Biophotonics Lecture #6, 2013 Signaling and Membrane Processes

Transkript

1 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 Jena

2 Signaling: exchange of information between & in cells Agenda: Extracellular signaling molecules (messengers): Transmitters, Hormones Receptors Signaling cascades Second messengers Electrical signaling Script available at: Lehrveranstaltungen user: Student passw: Biophysik13

3 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?

4 Cellular signaling Extracellular signal Important key words: Receptor (specificity of signal) Signal transduction (includes amplification and processing) Cell state (related to function) Crossing of plasma membrane Transport through cytosol Entering of nucleus Alteration of gene transcription change of cellular state Cellular response

5 Types of signaling Electrical signal Chemical signal Change in electrical transmembrane voltage Release of transmitters: specificity Soma Axon Synapse Very fast ( 100 m/s), directed, basis for complex processing in the brain

6 Synaptic transmission Excitatory synapse Na + K + Ca 2+ V m (t) Na + Inhibitory synapse Na + K + Ca 2+ V m (t) Cl

7 Types of signaling Hormones autocrine paracrine systemic Blood vessel

8 Types of signaling Diffusible extracellular signaling molecules: Transmitter, Messenger, Hormones, Neuropeptides Extracellular signaling molecule primary messenger Channels Serpentine receptors Receptor kinases Cytosolic receptors Nuclear receptors P P Intracellularly generated signaling molecule second messenger P P P Nucleus Signaling cascades (chains of phosphorylation reactions) Physiological response Alteration of gene transcription

9 Phosphorylation: Specific encoding of proteins ATP acetylation Protein oxidation nitration Kinase Phosphoprotein Phosphatase ADP P There are very many enzymes (kinases) that specifically attach phosphate groups to proteins. Specificity is obtained by so-called consensus sites. Phosphatases dephosphorylate phosphoproteins.

10 Tyrosine kinase Kinases CDK, MAPK, GSK3, CLK Casein kinase 1 Protein kinase families A, C, G: PKA, PKC, PKG Calcium/Calmodulin dependent kinases

11 Ras-GDP / Ras-GTP: Biomodal switches < ATP P ADP P ATP P P ADP P P Grb-2 Ras GDP Ras GEF Ras GTP

12 Phosphorylation cascades

13 Complexity of cellular signaling

14 How to visualize cell states? Antibodies can bind very specifically to antigens, i.e. epitopes on (mostly) proteins. Target protein Some antibodies can even distinguish between proteins in different states, e.g. not phosphorylated and phosphorylated. Polyclonal / monoclonal antibodies. Primary antibody (e.g rabbit) Secondary antibody Anti-rabbit (e.g goat) with specific label (e.g. fluorescent group)

15 Western blots using specific (mostly) radioactive antibodies How to visualize cell states?

16 Immunohistochemistry How to visualize cell states?

17 How to visualize cell states? GFP-tagged proteins Heterologous expression Target protein Chiu V K et al. J. Biol. Chem. 2004;279:

18 Second messengers Ligand (primary messenger) Receptor (Intracellular) Second messengers: lipid soluble, water soluble, gaseous IP 3 DAG Ca 2+ camp cgmp NO CO

19 Ca 2+ channel GPCR Agonist A 2nd messengers GPCR Agonist B Ca 2+ PIP 2 DAG Ca 2+ export PLC PKC AC IP 3 P camp AMP SERCA IP 3 R PKA P PDE P Ca 2+ store Ca 2+ DAG / IP 3 camp

20 Heterotrimeric G-Proteins R / GDP R / GTP R / GTP R GTP / GTP GDP Targets R s G s + AC G i R i PLC + G q R q PKA PI3K PKC

21 Ca 2+ signaling The intracellular Ca 2+ concentration is the most important measure of the cellular state. Ca 2+ ions trigger a large number of molecular processes. [Ca 2+ ] i 100 nm [Ca 2+ ] o 2 mm Ca 2+ is stored in organelles: ER/SR, mitochondria

22 Muscle contraction and relaxation Contraction Relaxation

23 Ca 2+ signaling Fura-2: Ca 2+ sensitive ratiometric dye Typically: 340 / 380 nm Fluorescence intensity Wavelength of excitation (nm)

24 Ca 2+ signaling

25 Insulin secretion in beta cells Pancreas Glucose Sulphonylureas K + Diazoxide K(ATP) channel =f([atp]) + 2 Metabolism 1 ATP MgADP Ca 2+ + Secretory granules 5 Insulin release =f(ca 2+ ) 4 Depolarization 3 + Ca 2+ channel =f(v m )

26 Membrane processes and transport Channels mediate the passive flux of ions according to the EC gradient. Pumps build-up EC gradients. U m Two properties of ions have to be considered: Chemical Element Electronic Charge The electro-chemical gradient is relevant.

27 Ion gradients and Nernst potentials E ion = ' '' = RT zf ln c'' c' Nernst-Equation Room temp: RT/F = 25.5 mv bzw. RT/F ln(10) = 58.7 mv (for log-10). The concentration gradient is compensated by electric voltage. E is termed: Nernst potential or Ion potential.

28 Channel vs. transporter Channel Transporter continuous flux no conformational change required high flux rates ( /s) quantal transport, coupled to conformational change lower transport rates ( /s)

29 Ion channel classes Two major channel classifications Gating ligands voltage mechanical stimulus not gated (leak channels) Selectivity potassium (K + ) sodium (Na + ) calcium (Ca 2+ ) cations anions (chloride, Cl )

30 Membrane voltage Voltmeter E1 E2 Cell, i Bath, a = ground Action potentials of mouse DRG neurons Time (s)

31 Control of membrane voltage and current Current clamp Voltage clamp V m I clamp V m =V clamp Time (s) I Membrane voltage is measured for a given current Measure current necessary to keep voltage at a given level

32 Variations of the patch-clamp method > 1 G R f = 50 G i(t,v) 1 pa 0.2 pa rms cell attached V command 5 pa 50 pa fast perfusion inside-out excised patch 1-3 pf I(t,V), C(t,V) 500 pa 0.5 pa rms flash photolysis fluorometry FCS h whole cell pf 10 ms outside-out patch

33 Experimental setup From Triggle et al., 2006

34 Experimental setup

35 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 ( ms): FRET between DiBACc4(3) and Coumarin 405 nm 570 nm 405 nm 460 nm donor acceptor

36 Channelrhodopsin (Optogenetics)

37 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, ; Nagel, G. et al. (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. U S A 100, Na + channel Cl pump

38 Optogenetics

39 Optogenetics tools

40 A External stimulus Node of Ranvier Schwann cell Soma Synapse (a) (c) (e) (b) (d) Trigger zone Transmitter B (a) (b) (c) (d) (e) Threshold Cellular signals Stimulus Receptor potential analog Action potential digital [Ca 2+ ] i analog Transmitter quantized Sensoric neuron = complex AD/DA converter with capability of differentiation and integration

41 A External stimulus Node of Ranvier Schwann cell Soma Synapse (a) (c) (e) (b) (d) Trigger zone Transmitter B (a) (b) (c) (d) (e) Threshold Cellular signals

42 Transport systems Ion channels ( ions/s) ATP-powered pumps ( ions/s) Transporters ( ions/s) a b c d ATP ADP passive primary active secondary active

43 Gating stimuli closed open closed open Ligand gated Voltage gated out in Ligand binding Depolarization Phosphorylation dependent Mechano sensitive P i Phosphorylation P Membrane stretch Ion transport systems are prime targets of drugs!

44 Example: Pacemaker Neuron Membrane potential (mv) Time (s) [Ca 2+ ] i

45 Heterologous expression Mammalian cell (e.g. HEK 293) Plasmid CMV-Promoter Transfection e.g. Lipofection whole-cell patch-clamp Xenopus laevis - Oocytes V(t) I(V,t) Plasmid T7/SP6-Promoter mrna In vitro synthesis mrna microinjection TEV Two-electrode voltage clamp

46 Gating mechanisms Diode Ion channel: Kir (inward rectifier) Current (na) Current ( A) Potential (mv) Potential (mv)

47 Intrinsic gating charge: Kv channels Transistor Ion channel: Kv (voltage-gated) G D S U + + m E I-gate (na) I-drain (ma) I-ion (pa) Q-gate (e 0 ) U GS (mv) U m (mv)

48 4 independent voltage sensors in K V channels out in Pore Voltage sensor Per subunit about 3 e 0 have to be effectively moved across the electric field.

49 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.

50 Voltage sensors: voltage-clamp fluorometry Dye attached via thiol reaction. This mutation eliminates K + current. Fluorescence quenching of a dye attached to a channel protein reports on voltage-driven protein conformational changes.

51 Further reading Heinemann, S.H., R. Schönherr, T. Hoshi 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 Ion Channels: Molecules in Action. The Rockefeller University Press Aidley, J., Stanfield, P.R. Ion Channels of Excitable Membranes, 3rd Ed. Sinauer, Sunderland Hille, B. Ion Channels and Disease. Academic Press, San Diego, 2000, Ashcroft, F.M.