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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.

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slide1

Michael Berger (Center for Brain Research, Medical University Vienna, Austria):

Ligand/Receptor Interaction

L*

http://cwx.prenhall.com/horton/medialib/media_portfolio/09.html

slide3

Wenn Du mit anderen ein Schiff bauen willst,

beginne nicht, mit Ihnen Holz zu sammeln,

Antoine de Saint Exupery

slide4

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

slide6

What is a receptor?

A physical target mediating the physiological effect of a drug.

slide7

What is a receptor?

A physical target mediating the physiological effect of a drug.

What is a ligand?

slide8

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.

slide9

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?

slide10

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.

slide11

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?

slide12

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

slide13

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?

slide14

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.

slide15

Association:

[BL]

B + L BL

KA =

[B] . [L]

slide16

Association:

[BL]

B + L BL

KA =

[B] . [L]

KA: association equilibrium constant

slide17

Association:

[BL]

B + L BL

KA =

[B] . [L]

KA: association equilibrium constant

Dissociation:

[B] . [L]

BL B + L

KD =

[BL]

KD: dissociation equilibrium constant

slide18

Association:

[BL]

B + L BL

KA =

[B] . [L]

KA: association equilibrium constant

dimension: (concentration)-1

Dissociation:

[B] . [L]

BL B + L

KD =

[BL]

KD: dissociation equilibrium constant

slide19

Association:

[BL]

B + L BL

KA =

[B] . [L]

KA: association equilibrium constant

dimension: (concentration)-1

Dissociation:

[B] . [L]

BL B + L

KD =

[BL]

KD: dissociation equilibrium constant

dimension: concentration

slide20

Association:

Dissociation:

B + L BL

BL B + L

slide21

Association:

Dissociation:

B + L BL

BL B + L

Strong binding: equilibrium is

on right side on left side

slide22

Association:

Dissociation:

B + L BL

BL B + L

Strong binding: equilibrium is

on right side on left side

[BL]

[B] . [L]

KA =

<< 1

KD =

>> 1

[BL]

[B] . [L]

slide23

Association:

Dissociation:

B + L BL

BL B + L

Strong binding: equilibrium is

on right side on left side

[BL]

[B] . [L]

KA =

<< 1

KD =

>> 1

[BL]

[B] . [L]

ln KD negativ

ln KA positiv

slide24

Association:

Dissociation:

B + L BL

BL B + L

Strong binding: equilibrium is

on right side on left side

[BL]

[B] . [L]

KA =

<< 1

KD =

>> 1

[BL]

[B] . [L]

ln KD negativ

ln KA positiv

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

slide25

The Van‘t Hoff equation allows the calculation of the free enthalpy change of a reaction from the reaction‘s equilibrium constant:

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

slide26

Examples for the change in free enthalpy Go in various reactions

ΔGo (kcal/Mol)

Glucose + 6 O2 6 CO2 + 6 H2O -686

H2 + ½ O2 H2O -46

ATP ADP + Pi -7.3

slide27

Examples for the change in free enthalpy Go in various reactions

ΔGo (kcal/Mol)

Glucose + 6 O2 6 CO2 + 6 H2O -686

H2 + ½ O2 H2O -46

ATP ADP + Pi -7.3

In these reactions, Go is reduced (exergonic processes)

slide28

Examples for the change in free enthalpy Go in various reactions

ΔGo (kcal/Mol)

Glucose + 6 O2 6 CO2 + 6 H2O -686

H2 + ½ O2 H2O -46

ATP ADP + Pi -7.3

In these reactions, Go is reduced (exergonic processes)

Bond dissociation energies

HO-H HO· + ·H 118

CH3CH2-H CH3CH2· + ·H 101

CH3-CH3 CH3· + ·CH3 90

slide29

The free enthalpy change ΔGo of a reaction is composed of 2 terms:

Gibbs & Helmholtz: ΔGo = ΔHo – T . ΔSo

slide30

The free enthalpy change ΔGo of a reaction is composed of 2 terms:

Gibbs & Helmholtz: ΔGo = ΔHo – T . ΔSo

change in enthalpy

slide31

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

slide32

! 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 !

slide33

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

ΔHo < 0: exotherm

(reaction mixture warms)

lg KD

0 -

55 °C

0 °C

ΔSo > 0

(order is decreased)

-3 -

-6 -

-9 -

1/T

0.001 0.002 0.003 0.004

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[0.434 = 1/ln10]

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide34

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

ΔHo < 0: exotherm

(reaction mixture warms)

lg KD

0 -

55 °C

0 °C

ΔSo > 0

(order is decreased)

-3 -

-6 -

-9 -

1/T

0.001 0.002 0.003 0.004

Most common case: The warmer (the lower 1/T), the weaker the affinity (the less negative lg KD).

Intersection with ordinate gives information about ΔSo.

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[0.434 = 1/ln10]

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide35

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

ΔHo < 0: exotherm

(reaction mixture warms)

lg KD

0 -

55 °C

0 °C

ΔSo > 0

(order is decreased)

-3 -

-6 -

-9 -

1/T

0.001 0.002 0.003 0.004

Most common case: The warmer (the lower 1/T), the weaker the affinity (the less negative lg KD).

Intersection with ordinate gives information about ΔSo.

ln KD = ΔGo / RT = ΔHo / RT - ΔSo / R

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[0.434 = 1/ln10]

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide36

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

ΔHo < 0: exotherm

(reaction mixture warms)

lg KD

0 -

55 °C

0 °C

ΔSo > 0

(order is decreased)

ΔSo < 0

(order is increased)

-3 -

-6 -

-9 -

1/T

0.001 0.002 0.003 0.004

lg KD

0 -

If order is increased, driving force is even more sensitive to high temperatures

-3 -

-6 -

-9 -

1/T

0.001 0.002 0.003 0.004

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 [0.434 = 1/ln10]

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide37

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

ΔHo < 0: exotherm

(reaction mixture warms)

lg KD

0 -

55 °C

0 °C

ΔSo > 0

(order is decreased)

ΔSo < 0

(order is increased)

-3 -

-6 -

-9 -

1/T

0.001 0.002 0.003 0.004

lg KD

0 -

If order is increased, driving force is even more sensitive to high temperatures

-3 -

-6 -

-9 -

1/T

0.001 0.002 0.003 0.004

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 [0.434 = 1/ln10]

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide38

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

ΔHo < 0: exotherm

(reaction mixture warms)

ΔHo > 0: endotherm

(reaction mixture cools)

lg KD

lg KD

0 -

0 -

55 °C

0 °C

ΔSo > 0

(order is decreased)

ΔSo < 0

(order is increased)

-3 -

-3 -

-6 -

-6 -

-9 -

-9 -

1/T

1/T

0.001 0.002 0.003 0.004

0.001 0.002 0.003 0.004

lg KD

0 -

Endotherm binding is driven by decrease in order only; here, driving force increases with temperature.

-3 -

-6 -

-9 -

1/T

0.001 0.002 0.003 0.004

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 [0.434 = 1/ln10]

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide39

Mechanisms contributing to ligand/receptor interaction:

  • Ionic interaction
  • Hydrogen bonds
  • Hydrophobic interaction
  • Cation/p interaction
  • Van der Waals interaction
slide40

ionic interaction

e1. e2

attraction between 2 charges depends on

D . r2

r ... distance

D ... dielectric constant

slide41

ionic interaction

e1. e2

attraction between 2 charges depends on

D . r2

r ... distance

D ... dielectric constant

vacuum ... 1.0

hexane ... 1.9

H2O ... 78

slide42

ionic interaction

e1. e2

attraction between 2 charges depends on

D . r2

r ... distance

D ... dielectric constant

vacuum ... 1.0

hexane ... 1.9

H2O ... 78

In water, ionic interaction is hindered by shells of water molecules surrounding each ion.

slide43

hydrogen bonds

B + L BL

Formation of a hydrogen bond is highly exergonic, yields 3-7 kcal/mol

slide44

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

slide45

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

1. Break this bond.

slide46

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.

slide47

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.

slide48

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

4. Form this bond.

2. Break this bond.

1. Break this bond.

3. Form this bond.

slide49

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

4. Form this bond.

2. Break this bond.

1. Break 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).

slide50

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.

slide51

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.

slide52

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.

slide53

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.
slide54

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

slide55

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).
slide56

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)

slide57

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

slide58

Van der Waals interaction

range 3-4 Ǻ, turns into repulsion at shorter distances

contribution to ΔGo 0.5-1.0 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

slide59

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

slide61

Example for the interaction of a hypothetical ligand with its receptor:

kcal/mol

formation of a hydrogen bond ... - 5.0

loss of hydrogen bond with H2O... + 5.0

slide62

Example for the interaction of a hypothetical ligand with its receptor:

kcal/mol

formation of a hydrogen bond ... - 5.0

loss of hydrogen bond with H2O... + 5.0

preliminary balance: ± 0

slide63

Example for the interaction of a hypothetical ligand with its receptor:

kcal/mol

formation of a hydrogen bond ... - 5.0

loss of hydrogen bond with H2O ... + 5.0

2 H2O set free … - 4.0

Hydrophobic interaction … - 2.0

8 van der Waals contacts … - 4.7

slide64

Example for the interaction of a hypothetical ligand with its receptor:

kcal/mol

formation of a hydrogen bond ... - 5.0

loss of hydrogen bond with H2O ... + 5.0

2 H2O set free … - 4.0

Hydrophobic interaction … - 2.0

8 van der Waals contacts … - 4.7

balance: -10.7

slide65

Example for the interaction of a hypothetical ligand with its receptor:

kcal/mol

formation of a hydrogen bond ... - 5.0

loss of hydrogen bond with H2O ... + 5.0

2 H2O set free … - 4.0

Hydrophobic interaction … - 2.0

8 van der Waals contacts … - 4.7

balance: -10.7

slide66

How many receptors do we expect in a responsive tissue?

Which analytical tools will be necessary to detect them?

slide67

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
slide68

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
slide69

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

slide70

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

slide71

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

slide72

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

slide73

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

slide74

Which analytical tools will be necessary to detect them?

Comparison of 3H with other nuclides (1 radioactive atom / molecule)

The shorter the half-life, the hotter the radioligand.

slide75

Which analytical tools will be necessary to detect them?

How many dpm can be expected from 1 fMol 3H?

slide76

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)

slide77

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
slide78

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
slide79

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.

slide80

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.

slide81

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

0.5 would be correct, if the decay rate would be the same for the whole decay period.

slide82

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

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.

slide84

! attention ℮ ∫ ∑mathematics∂ ∞ √ % 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

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide85

! attention ℮ ∫ ∑mathematics∂ ∞ √ % 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

A = Ao . e-k.Δt

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide86

! attention ℮ ∫ ∑mathematics∂ ∞ √ % 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

A = Ao . e-k.Δt

k is related to t½: ln(½) = -k .t½

k = ln2/ t½

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide87

! attention ℮ ∫ ∑mathematics∂ ∞ √ % 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

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

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide88

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 = 64electrons / min.

A molecule labeled with one single 3H has a specific radioactivity (short: specific activity) of 64 dpm / fMol.

slide89

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 = 64electrons / min.

A molecule labeled with one single 3H has a specific radioactivity (short: specific activity) of 64 dpm / fMol.

1 µCi = 2 220 000 dpm

1 nCi = 2 220 dpm

1 pCi = 2.22 dpm

Remember:

64 dpm / fMol = 28.8 pCi / fMol = 28.8 Ci / mMol

slide90

Which analytical tools will be necessary to detect them?

64 dpm / fMol = 28.8 pCi / fMol = 28.8 Ci / mMol

slide91

Which analytical tools will be necessary to detect them?

64 dpm / fMol = 28.8 pCi / fMol = 28.8 Ci / mMol

slide92

Which analytical tools will be necessary to detect them?

Comparison of 3H with other nuclides (1 radioactive atom / molecule)

dpm / mg tissue

slide93

Which analytical tools will be necessary to detect them?

Comparison of 3H with other nuclides (1 radioactive atom / molecule)

dpm / mg tissue

most common experimental condition

slide94

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:
slide95

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
slide96

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
slide97

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
slide98

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
slide99

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
slide100

Analytical techniques

B + L* BL*

equilibrium dialysis

non-equilibrium techniques for receptors in solution

  • gel filtration
  • charcoal adsorption
  • precipitation
  • adsorption to glass fiber filters

polyethylene glycol

n = 6 000 – 8 000

non.-equilibrium techniques for particulate receptors

  • centrifugation
  • filtration
  • slice autoradiography
slide101

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
slide102

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
slide103

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
slide104

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
slide105

Saturation & non-specific binding

Saturability: a radioligand can only be displaced if the target density is low.

Other examples for saturability: Langmuir isotherme (mono-molecular layer on a surface), enzyme reaction rate (Michaelis-Menten).

http://www.steve.gb.com/science/membranes.html

slide107

Saturation & non-specific binding

At low nM concentrations,

most of the radioligand L

is bound to saturable

high affinity sites.

slide108

Saturation & non-specific binding

At high concentrations,

the linearly rising

non-specific binding

will dominate, and

specific binding

can no longer

be detected.

At low nM concentrations,

most of the radioligand L

is bound to saturable

high affinity sites.

slide109

Saturation & non-specific binding

[B] . [L]

BL B + L

KD =

[BL]

KD: dissociation equilibrium constant

With increasing [L] more binding sites are occupied (BL) and free sites (B) are lost. The sum

BL + B = BM

remains constant.

slide110

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

slide111

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . [L]

KD =

[BL]

solve for [BL]:

slide112

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . [L]

KD =

[BL]

solve for [BL]:

KD. [BL] = BM. [L] – [BL] . [L]

slide113

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . [L]

KD =

[BL]

solve for [BL]:

KD. [BL] = BM. [L] – [BL] . [L]

[BL] . ([L] + KD) = BM . [L]

slide114

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . [L]

KD =

[BL]

solve for [BL]:

KD. [BL] = BM. [L] – [BL] . [L]

[BL] . ([L] + KD) = BM . [L]

[L]

BM.

Langmuir isotherm

[BL] =

[L] + KD

slide115

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . [L]

KD =

[BL]

solve for [BL]:

KD. [BL] = BM. [L] – [BL] . [L]

[BL] . ([L] + KD) = BM . [L]

[L]

BM.

Langmuir isotherm

[BL] =

Irving Langmuir

1881-1957

Nobel price 1932

[L] + KD

slide116

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . [L]

wrong!

KD =

[BL]

solve for [BL]:

KD. [BL] = BM. [L] – [BL] . [L]

[BL] . ([L] + KD) = BM . [L]

[L]

BM.

Langmuir isotherm

[BL] =

Irving Langmuir

1881-1957

Nobel price 1932

[L] + KD

slide117

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . (Lo – [BL])

correct

KD =

[BL]

slide118

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . (Lo – [BL])

KD =

[BL]

solve for [BL]:

KD. [BL] = BM. Lo – BM. [BL] – [BL] . Lo + [BL]2

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide119

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

Saturation & non-specific binding

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . (Lo – [BL])

KD =

[BL]

solve for [BL]:

KD. [BL] = BM. Lo – BM. [BL] – [BL] . Lo + [BL]2

[BL]2 – [BL] . (BM + Lo + KD) + BM. Lo = 0

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide120

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

Saturation & non-specific binding

Sweet memories…

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . (Lo – [BL])

KD =

[BL]

solve for [BL]:

KD. [BL] = BM. Lo – BM. [BL] – [BL] . Lo + [BL]2

[BL]2 – [BL] . (BM + Lo + KD) + BM. Lo = 0

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide121

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

Saturation & non-specific binding

Sweet memories…

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . (Lo – [BL])

KD =

[BL]

solve for [BL]:

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}

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide122

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

Saturation & non-specific binding

Sweet memories…

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . (Lo – [BL])

KD =

[BL]

solve for [BL]:

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 BM.

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide123

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

Saturation & non-specific binding

Sweet memories…

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . (Lo – [BL])

KD =

[BL]

solve for [BL]:

3 times more ligand than receptors at KD concentration (8% loss)

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 BM.

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide124

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

Saturation & non-specific binding

Sweet memories…

[B] . [L]

KD =

[BL]

replace [B] by BM – [BL]:

(BM - [BL]) . (Lo – [BL])

KD =

[BL]

solve for [BL]:

3 times more receptor than ligand at KD concentration (57% loss)

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 BM.

! attention ℮ ∫ ∑mathematics∂ ∞ √ % attention !

slide125

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 (*).

slide126

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 (*).

2: specific binding

... Is sitting on the non-specific binding, obtained as difference between total and non-specific binding.

slide127

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 (*).

2: specific binding

... Is sitting on the non-specific binding, obtained as difference between total and non-specific binding (†).

slide128

Saturation & non-specific binding

Mathematical combination of both processes:

slide129

Saturation & non-specific binding

Mathematical combination of both processes:

1: non-specific binding

[L]

BU.

[BL] =

[L] + KU

slide130

Saturation & non-specific binding

Mathematical combination of both processes:

1: non-specific binding

[L]

BU.

[BL] =

[L] + KU

2: specific binding

[L]

BS.

[BL] =

[L] + KS

slide131

Saturation & non-specific binding

Mathematical combination of both processes:

1: non-specific binding

[L]

BU.

[BL] =

[L] + KU

2: specific binding

[L]

BS.

[BL] =

[L] + KS

KU (~mM) >> Ks (nM)

slide132

Saturation & non-specific binding

Mathematical combination of both processes:

1: non-specific binding

[L]

BU.

[BL] =

[L] + KU

2: specific binding

[L]

BS.

[BL] =

[L] + KS

KU (~mM) >> Ks (nM)

At reasonable ligand concentrations, [L] + KU ~ KU and non-specific binding is a linear function of [L]:

[L]

BU

. [L]

BS.

+

[BL] =

[L] + KS

KU

slide133

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.

slide134

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.

The non-specific binding NB is measured as bound ligand that is impossible to displace, even by high concentrations of potent displacers.

slide135

Saturation & non-specific binding

Strategies to keep non-specific binding low:

slide136

Saturation & non-specific binding

Strategies to keep non-specific binding low:

  • choose a biological source with a high density of high-affinity binding sites
slide137

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)
slide138

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
slide139

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
slide140

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
slide141

Radioligands for

excitatory amino acid (EAA) receptors

Classification of glutamate receptors

ionotropic receptors metabotropic receptors

slide142

Radioligands for

excitatory amino acid (EAA) receptors

Classification of glutamate receptors

ionotropic receptors metabotropic receptors

NMDA

receptors

non-NMDA

receptors

Group I Group II Group III

slide143

Radioligands for

excitatory amino acid (EAA) receptors

Classification of glutamate receptors

ionotropic receptors metabotropic receptors

NMDA

receptors

non-NMDA

receptors

Group I Group II Group III

AMPA

receptors

kainate

receptors

slide144

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

slide145

Radioligands for

excitatory amino acid (EAA) receptors

L-glutamic acid

(S)-1-aminopropane-1,3-dicarboxylic acid

N-methyl-D-aspartic acid (NMDA)

D-Aminophosphonovaleric acid

CGP 39653

(E)-2-Amino-4-propyl-5-phosphono-3-pentenoic acid

slide152

Radioligands for

excitatory amino acid (EAA) receptors

Glycine

L-701.324 ( a phenyl quinolinone)

MK-801

MDL-105.519 (an indole carboxylic acid)

slide153

Radioligands for

excitatory amino acid (EAA) receptors

Glycine

L-701.324 ( a phenyl quinolinone)

MK-801

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)

slide154

Radioligands for

excitatory amino acid (EAA) receptors

kainic acid ( a pyrrolidine)

AMPA (a-Amino-3-hydroxy-5-methylisoxazol-4-propionic acid)

LY-354.740 (a bicyclo[3.1.0]hexan)

slide155

Radioligands for

excitatory amino acid (EAA) receptors

kainic acid ( a pyrrolidine)

AMPA (a-Amino-3-hydroxy-5-methylisoxazol-4-propionic acid)

LY-354.740 (a bicyclo[3.1.0]hexan)

Grant et al (2010) Neurotox Terat 32:132

slide156

Radioligands for

excitatory amino acid (EAA) receptors

kainic acid ( a pyrrolidine)

AMPA (a-Amino-3-hydroxy-5-methylisoxazol-4-propionic acid)

Muscimol Ibotensäure

LY-354.740 (a bicyclo[3.1.0]hexan)

slide157

Radioligands for

excitatory amino acid (EAA) receptors

kainic acid ( a pyrrolidine)

AMPA (a-Amino-3-hydroxy-5-methylisoxazol-4-propionic acid)

LY-354.740 (a bicyclo[3.1.0]hexan)

LY-404.039 LY-379.268

slide158

B + L* BL*

The most important binding techniques

... are all non-equilibrium techniques for particulate receptor preparations:

  • Centrifugation
  • Filtration over glass fiber filters
  • Slice autoradiography
slide159

B + L* BL*

The most important binding techniques

... 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.

slide160

B + L* BL*

The most important binding techniques

... 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

slide161

B + L* BL*

The most important binding techniques

... 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 ●

slide162

B + L* BL*

The most important binding techniques

... 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 ● for best results, filter should be shaken in scintillation cocktail for 30 min.

slide163

B + L* BL*

The most important binding techniques

... 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..

slide164

B + L* BL*

The most important binding techniques

... 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...

slide165

B + L* BL*

The most important binding techniques

... 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 co-exposure of stripes containing known amounts of radioactivity.