1. Regulation and measurement of intracellular calcium May 12, 2006

2. The four units of the Ca signaling network. Stimuli act by generating Ca-mobilizing signals that act on various ON mechanisms to trigger an increase in the intracellular concentration of Ca. The increased level of Ca stimulates various Ca-sensitive processes to trigger many different cellular pathways. The response is terminated by OFF mechanisms that restore Ca to its resting level. Nature reviews (MCB) 2000, 1, 11- 21

3. Nat. Rev. MCB (2003) 4, 552 The regulation of intracellular calcium compartmentalization

5. The four units of the Ca signaling network. Stimuli act by generating Ca-mobilizing signals that act on various ON mechanisms to trigger an increase in the intracellular concentration of Ca. The increased level of Ca stimulates various Ca-sensitive processes to trigger many different cellular pathways. The response is terminated by OFF mechanisms that restore Ca to its resting level. Nature reviews (MCB) 2000, 1, 11- 21

6. Ca ATPase 2Ca/1ATP

7. Ca ATPase (Ca pump) 2Ca/1ATP

9. Structure of the catalytic alpha subunit of the muscle Ca-ATPase

10. K Na Ca K H Na Glucose Na Ca H Glucose H Cl HCO 3 lysosome ER mitochondria Na = 145 mM Ca = 2 mM K = 150 mM Ca = 0.1 μM

11. The free energy change of moving solute X, from one side of a membrane to the otherside with concentration of C1 and C2, respectively, is Delta G = RT ln C2/C1 = 2.3 RT log C2/C1 C1 < C2, delta G is positive, need energy to occur, so called “active transport” C1 > C2, delta G is negative, spontaneously occur, “passive diffusion” or “passive Transport” For example, 10 fold concentration gradient across the membrane Delta G = 2.3 RT log 0.1/1 = -1,359 cal/mol (energy released in the process) If X is a charged compound, both the chemical concentration and the electric Potential have to be consider. Delta G = 2.3 RT log C2/C1 + ZF delta V Z: the number of charge F: the Faraday constant Delta V: the difference of electric potential across the membrane

12. ATP Na K Ca Ouabain digitoxigenin 3Na/1Ca Electrogenic Reversible - - - - - - - + + + + + Na Ca Na = 145 mM Na = 5 mM

13. Nat. Rev. MCB (2003) 4, 552 The regulation of intracellular calcium compartmentalization

14. The four units of the Ca signaling network. Stimuli act by generating Ca-mobilizing signals that act on various ON mechanisms to trigger an increase in the intracellular concentration of Ca. The increased level of Ca stimulates various Ca-sensitive processes to trigger many different cellular pathways. The response is terminated by OFF mechanisms that restore Ca to its resting level. Nature reviews (MCB) 2000, 1, 11- 21

15. Nat. Rev. MCB (2003) 4, 552 The regulation of intracellular calcium compartmentalization

17. Ion channels Voltage-gated ion channels Ligand-gated ion channels

18. Annu. Rev. Cell Dev. Biol. (2000) 16: 521-555 Skeletal muscle

19. Annu. Rev. Cell Dev. Biol. (2000) 16: 521-555 Cardiac muscle

20. Membrane 去極化超過 threshold Channel 才 open

21. Structure and function of the voltage-gated ion channels.

24. Nat. Rev. MCB (2003) 4, 552 The regulation of intracellular calcium compartmentalization

25. Acetylcholine receptor

27. Structure of the acetylcholine receptor ion channel.

28. An action potential is generated about every 4 ms. Action potentials move down the axon at speeds up to 100 meters per second. Their arrival at a synapse causes release of neurotransmitters that bind to receptors in the postsynaptic cells, generally depolarizing the membrane (making the potential less negative) and tending to induce an action potential on it.

29. The threshold potential for generation of an action potential in a postsynaptic cell.

30. ATP K Na K K Ca Ach CaSecretion contraction Memb. potential Ligand-gated ion channel Voltage-gated ion channel E K = - 91 mV E Na = 64 mV [K] = 140 mM [Na] = 145 mM

31. Nat. Rev. MCB (2003) 4, 552 The regulation of intracellular calcium compartmentalization

32. Activate PKC Release calcium from ER Hormone-activated phospholipase C and IP3

33. IP3 receptor was cloned in 1989. Ryanodine receptor IP3 receptor

34. Molecular and Cellular Biology (1999) 190, 185-190 IP3 receptor

35. Molecular and Cellular Biology (1999) 190, 185-190 Ryanodine receptor Voltage gated Ca channel

36. Sequential activation of gated ion channels at a neuromuscular junction. 1 voltage-gated Ca channel 2 ligand-gated nicotinic receptors 3 voltage-gated Na channel to generated action potential 4 voltage-gated Ca channel and Ca induced Ca release channel (ryanodine receptor)

37. Role of voltage-gated and ligand-gated ion channels in neural transmission

38. Annu. Rev. Cell Dev. Biol. (2000) 16: 521-555

42. Troponin and tropomyosin block the interaction between myosin and actin. Troponin C: binds Ca Troponin I: bind actin Troponin T: bind tropomyosin Tropomyosin: double helix polypeptide

43. Cardiac muscle Ca entry (L channel) Ca induced Ca release from SR via ryanodine receptor Skeletal muscle L channel directly activates Ca release from SR, extracellular Ca is not required. Smooth muscle myosin light chain phosphorylation catalyzed by Ca/CaM MLC kinase. + Ca - Ca Myosin binding site exposed.

44. neuron myocyte Na L (skeletal muscle) (cardiac muscle) L nonexcitable cell Ca R G E IP3 Ca depletion (SOC) (VOC) Ca Na (ROC) Na IP3 Ca cAMP Ryanodine receptor Ca RGE R G E (IP3 receptor) (1983) (1996) ?

47. Nat. Rev. MCB (2003) 4, 552 The regulation of intracellular calcium compartmentalization

48. Capacitative Ca entry Nature reviews (MCB) 2000, 1, 11- 21

49. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature (`1992) 355, 353-356. Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca-permeable channel. Nature (1992) 355, 356-358. Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature (1993) 364, 809-814. Depletion of InsP3 stores activates a Ca and K current by means of a phosphatase and a diffusible messenger. Nature (1993) 364, 814-818 Ca influx factor (CIF) Activation of store-operated Ca current in xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell (1999) 98, 475-485. Store-operated Ca entry: evidence for a secretion-like coupling model. Cell (1999) 98, 487-499

50. Cell (1999) 98, 487

51. Cell (1999) 99, 5

52. TINS (2002) 23:63-70

53. Nat. Rev. MCB (2003) 4, 552 The regulation of intracellular calcium compartmentalization

54. Science’s STKE 2004, January 13

56. Biochim. Biophys. Acta (2004) 1742:119-131

57. The four units of the Ca signaling network. Stimuli act by generating Ca-mobilizing signals that act on various ON mechanisms to trigger an increase in the intracellular concentration of Ca. The increased level of Ca stimulates various Ca-sensitive processes to trigger many different cellular pathways. The response is terminated by OFF mechanisms that restore Ca to its resting level. Nature reviews (MCB) 2000, 1, 11- 21

58. Elements of the Ca signaling toolkit. Nature reviews (MCB) 2000, 1, 11- 21

59. Catalytic domain T286 CaM-inhibitory association C CaM kinase Catalytic subunit Inhibitory cAMP PKA Catalytic domain inhibitory Lipid/Ca PKC N

60. Calcium-calmodulin complex mediates many cellular responses.

62. Ca 2+ /calmodulin is activated by increased cytosolic Ca 2+ level. Dedicated CaM kinases myosin light chain kinase (myosin light chain) phosphorylase kinase (phosphorylase) CaMKIII (eukaryotic elongation factor II) Multifunctional CaM kinases CaM kinases I, II and IV (broad substrates)

63. N C Catalytic domain Inhibitory domain Calmodulin binding Association domain 0% 100% 20 – 80% thr286 Thr305, 306 CaMKII Ca/CaM Ca increase Ca decrease ATP - CaM (trapped) (capped)

64. Calcium frequency decoding mechanism by CaM-K II autophosphorylation

65. Biochem. J. (2002) 364, 593-611

66. Calcium regulation Proteins involved in Ca signaling (mobilization) “off”Ca pump (plasma membrane and endoplasmic reticulum) Na/Ca exchanger “on”Ca entry (VOC, ROC and SOC) Ca release (IP3 receptor and ryanodine receptor) Calcium sensitive cellular function secretion contraction activating calmodulin and CaMKII Role of mitochondria in calcium regulation Calcium measurement

67. [Ca]i Ca ATP IP3 Saponine or digitonin is used to permeabilized cells. Ca electrode IP3 was shown to be the second messenger to induce calcium release. Nature (1983) 306, 67-68

68. Fura-2 acid Fura-2/acetoxymethyl (AM) (membrane permeable)

70. Ratioing method fura-2 + Ca ---- > fura-2-Ca (Cf) (Cb) Kd = Ca x Cf/Cb Cb = Cf x Ca/Kd F = SC S = I Φεl while I, incident intensity, Φ, quantum yield, ε, extinction coefficient, and l, length of light path. C = concentration of fura-2 F1 (fluorescence at 340 nm) = Sf1Cf + Sb1Cb F2 (fluorescence at 380 nm) = Sf2Cf + Sb2Cb R = F1/F2 = (Sf1Cf + Sb1Cf x Ca/Kd) / (Sf2Cf + Sb2Cf x Ca/Kd) Ca = Kd x [(R – (Sf1/Sf2)/(Sb1/Sb2) – R] x (Sf2/Sb2) While Ca = 0, no Cb, R = Sf1/Sf2 designated Rmin While Ca = saturating conc., no Cf, R = Sb1/Sb2 designated Rmax Ca = Kd x [(R – Rmin)/(Rmax – R)] x (Sf2/Sb2) Sf2/Sb2 = F of fura-2 at zero Ca (380 nm) / F of fura-2 at saturating Ca (380 nm) = Sf2 Cf / Sb2 Cb Ca = Kd x [(R – Rmin)/(Rmax – R)] x (Sf2/Sb2) Peak at 340 nm, Ca under saturating conc. Ca-bound Peak at 365nm, Ca = 0 unbound 340380 360 (isosbestic point) Excitation 340 and 380 nm Emission 505 nm JBC (1985) 260, 3440-3450

71. Calcium sensors Ratiometric dyes Non-ratiometric dyes Nat. Rev. MCB (2003) 4, 579

72. Protein-based calcium sensors Nat. Rev. MCB (2003) 4, 579 aequorin cameleon camgaroo pericam coelenterazine Tyr145 146

73. Nat. Rev. MCB (2003) 4, 579