ms [Ca] i (nM) 10 m Ca 2+ Signaling Mark T Ziolo, PhD Department of Physiology & Cell Biology 019 Hamilton Hall Signal Transduction Mechanism to convert a stimulus (mechanical/chemical) to a specific cellular response Signaling moleculeReceptor2 nd messengerCellular Response 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) 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 Ca 2+ Signaling High [Ca 2+ ]: M Mitochondrion High [Ca 2+ ] Low [Ca 2+ ]: 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 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 = M [Ca 2+ ] i = M Ca 2+ [Ca 2+ ] ER = ~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 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 = M [Ca 2+ ] i = M Ca 2+ [Ca 2+ ] ER = ~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 On and Off mechanisms of intracellular Ca 2+ signal Berridge et al., 2000, Nature Rev. Mol. Cell. Biol. 1, 1121. Cytoplasm Plasma membrane Sphingosine Ca 2+ 1 mM NAD NADP PtdIns(4,5)P 2 RTK Stimulus R VV 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 M 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 Store-operated channels (SOCs) Store-operated channels (SOCs) - The phenomenon Time (sec) [Ca 2+ ] i (nM) 2 mM Ca M TG M TG Ca 2+- free2 mM Ca 2+ Time (sec) [Ca 2+ ] i (nM) A. Agonist stimulation B. Store depletion 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 Internal Ca 2+ releaseCa 2+ influx Global Ca 2+ changes ms [Ca] i (nM) 10 m 10m i [Ca] (nM) 200 ms 20m 200 ms Bers, DM Localized Ca 2+ signal Ca 2+ waves Kaneko, T. et al. Circ Res 2000;86: 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 Ca 2+ oscillations Weak stimulation (3 M phenylephrine) [Ca 2+ ] i (M) [Ca 2+ ] i (1/ F380 nm ) [Ca 2+ ] m (F rhod2, % Basal) Time (s) [Ca 2+ ] m (F rhod2, % Basal) Strong stimulation (50 nM vasopressin) Robb-Gaspers et al., 1998 Biochim. Biophys. Acta 1366, Ca 2+ signals in Fura2- and Rhod2-coloaded single hepatocytes Time (s) 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 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 Wang et al., JMCC, 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 2) Measurement of ER Ca 2+ release channels (IP 3 Rs and RyRs) in planar lipid bilayers. Wang et al., J Physiol, Experimental methods for studying Ca 2+ signaling Fluorescence Ca 2+ indicators Fluorescence intensity Emission wavelength (nm) 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: Single wavelength dyes Fluo3 Dual wavelength dyes Fluorescence intensity Excitation wavelength (NM) Emission wavelength (nm) 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 M free Ca 2+ 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 * Ratio (F340/380) S f2 CCh S b2 R max R min [Ca 2+ ] i (nM) Time (min) CCh Permeability issue Fura2 pentapotassium salt: cell-impermeant 5 K + Acetoxymethyl (AM) esters Fura2 AM: cell-permeant GFP-based probes Maier at el., JMCC, 2006. Cardiac myocyte