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
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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.
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