1. 100 300 200 ms [Ca] i (nM) 10 µm Ca 2+ Signaling Mark T Ziolo, PhD Department of Physiology & Cell Biology 019 Hamilton Hall 8-7905 ziolo.1@osu.edu

2. Signal Transduction Mechanism to convert a stimulus (mechanical/chemical) to a specific cellular response Signaling molecule→Receptor→2 nd 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., TNF  ) Neurotransmitters (e.g., acethylcholine) ROS (e.g., O 2 - ) Receptors Cell Surface Receptors (e.g.,  -adrenergic receptor) Intracellular Receptors (e.g., steroid hormones)

4. 2 nd Messengers Cyclic nucleotides (cAMP, cGMP) Lipophilic (e.g., diacylglycerol) Nitric Oxide Inositol 1,4,5-triphosphate (IP 3 ) Arachidonic Acid Ca 2+ Cellular Response Contraction Secretion Plasticity Differentiation Proliferation Apoptosis

5. Ca 2+ Signaling High [Ca 2+ ]: 10 -3 M Mitochondrion High [Ca 2+ ] Low [Ca 2+ ]: 10 -7 M ER Ca 2+ Store High [Ca 2+ ] Ca 2+ Ca 2+ -regulated kinases Ca 2+ -regulated phosphatases Ca 2+ -regulated channels Ca 2+ -regulated transcription factors Contraction Secretion Proliferation Differentiation Plasticity Apoptosis

6. Ca 2+ signaling: sources voltage-gated Ca 2+ channels ionotropic receptor/cation channels cyclic nucleotide-gated channels store-operated channels other non-selective cation channels + + + + + + + + + + + - - - - - - - - - - - Ca 2+ Ca 2+v Ca 2+ [Ca 2+ ] o = 10 -3 M [Ca 2+ ] i = 10 -7 M Ca 2+ [Ca 2+ ] ER = 10 -4 ~10 -3 M Plasma membrane Ca 2+ permeable channels endoplasmic reticulum cytosol extracellular space IP 3 receptors ryanodine receptors NAADP receptor PTP? (MT) Intracellular Ca 2+ channels

7. Ca 2+ signaling: removal Ca 2+ -ATPases (PMCA1-4) Na + /Ca 2+ exchanger (NCX1-3) + + + + + + + + + + + - - - - - - - - - - - Ca 2+ Ca 2+v Ca 2+ [Ca 2+ ] o = 10 -3 M [Ca 2+ ] i = 10 -7 M Ca 2+ [Ca 2+ ] ER = 10 -4 ~10 -3 M Plasma membrane extrusion endoplasmic reticulum cytosol extracellular space Ca 2+ -ATPases (SERCA1-3) mitochondrial uniporter Intracellular Ca 2+ reuptake Cytosolic Ca 2+ -binding proteins: Calmodulin, Neuronal Ca 2+ sensors, Phospholipase C, Protein kinase C, Troponin C, Calbindin, Calretinin Parvalbumin Calreticulin (CRT), Calsequestrin (CSQ) Calnexin, Junctate, Bip, Grp94, Reticulocalbin, ERC55, Cab55 Calumenin, Crocalbin

8. “On” and “Off” mechanisms of intracellular Ca 2+ signal Berridge et al., 2000, Nature Rev. Mol. Cell. Biol. 1, 11–21. Cytoplasm Plasma membrane Sphingosine Ca 2+ 1 mM NAD NADP PtdIns(4,5)P 2 RTK Stimulus R VV R R Buffers/chaperones Ca 2+ buffers Sphingosine kinase ADP ribosyl cyclase PLC  PLC  G ER/SR ? ? Na + /Ca 2+ Exchanger Ca 2+ Na + PMCA SERCA - - Ca 2+ ~100 µM Mitochondrion Na + /Ca 2+ Exchanger Na + PTP Ca 2+ Plasma membrane Ca 2+ channels IP 3 R, RyR, SCaMPER NAADPR Ca 2+ ~1,000 nM Ins(1,4,5)P 3 cADPR NAADP S1P Ca 2+ 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 CAMMLCK CAMKs Calcineurin Transcription factors Adenylyl cyclase Cyclic AMP PDE NOS PKC PYK2 IP 3 3-kinase Ion channels Synaptotagmin Phosphorylase kinase Annexin family S100 family Cytochrome c Mitochondrial enzymes Caspases ATP synthesis Steroid synthesis Apoptosis Ca 2+ ~100 nM

9. Store-operated channels (SOCs)

10. Store-operated channels (SOCs) - The phenomenon Time (sec) [Ca 2+ ] i (nM) 2 mM Ca 2+ 0.5 µM TG 0 100 200 300 400 0200400600800 0 0200400600800 100 200 300 400 0.5 µM TG Ca 2+- free2 mM Ca 2+ Time (sec) [Ca 2+ ] i (nM) A. Agonist stimulation B. Store depletion

11. Time (ms, s or min) Time (min to hr) Phenotypes of cytosolic Ca 2+ changes Local / Regional / Global [Calcium] (nM to  M) Basal [Calcium] (nM) Transient Sustained Propagated Dr. Sarvazyan (Texas Tech) Haag & Barst J Neurophys 2000 Whitaker Phys Rev 2006

12. Internal Ca 2+ releaseCa 2+ influx Global Ca 2+ changes

13. 100 300 200 ms [Ca] i (nM) 10 µm 10µm i 100 300 [Ca] (nM) 200 ms 20µm 200 ms Bers, DM. 2001. Localized Ca 2+ signal

14. Ca 2+ waves Kaneko, T. et al. Circ Res 2000;86:1093-1099

15. Modes of intracellular [Ca 2+ ] changes: oscillation Time [Ca 2+ ] i Time [Ca 2+ ] i Time [Ca 2+ ] i Sustained elevation Low-frequency oscillation High-frequency oscillation stimulus Frequency-dependent cellular function

16. Ca 2+ oscillations Weak stimulation (3 µM phenylephrine) 0 200 400 600 800 [Ca 2+ ] i (µM) [Ca 2+ ] i (1/ F380 nm ) 100 150 200 [Ca 2+ ] m (F rhod2, % Basal) 100 120 80 90 100 110 300600900 200400600 0 0 Time (s) [Ca 2+ ] m (F rhod2, % Basal) Strong stimulation (50 nM vasopressin) Robb-Gaspers et al., 1998 Biochim. Biophys. Acta 1366, 17-32 Ca 2+ signals in Fura2- and Rhod2-coloaded single hepatocytes Time (s)

17. Transcription: Contraction: Ca 2+ Homeostasis: Dream Troponin C Parvalbumin Cytoskeletal Dynamics: S100 Phosphorylation: Calmodulin Kinases + Dephosphorylation: Calcineurin Degradation: Calpain Ionic Balance: KChIPs Ca 2+ -Binding Proteins & the EF-Hand Family: Decoders of Ca 2+ Signals Helix Loop Ca 2+ Signaling: targets

20. Experimental methods for studying Ca 2+ signaling Electrophysiology 1)Direct measurement of conductivity of plasma membrane channels in whole-cell, cell-attached, inside-out or outside-out configurations

21. Wang et al., JMCC, 2009. Experimental methods for studying Ca 2+ signaling

22. 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 Ca 2+ release channels (IP 3 Rs and RyRs) in planar lipid bilayers.

23. Wang et al., J Physiol, 2010. Experimental methods for studying Ca 2+ signaling

24. Fluorescence Ca 2+ indicators Fluorescence intensity Emission wavelength (nm) 500 550 600 650 Ca 2+ -dependent fluorescence emission spectra of Fluo3. The spectrum for the Ca 2+ -free solution is indistinguishable from the baseline. Fluo3, pentaammonium salt Molecular Formula: C 36 H 45 C l2 N 7 O 13 Molecular Weight: 854.7 Single wavelength dyes Fluo3

25. Dual wavelength dyes Fluorescence intensity Excitation wavelength (NM) 250300 350 400 450 Emission wavelength (nm) 350 400 450 500 550 600 Fura2 Indo1 5 K + C 29 H 22 N 3 K 5 O 14 C 32 H 26 K 5 N 3 O 12 5 K + Fluorescence excitation spectra (Fura2) and emission spectra (Indo1) in solutions containing zero to 39.8 µM free Ca 2+

26. Relationship between [Ca 2+ ] i and the ratio of a ratiometric dye ()() [Ca 2+ ] i = K d R - R min S f2 R max - R S b2 * 0.2 1.2 2.2 0123456 Ratio (F340/380) S f2 CCh S b2 R max R min [Ca 2+ ] i (nM) Time (min) CCh

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

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

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

30. Cardiac myocyte