Biophotonics - nanoimaging.de€¦ · Biophotonics Lecture #6, 2013 Signaling and Membrane Processes Prof. Dr. Stefan H. Heinemann Zentrum für molekulare Biomedizin, CMB Lehrstuhl

Biophotonics Lecture #6, 2013

Signaling and Membrane Processes

Prof. Dr. Stefan H. Heinemann

Zentrum für molekulare Biomedizin, CMB

Lehrstuhl für Biophysik FSU Jena

Hans-Knöll-Straße 2 D-07745 Jena

Signaling: exchange of information between & in cells

Agenda:

Extracellular signaling molecules (messengers):

Transmitters, Hormones

Receptors

Signaling cascades

Second messengers

Electrical signaling

Script available at:

http://www.biophysik.uni-jena.de/ Lehrveranstaltungen

user: Student

passw: Biophysik13

Cellular signaling

How do cells communicate?

Exchange of “signals”

How are signals encoded? How are signals decoded?

How is the message stored? How is the message processed?

What is the final outcome?

?

Cellular signaling

Extracellular signal

Cellular response • Crossing of plasma membrane

• Transport through cytosol • Entering of nucleus

• Alteration of gene transcription change of cellular “state”

Important key words:

• Receptor (specificity of signal) • Signal transduction (includes

amplification and processing) • Cell “state” (related to function)

Types of signaling

Synapse

Electrical signal

Change in electrical

transmembrane voltage

Chemical signal

Release of transmitters:

specificity

Soma Axon

Very fast ( 100 m/s), directed,

basis for complex processing in the brain

Synaptic transmission

Ca2+

Na+K+

Na+

Vm (t)

Excitatory synapse

Ca2+

Na+K+

Cl–

Vm (t)

Inhibitory synapse

Types of signaling

Hormones

autocrine paracrine

Blood vessel

systemic

Types of signaling

Diffusible extracellular signaling molecules: Transmitter, Messenger, Hormones, Neuropeptides

Extracellular signaling molecule – primary messenger

Intracellularly generatedsignaling molecule –second messenger

Nucleus

Channels Serpentine

receptors

P

P

P

P

P

Receptor

kinases

Signaling cascades

(chains of phosphorylation reactions)

Cytosolic

receptors

Nuclear

receptors

Physiological response

Alteration of gene transcription

Phosphorylation: Specific encoding of proteins

acetylation

oxidation

nitration

Protein

Kinase

Phospho-

protein

P

ADP ATP

Phos-

phatase

• There are very many enzymes (kinases) that

specifically attach phosphate groups to proteins.

• Specificity is obtained by so-called consensus sites.

• Phosphatases dephosphorylate phosphoproteins.

Kinases

Protein kinase

families A, C, G:

PKA, PKC, PKG

Calcium/Calmodulin dependent kinases

Casein kinase 1

Tyrosine kinase

CDK,

MAPK,

GSK3, CLK

Ras-GDP / Ras-GTP: Biomodal switches

<

1 2 3

P

ATP ADP

4

P

5

P

P

P

P

ATP ADP Grb-2

Ras GEF

GDP

Ras GTP

Ras

6

Phosphorylation cascades

Complexity of cellular signaling

How to visualize “cell states“?

Antibodies can bind very specifically to

antigens, i.e. epitopes on (mostly) proteins.

Some antibodies can even distinguish

between proteins in different states, e.g.

not phosphorylated and phosphorylated.

Polyclonal / monoclonal antibodies.

Target protein

Primary antibody

(e.g rabbit)

Secondary antibody

Anti-rabbit (e.g goat) with specific label

(e.g. fluorescent group)

How to visualize “cell states“?

Western blots using specific

(mostly) radioactive antibodies

How to visualize “cell states“?

Immunohistochemistry

How to visualize “cell states“?

GFP-tagged proteins Heterologous expression

Target protein

Chiu V K et al. J. Biol. Chem. 2004;279:7346-7352

Second messengers

Ligand (primary messenger)

Receptor

(Intracellular)

Second messengers: lipid soluble,

water soluble, gaseous

Ca2+

cAMP

cGMP

NO

CO

IP3

DAG

2nd messengers

Ca2+ channel

Ca2+

SERCA

Ca2+ store

Ca2+ DAG / IP3 cAMP

Ca2+

export

GPCR

Agonist A

PLC

DAG

IP3

PIP2

IP3R

PKC

P

P P PKA

GPCR

Agonist B

AC

PDE

AMP

cAMP

Heterotrimeric G-Proteins

R

GDP

/

1

R

/

GTP

GTP GDP

2

R

/

GTP

3

R

/

GTP

Targets

4

PI3K

Rs AC

+

Gs Ri

–

Gi

PKA

PLC

PKC

Rq

+

Gq

Ca2+ signaling

The intracellular Ca2+ concentration is the most important measure of the

cellular state. Ca2+ ions trigger a large number of molecular processes.

[Ca2+]o

2 mM

Ca2+ is stored in organelles: ER/SR, mitochondria

[Ca2+]i

100 nM

Muscle contraction and relaxation

•

Contraction Relaxation

Ca2+ signaling

Fura-2:

Ca2+ sensitive ratiometric dye

Typically: 340 / 380 nm

Wavelength of excitation (nm)

Flu

ore

scence inte

nsity

Ca2+ signaling

Insulin secretion in beta cells

Pancreas Glucose

4

+

Ca2+

Ca2+ channel

=f(Vm)

2

–

K+

K(ATP)

channel =f([ATP])

Secretory

granules

+ –

Sulphonylureas

Diazoxide

Metabolism

ATP

MgADP

1

3 Depolarization

5

+

Insulin

release =f(Ca2+)

Membrane processes and transport

Pumps build-up EC gradients.

Channels mediate the passive flux of

ions according to the

EC gradient.

Two properties of ions have to be considered:

• Chemical Element • Electronic Charge

The electro-chemical gradient is relevant.

Um

Ion gradients and Nernst potentials

Nernst-Equation

Room temp: RT/F = 25.5 mV bzw. RT/F ln(10) = 58.7 mV (for log-10).

Eion = ' ' ' =RT

z Fln

c' '

c'

The concentration gradient

is compensated by electric voltage.

E is termed:

Nernst potential or

Ion potential.

Channel vs. transporter

continuous flux

no conformational change required

high flux rates (107-108 /s)

Channel

quantal transport, coupled to

conformational change

lower transport rates (102-104 /s)

Transporter

Ion channel classes

Gating ligands

voltage

mechanical stimulus

not gated (leak channels)

Two major channel classifications

Selectivity potassium (K+)

sodium (Na+)

calcium (Ca2+)

cations

anions (chloride, Cl–)

Membrane voltage

Cell, i Bath, a = ground

Voltmeter

E1 E2

Action potentials of mouse DRG neurons

Time (s)

Control of membrane voltage and current

Current clamp

Vm Iclamp

Membrane voltage is measured

for a given current

Voltage clamp

Vm=Vclamp I

Measure current necessary to keep

voltage at a given level

Time (s)

i(t,V)

cell attached

> 1 G

V command

inside-out

excised patch 1-3 pF fast perfusion I(t,V), C(t,V)

h

whole cell 10-100 pF

flash photolysis

fluorometry

FCS

5 pA

1 pA

0.2 pA rms

50 pA

500 pA

10 ms

0.5 pA rms

outside-out patch

Rf = 50 G

Variations of the patch-clamp method

Experimental setup

From Triggle et al., 2006

Experimental setup

Voltage sensitive dyes

Lipophilic substances with delocalized charge. Change in fluorescence properties with alteration of

membrane voltage.

Fast (action potential, small changes in fluorescence):

Di-ANEPPS (Amino Naphthyl Ethenyl Pyridinium)

Slow (> minutes): DiBAC (Dibutyl-barbituric Acid - Trimethine Oxonol)

Medium (20-200 ms): FRET between DiBACc4(3) and Coumarin

donor

acceptor

405 nm 405 nm 570 nm 460 nm

Channelrhodopsin (Optogenetics)

Channelrhodopsin (Optogenetics)

Channelrhodopsin is a 7-helix receptor from green algae; it is activated by light and acts as a proton

channel (ChR1) or a non-selective cation channel (ChR2), respectively. Nagel, G. et al. (2002) Channelrhodopsin-1: a light-gated proton channel in green algae. Science 296, 2395-239; Nagel, G. et al. (2003)

Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. U S A 100, 13940-13945.

Cl– pump Na+ channel

Optogenetics

Optogenetics tools

A

Trigger zone

Node of Ranvier Soma Synapse

Transmitter

B(a) (b) (c) (d) (e)

External stimulus Schwann cell

(a)

(b)

(c)

(d)

(e)

Threshold

Cellular signals

Stimulus Receptor potential

“analog“

Action potential

“digital“

[Ca2+]i

“analog“

Transmitter

“quantized“

Sensoric neuron =

complex AD/DA converter with capability of differentiation and integration

A

Trigger zone

Node of Ranvier Soma Synapse

Transmitter

B(a) (b) (c) (d) (e)

External stimulus Schwann cell

(a)

(b)

(c)

(d)

(e)

Threshold

Cellular signals

Transport systems

a b d

ATP ADP

c

passive primary active secondary active

ATP-powered pumps (100 – 103 ions/s)

Ion channels (107 – 108 ions/s)

Transporters (102 – 104 ions/s)

Gating stimuli

Ion transport systems are prime targets of drugs!

closed open

out

in

Ligand gated

Ligand binding

+ + + + + +

closed open

Voltage gated

+ +

– –

+ +

– –

+ +

– –

+ +

– –

Depolarization

Phosphorylation dependent

P Pi

Phosphorylation

Mechano sensitive

Membrane stretch

1

3

4 5

2

[Ca2+]i

Example: Pacemaker Neuron

Time (s)

Mem

bra

ne p

ote

ntial (m

V)

Heterologous expression

Mammalian cell (e.g. HEK 293)

Plasmid

CMV-Promoter

Transfection

e.g. Lipofection

whole-cell

patch-clamp

Xenopus laevis - Oocytes

Plasmid

T7/SP6-Promoter

mRNA

In vitro synthesis

mRNA

microinjection

V(t) I(V,t)

TEV

Two-electrode voltage clamp

-80

-60

-40

-20

0

-100 -50 0 50 100 Potential (mV)

Diode

Curr

ent

(nA

)

Gating mechanisms

Ion channel: Kir (inward rectifier)

-20

-15

-10

-5

0

-100 -50 0 50 100 Potential (mV)

Curr

ent

(A

)

+

+ + +

+

– + +

+

+

–

+ +

D S

G

Transistor

1.5

1.0

0.5

0.0

20 0 -100 -50 0 50 100

U GS (mV)

I-gate

(nA

) I-

dra

in (

mA

)

Intrinsic gating charge: Kv channels

Ion channel: Kv (voltage-gated)

1

0

10

0 -100 -50 0 50 100

U m (mV)

I-io

n (

pA

) Q

-gate

(e

0)

2

E U + + + m

Per subunit about 3 e0 have to be effectively moved across the electric field.

4 “independent“ voltage sensors in KV channels

Pore

Voltage

sensor

out

in

Intrinsic charge movement: gating currents

“Gating currents“ report on protein conformational changes associated with

charge translocation across the transmembrane electric field. Typically, they are much smaller than the current associated with ion flux through an open channel and,

hence, such ion currents have to be eliminated.

The latter can be achieved by a mutation in the pore, pharmacological pore block, or removal of permeant ions.

Voltage sensors: voltage-clamp fluorometry

“Fluorescence quenching“ of a dye attached to a channel protein reports on

voltage-driven protein conformational changes.

Dye attached via

thiol reaction. This mutation

eliminates K+ current.

Further reading

• Heinemann, S.H., R. Schönherr, T. Hoshi. 2011. Biology.

In: J. Popp, V.V. Tuchin, A. Chiou, S.H. Heinemann (edts), Handbook of Biophotonics, Vol. 1: Basics and Techniques,

WILEY-VCH Verlag & Co. KGaA, Weinheim, p. 489–648

• Ion Channels: Molecules in Action. The Rockefeller University Press.

1996. Aidley, J., Stanfield, P.R.

• Ion Channels of Excitable Membranes, 3rd Ed. Sinauer, Sunderland. 2001. Hille, B.

• Ion Channels and Disease. Academic Press, San Diego, 2000, Ashcroft, F.M.