √ % attention !
! attention
℮ ∫ ∑ mathematics
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Saturation & non-specific binding
Sweet memories…
[B] . [L]
KD =
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . (Lo – [BL])
[BL]
solve for [BL]:
50
0
3 times more ligand than
receptors at KD
concentration (8% loss)
0
10
20
L
KD . [BL] = BM . Lo – BM . [BL] – [BL] . Lo + [BL]2
30
[BL]2 – [BL] . (BM + Lo + KD) + BM . Lo = 0
[BL] = ½ . {BM + Lo + KD - [(BM + Lo + KD)2 – 4 . BM . Lo]½}
In this case, quantities Lo and KD are not entered as concentrations, but
as moles in the respective volume chosen, in the same units as B M.
! attention
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∞ √ % attention !
! attention
℮ ∫ ∑ mathematics
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∞ √ % attention !
Saturation & non-specific binding
Sweet memories…
[B] . [L]
KD =
[BL]
replace [B] by BM – [BL]:
BL
150
KD =
100
(BM - [BL]) . (Lo – [BL])
[BL]
solve for [BL]:
50
0
3 times more receptor than
ligand at KD concentration
(57% loss)
0
20
10
L
30
KD . [BL] = BM . Lo – BM . [BL] – [BL] . Lo + [BL]2
[BL]2 – [BL] . (BM + Lo + KD) + BM . Lo = 0
[BL] = ½ . {BM + Lo + KD - [(BM + Lo + KD)2 – 4 . BM . Lo]½}
In this case, quantities Lo and KD are not entered as concentrations, but
as moles in the respective volume chosen, in the same units as B M.
! attention
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∂
∞ √ % attention !
Saturation & non-specific binding
A realistic saturation function is a composite of 2
simultaneous processes:
1: non-specific binding
It is sufficient to measure 2 points;
extrapolation of L  0 results in the
blank of the measuring method (*).
BL
150
100

50
0

0
*
10
20
L
30
1
Saturation & non-specific binding
A realistic saturation function is a composite of 2
simultaneous processes:
1: non-specific binding
BL
150
2
2: specific binding
100

50
0
It is sufficient to measure 2 points;
extrapolation of L  0 results in the
blank of the measuring method (*).

0
*
10
20
L
30
1
... Is sitting on the non-specific
binding, obtained as difference
between total and non-specific
binding.
Saturation & non-specific binding
A realistic saturation function is a composite of 2
simultaneous processes:
1: non-specific binding
BL
150
2
2: specific binding
100
†

50
0
It is sufficient to measure 2 points;
extrapolation of L  0 results in the
blank of the measuring method (*).

0
*
10
20
L
30
1
... Is sitting on the non-specific
binding, obtained as difference
between total and non-specific
binding (†).
Saturation & non-specific binding
Mathematical combination of both processes:
BL
150
2
100
†

50
0

0
*
10
20
L
30
1
Saturation & non-specific binding
Mathematical combination of both processes:
1: non-specific binding
[L]
BL
150
2
100
†

50
0

0
*
10
20
L
30
1
[BL] = BU .
[L] + KU
Saturation & non-specific binding
Mathematical combination of both processes:
1: non-specific binding
[L]
BL
150
2
[L] + KU
2: specific binding
100
†
[L]

50
0
[BL] = BU .

0
*
10
[BL] = BS .
[L] + KS
20
L
1
30
Saturation & non-specific binding
Mathematical combination of both processes:
1: non-specific binding
[L]
BL
150
2
[L] + KU
2: specific binding
100
†
[L]

50
0
[BL] = BU .
1
[BL] = BS .

0
*
10
[L] + KS
20
L
30
KU (~mM) >> Ks (nM)
Saturation & non-specific binding
Mathematical combination of both processes:
1: non-specific binding
[L]
BL
150
2
[L] + KU
2: specific binding
100
†
[L]

50
0
[BL] = BU .
1
[BL] = BS .

0
*
10
[L] + KS
20
30
KU (~mM) >> Ks (nM)
L
At reasonable ligand concentrations, [L] + KU ~ KU and non-specific binding is
a linear function of [L]:
[L]
BU
. [L]
+
[BL] = BS .
[L] + KS
KU
Saturation & non-specific binding
The most important value, the specific
binding, is not directly accessible. It
must be calculated by substracting the
non-specific binding from total binding.
BL
100
50
NB
0
-9
-8
-7
-6
-5
log[I]
Saturation & non-specific binding
The most important value, the specific
binding, is not directly accessible. It
must be calculated by substracting the
non-specific binding from total binding.
BL
100
The non-specific binding NB
is measured as bound ligand
that is impossible to
displace, even by high
concentrations of potent
displacers.
50
NB
0
-9
-8
-7
-6
-5
log[I]
Saturation & non-specific binding
Strategies to keep non-specific binding low:
Saturation & non-specific binding
Strategies to keep non-specific binding low:
• choose a biological source with a high density of
high-affinity binding sites
Saturation & non-specific binding
Strategies to keep non-specific binding low:
• choose a biological source with a high density of
high-affinity binding sites
• select a radioligand concentration around the
expected KD ( a few hundred to a few thousand
dpm will be sufficient as result)
Saturation & non-specific binding
Strategies to keep non-specific binding low:
• choose a biological source with a high density of
high-affinity binding sites
• select a radioligand concentration around the
expected KD ( a few hundred to a few thousand
dpm will be sufficient as result)
• use a clean radioligand; if necessary, any
radioligand can be purified easily by thin layer
chromatography
Saturation & non-specific binding
Strategies to keep non-specific binding low:
• choose a biological source with a high density of
high-affinity binding sites
• select a radioligand concentration around the
expected KD ( a few hundred to a few thousand
dpm will be sufficient as result)
• use a clean radioligand; if necessary, any
radioligand can be purified easily by thin layer
chromatography
• If you filter your samples and if you use a
radioligand with an amino group, pre-treat the
glass fiber filters with polyethylene imine
Saturation & non-specific binding
Strategies to keep non-specific binding low:
• choose a biological source with a high density of
high-affinity binding sites
• select a radioligand concentration around the
expected KD ( a few hundred to a few thousand
dpm will be sufficient as result)
• use a clean radioligand; if necessary, any
radioligand can be purified easily by thin layer
chromatography
• If you filter your samples and if you use a
radioligand with an amino group, pre-treat the
glass fiber filters with polyethylene imine
• optimise the rinsing procedure of pellets and
filters, respectively
Radioligands for
excitatory amino acid (EAA) receptors
Classification of glutamate receptors
ionotropic receptors
metabotropic receptors
Radioligands for
excitatory amino acid (EAA) receptors
Classification of glutamate receptors
ionotropic receptors
NMDA
receptors
non-NMDA
receptors
metabotropic receptors
Group I
Group II
Group III
Radioligands for
excitatory amino acid (EAA) receptors
Classification of glutamate receptors
ionotropic receptors
NMDA
receptors
non-NMDA
receptors
AMPA
receptors
metabotropic receptors
Group I
kainate
receptors
Group II
Group III
Radioligands for
excitatory amino acid (EAA) receptors
Classification of glutamate receptors
ionotropic receptors
NMDA
receptors
non-NMDA
receptors
Schmid et al (2009) PNAS 106:10320
AMPA
receptors
kainate
receptors
Radioligands for
excitatory amino acid (EAA) receptors
COOH
H2N
L-glutamic acid
(S)-1-aminopropane-1,3dicarboxylic acid
COOH
COOH
N
H
HOOC
N-methyl-Daspartic acid
(NMDA)
COOH
H2N
D-Aminophosphonovaleric
acid
PO3H2
COOH
H2N
PO3H2
CGP 39653
(E)-2-Amino-4-propyl-5phosphono-3-pentenoic acid
Ca2+
Mg2+
Na+
Mg2+
Mg2+
outside
Mg2+
membrane
inside
-70 mV
Ca2+
Mg2+
Na+
Mg2+
Mg2+
glu
gly
glu
outside
Mg2+
membrane
inside
-70 mV
AMPAreceptor
Ca2+
Mg2+
Na+
Mg2+
Mg2+
glu
Mg2+
gly
glu
outside
membrane
inside
-30 mV
Ca2+
Mg2+
Na+
Mg2+
Mg2+
Mg2+
glu
gly
glu
outside
membrane
inside
-10 mV
Ca2+
Mg2+
Na+
Mg2+
Mg2+
Mg2+
glu
gly
glu
outside
membrane
inside
0 mV
Ca2+
Mg2+
Na+
Mg2+
Mg2+
Mg2+
glu
gly
glu
outside
membrane
inside
0 mV
Radioligands for
excitatory amino acid (EAA) receptors
COOH
Glycine
H2N
NH
*
OH
O
Cl
N
H
O
L-701.324 ( a
phenyl
quinolinone)
HOO C
Cl
COOH
Cl
N
H
MDL-105.519 (an
indole carboxylic
acid)
MK-801
Radioligands for
excitatory amino acid (EAA) receptors
COOH
Glycine
H2N
NH
*
OH
O
Cl
N
H
O
L-701.324 ( a
phenyl
quinolinone)
MK-801
*
HOO C
COOH
N
H
N
O
Cl
Cl
N
MDL-105.519 (an
indole carboxylic
acid)
[3H]GSK-931.145
radioligand for the glycine
transporter GlyT-1
(Herdon et al 2010 Neuropharmacol 59:558)
Radioligands for
excitatory amino acid (EAA) receptors
O
OH
H
H
COOH
kainic acid ( a pyrrolidine)
N
H
COOH
H2N
**
O
N
OH
COOH
H2N
H
COOH
AMPA (a-Amino-3-hydroxy5-methylisoxazol-4-propionic
acid)
LY-354.740 (a
bicyclo[3.1.0]hexan)
Radioligands for
excitatory amino acid (EAA) receptors
HOOC
H
O
O
OH
H
OH
H
H
H
COOH
COOH
N
H
kainic acid ( a pyrrolidine)
N
H
COOH
H2N
**
O
N
OH
COOH
H2N
H
Grant et al (2010) Neurotox Terat 32:132
COOH
AMPA (a-Amino-3-hydroxy5-methylisoxazol-4-propionic
acid)
LY-354.740 (a
bicyclo[3.1.0]hexan)
Radioligands for
excitatory amino acid (EAA) receptors
O
OH
H
H
COOH
kainic acid ( a pyrrolidine)
N
H
COOH
H2N
COOH
H2N
H2N
O
N
OH
Muscimol
O
N
OH
**
O
N
OH
AMPA (a-Amino-3-hydroxy5-methylisoxazol-4-propionic
acid)
Ibotensäure
COOH
H2N
H
COOH
LY-354.740 (a
bicyclo[3.1.0]hexan)
Radioligands for
excitatory amino acid (EAA) receptors
O
OH
H
H
COOH
kainic acid ( a pyrrolidine)
N
H
COOH
H2N
**
O
COOH
H2N
S
O
H
COOH
LY-404.039
H2N
O
H
COOH
LY-379.268
OH
COOH
COOH
O
N
H2N
H
COOH
AMPA (a-Amino-3-hydroxy5-methylisoxazol-4-propionic
acid)
LY-354.740 (a
bicyclo[3.1.0]hexan)
The most important binding
techniques
... are all non-equilibrium
techniques for particulate
receptor preparations:
• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography
B + L*
BL*
The most important binding
techniques
B + L*
BL*
... are all non-equilibrium
techniques for particulate
receptor preparations:
• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography
... applied to weak ligands (KD > 20
nM) ● you need a high speed
refrigerated certrifuge ● plastic vials
must support 40 000 x g ● after
centrifugation, pellet and inner wall
needs rinsing ● scintillation cocktail
added directly to the rinsed
incubation vials.
The most important binding
techniques
B + L*
BL*
... are all non-equilibrium
techniques for particulate
receptor preparations:
• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography
... Can only be applied to high
affinity ligands (KD < 20 nM) ● you
need a vacuum filter box
The most important binding
techniques
B + L*
BL*
... are all non-equilibrium
techniques for particulate
receptor preparations:
• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography
... Can only be applied to high
affinity ligands (KD < 20 nM) ● you
need a vacuum filter box or better a
harvester ● for radioligands with
amino group, the glass fiber filter
must
be
soaked
in
0.3%
polyethylenimine ●
B + L*
The most important binding
techniques
... are all non-equilibrium
techniques for particulate
receptor preparations:
BL*
scintillator
L*
L*
L*
L*
• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography
... Can only be applied to high
affinity ligands (KD < 20 nM) ● you
need a vacuum filter box or better a
harvester ● for radioligands with
amino group, the glass fiber filter
must
be
soaked
in
0.3%
polyethylenimine ● for best results,
filter should be shaken in scintillation
cocktail for 30 min.
B + L*
The most important binding
techniques
BL*
L*
L*
L*
L*
... are all non-equilibrium
techniques for particulate
receptor preparations:
L*
L*
L*
L*
L*
L*
L*
L*
coating
glass
• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography
... applied to frozen slices prepared
in a cryostat / microtom (10-20 µm)
● tissue must be shock-frozen (-40
°C) in dry ice / isopentane ● slices
taken up to coated glass slides ● for
incubation, you can use..
The most important binding
techniques
B + L*
BL*
... are all non-equilibrium
techniques for particulate
receptor preparations:
• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography
... applied to frozen slices prepared
in a cryostat / microtom (10-20 µm)
● tissue must be shock-frozen (-40
°C) in dry ice / isopentane ● slices
taken up to coated glass slides ● for
incubation, you can use a jar or...
The most important binding
techniques
B + L*
BL*
... are all non-equilibrium
techniques for particulate
receptor preparations:
• Centrifugation
• Filtration over glass fiber filters
• Slice autoradiography
... applied to frozen slices prepared
in a cryostat / microtom (10-20 µm)
● tissue must be shock-frozen (-40
°C) in dry ice / isopentane ● slices
taken up to coated glass slides ● for
incubation, you can use a jar or
simply a droplet on the slide ●
expose dried slices to film or
phosphoscreen ● evaluation by coexposure of stripes containing
known amounts of radioactivity.