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Ca 2+ Signaling. Mark T Ziolo, PhD Department of Physiology & Cell Biology 019 Hamilton Hall 8-7905 ziolo.1@osu.edu. 300. [Ca. ]. i. (nM). 100. 10. µm. ms. 200. Signal Transduction. Mechanism to convert a stimulus (mechanical/chemical) to a specific cellular response

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  1. Ca2+ Signaling Mark T Ziolo, PhD Department of Physiology & Cell Biology 019 Hamilton Hall 8-7905 ziolo.1@osu.edu 300 [Ca ] i (nM) 100 10 µm ms 200

  2. Signal Transduction • Mechanism to convert a stimulus (mechanical/chemical) to a specific cellular response • Signaling molecule→Receptor→2nd messenger→Cellular Response

  3. Signaling Molecule • Hormones (e.g., vasopressin) • Growth Factors (e.g., epidermal growth factor) • ECM components (e.g., fibronectin) • Cytokines (e.g., TNFa) • Neurotransmitters (e.g., acethylcholine) • ROS (e.g., O2-) Receptors • Cell Surface Receptors (e.g., b-adrenergic receptor) • Intracellular Receptors (e.g., steroid hormones)

  4. 2nd Messengers • Cyclic nucleotides (cAMP, cGMP) • Lipophilic (e.g., diacylglycerol) • Nitric Oxide • Inositol 1,4,5-triphosphate (IP3) • Arachidonic Acid • Ca2+ Cellular Response • Contraction • Secretion • Plasticity • Differentiation • Proliferation • Apoptosis

  5. Contraction Secretion Proliferation Differentiation Plasticity Apoptosis Ca2+ Signaling High [Ca2+]: 10-3 M Ca2+-regulated kinases Ca2+-regulated phosphatases Ca2+-regulated channels Ca2+-regulated transcription factors Ca2+ ER Ca2+ Store Mitochondrion High [Ca2+] High [Ca2+] Low [Ca2+]: 10-7 M

  6. Plasma membrane Ca2+ permeable channels Ca2+ Ca2+ signaling: sources Ca2+ Ca2+ voltage-gated Ca2+ channels ionotropic receptor/cation channels cyclic nucleotide-gated channels store-operated channels other non-selective cation channels Ca2+ Ca2+ Ca2+ Ca2+ extracellular space Ca2+ Ca2+ [Ca2+]o = 10-3 M Ca2+ Ca2+ Ca2+v + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - - - - - - - - cytosol [Ca2+]i = 10-7 M Ca2+ Intracellular Ca2+ channels IP3 receptors ryanodine receptors NAADP receptor PTP? (MT) Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ endoplasmic reticulum Ca2+ [Ca2+]ER = 10-4 ~10-3 M

  7. Plasma membrane extrusion Ca2+ Ca2+ signaling: removal Ca2+ Ca2+ Ca2+-ATPases (PMCA1-4) Na+/Ca2+ exchanger (NCX1-3) Ca2+ Ca2+ Ca2+ Ca2+ extracellular space Ca2+ Ca2+ [Ca2+]o = 10-3 M Ca2+ Ca2+ Ca2+v + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - - - - - - - - cytosol [Ca2+]i = 10-7 M Ca2+ Cytosolic Ca2+-binding proteins: Calmodulin, Neuronal Ca2+ sensors, Phospholipase C, Protein kinase C, Troponin C, Calbindin, Calretinin Parvalbumin Ca2+ Calreticulin (CRT), Calsequestrin (CSQ) Calnexin, Junctate, Bip, Grp94, Reticulocalbin, ERC55, Cab55 Calumenin, Crocalbin Ca2+ Ca2+ Intracellular Ca2+ reuptake Ca2+-ATPases (SERCA1-3) mitochondrial uniporter Ca2+ Ca2+ Ca2+ endoplasmic reticulum Ca2+ Ca2+ [Ca2+]ER = 10-4 ~10-3 M

  8. Plasma membrane Ca2+ channels Ca2+ sensitive processes Contraction Proliferation Fertilization Learning and memory Crosstalk with other signaling pathways Membrane excitability Secretion Metabolism Vesicle trafficking Cell proliferation Cell proliferation, cancer and metastasis TnC CAM MLCK CAMKs Calcineurin Transcription factors Adenylyl cyclase Cyclic AMP PDE NOS PKC PYK2 IP3 3-kinase Ion channels Synaptotagmin Phosphorylase kinase Annexin family S100 family SERCA PMCA Ca2+ ~1,000 nM Ca2+ ~100 nM - - Ca2+ IP3R, RyR, SCaMPER NAADPR Na+ Ca2+ Na+/Ca2+ Exchanger Na+/Ca2+ Exchanger Na+ ATP synthesis Steroid synthesis Apoptosis Mitochondrial enzymes Caspases Ca2+ Cytochrome c PTP “On” and “Off” mechanisms of intracellular Ca2+ signal Stimulus Ca2+ 1 mM Plasma membrane RTK R R R DV G ? ? Cytoplasm PLCb PtdIns(4,5)P2 NAD NADP PLCg Sphingosine Ins(1,4,5)P3 cADPR NAADP S1P ADP ribosyl cyclase Sphingosine kinase Ca2+ ~100 µM ER/SR Buffers/chaperones Ca2+ buffers Mitochondrion Berridge et al., 2000, Nature Rev. Mol. Cell. Biol. 1, 11–21.

  9. Store-operated channels (SOCs)

  10. 2 mM Ca2+ Ca2+-free 2 mM Ca2+ 0.5 µM TG 0.5 µM TG 400 400 300 300 [Ca2+]i (nM) [Ca2+]i (nM) 200 200 100 100 0 0 0 200 400 600 800 0 200 400 600 800 Time (sec) Time (sec) Store-operated channels (SOCs) - The phenomenon A. Agonist stimulation B. Store depletion

  11. Phenotypes of cytosolic Ca2+ changes Propagated Local / Regional / Global Dr. Sarvazyan (Texas Tech) Haag & Barst J Neurophys 2000 Whitaker Phys Rev 2006 Sustained Transient Basal [Calcium] (nM) [Calcium] (nM to M) Time (min to hr) Time (ms, s or min)

  12. Internal Ca2+ release Ca2+ influx Global Ca2+ changes

  13. 300 [Ca] (nM) i 100 200 ms Localized Ca2+ signal 10 µm 200 ms 20 µm 300 [Ca ] i (nM) 100 10 µm ms Bers, DM. 2001. 200

  14. Ca2+ waves Kaneko, T. et al. Circ Res 2000;86:1093-1099

  15. [Ca2+]i [Ca2+]i [Ca2+]i Time Time Time Modes of intracellular [Ca2+] changes: oscillation Frequency-dependent cellular function High-frequency oscillation Sustained elevation Low-frequency oscillation stimulus stimulus stimulus

  16. Ca2+ oscillations Ca2+ signals in Fura2- and Rhod2-coloaded single hepatocytes Weak stimulation (3 µM phenylephrine) Strong stimulation (50 nM vasopressin) 800 200 110 120 600 100 [Ca2+]i (µM) [Ca2+]i (1/F380 nm) 150 400 100 [Ca2+]m (Frhod2, % Basal) [Ca2+]m (Frhod2, % Basal) 90 200 80 100 80 0 0 300 600 900 0 200 400 600 Time (s) Time (s) Robb-Gaspers et al., 1998 Biochim. Biophys. Acta 1366, 17-32

  17. Ca2+ Signaling: targets Phosphorylation: Dephosphorylation: Calmodulin Kinases Calcineurin Cytoskeletal Dynamics: + S100 Ca2+ -Binding Proteins & the EF-Hand Family: Decoders of Ca2+ Signals Degradation: Calpain Loop Helix Contraction: Troponin C Helix Transcription: Ionic Balance: Ca2+ Homeostasis: KChIPs Dream Parvalbumin

  18. Experimental methods for studying Ca2+ signaling • Electrophysiology • Direct measurement of conductivity of plasma membrane channels in whole-cell, cell-attached, inside-out or outside-out configurations

  19. Experimental methods for studying Ca2+ signaling Wang et al., JMCC, 2009.

  20. Experimental methods for studying Ca2+ signaling Electrophysiology 1) Direct measurement of conductivity of plasma membrane channels in whole-cell, cell-attached, inside-out or outside-out configurations 2) Measurement of ER Ca2+ release channels (IP3Rs and RyRs) in planar lipid bilayers.

  21. Experimental methods for studying Ca2+ signaling Wang et al., J Physiol, 2010.

  22. Fluorescence Ca2+ indicators Single wavelength dyes Fluo3 Fluorescence intensity Fluo3, pentaammonium salt Molecular Formula: C36H45Cl2N7O13 Molecular Weight: 854.7 500 550 600 650 Emission wavelength (nm) Ca2+-dependent fluorescence emission spectra of Fluo3. The spectrum for the Ca2+-free solution is indistinguishable from the baseline.

  23. Dual wavelength dyes Fluorescence excitation spectra (Fura2) and emission spectra (Indo1) in solutions containing zero to 39.8 µM free Ca2+ Indo1 Fura2 Fluorescence intensity 350 400 450 500 550 600 250 300 350 400 450 Excitation wavelength (NM) Emission wavelength (nm) 5 K+ 5 K+ C29H22N3K5O14 C32H26K5N3O12

  24. EGTA 5 Triton X100 340 nm 380 nm CCh 4 F510 (arbitrary unit x 10-4) ( ) 3 R - Rmin Sf2 500 [Ca2+]i = Kd * * Rmax - R Sb2 400 2 300 1 200 0 1 2 3 4 5 6 100 0 0 1 2 3 4 5 Relationship between [Ca2+]i and the ratio of a ratiometric dye Sf2 CCh Sb2 [Ca2+]i (nM) CCh Rmax 2.2 Ratio (F340/380) Time (min) 1.2 Rmin 0.2 0 1 2 3 4 5 6 Time (min)

  25. Permeability issue 5 K + Fura2 pentapotassium salt: cell-impermeant

  26. Acetoxymethyl (AM) esters Fura2 AM: cell-permeant

  27. GFP-based probes Maier at el., JMCC, 2006.

  28. Cardiac myocyte Maier at el., JMCC, 2006.

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