1. 10 th PBL in calcium- and phospholipid signaling May 3-14, 2010 Md. Shahidul Islam, M.D., Ph.D. Associate Professor Department of Clinical Sciences and Education, Södersjukhuset Karolinska Institutet Forskningscentrum, Södersjukhuset 118 83 Stockholm, Sweden Shaisl@ki.se

3. Sydney Ringer (1883) Survival of Fish Muscle contraction Fertilization of eggs Development of tadpole Locke and Overton (1894) Impulse transmission

4. Why Ca 2+ is chosen by nature for diverse regulations?

5. Specific and tight binding to effector proteins Suitable coordination chemistry Larger diameter and flexible coordination number Can bind in to irregularly shaped protein cavities

6. Evolutionary history of calcium Ancient sea water was free of calcium Water contaminated by rocks Protection against calcium toxicity evolved Cells chose calcium as a signaling ion

7. Total Ca 2+ and free Ca 2+ Total cell calcium: 2.5 mmol/Kg wet weight. Higher total calcium in tumors 60-80% of it is bound to the extracellular coat Large amount of calcium in secretory granules and ER Free Ca 2+ concentration is important for regulatory purposes

8. What are the functions of Ca 2+ ? Structural. –Bone –Membrane fluidity and integrity (Ca 2+ phospholipid) –Protein structure and function –Chromatin structure

9. What are the functions of Ca 2+ ? Co-factor for enzymes –Protein Kinase C –Phospholipase A 2 –Prothrombin –Calpain –DNAse 1 Electrical –Ca 2+ current during action potential

10. Intracellular Regulation Second messenger Muscle contraction Secretion Metabolism Gene expression

11. Ca 2+ is a two-edged sword Excessive rise of [Ca 2+ ] i –Activation of Proteases Phosphatases Endonucleases

12. Excessive rise of [Ca 2+ ] i Impaired mitochondrial function Perturbation of cytoskeletal organization Apoptosis, cell death

13. Cellular components that determine Ca 2+ fluxes and Ca 2+ homeostasis: Plasma membrane Ca 2+ channels –Voltage-gated –Receptor operated –TRPs Plasma membrane Ca 2+ ATPase Na + /Ca 2+ Exchanger

14. Na + /Ca 2+ exchanger vs PM Ca 2+ ATPase Na + /Ca 2+ exchanger: Low affinity, high capacity. Takes care of large Ca 2+ loads PM Ca2+ ATPase. High affinity low capacity Three Na + /Ca 2+ exchanger genes. Gene products NCX1, NCX2, NCX3; several splice variants

15. PM Ca2+ ATPase P-type family of transport ATPases Four genes PMCA1, PMCA2, PMCA3, PMCA4 Four splice variants Calmodulin-dependent

16. Cellular components that determine Ca 2+ fluxes and Ca 2+ homeostasis: ER Serco-Endoplasmic Reticulum Ca 2+ ATPase (SERCA) IP3 Recepotors Ryanodine receptors TRIC (trimeric intracellular cation) channel (Yazawa M, Nature 2007) Luminal Ca 2+ -binding proteins

17. Cellular components that determine Ca 2+ fluxes and Ca 2+ homeostasis: Mitochondria –Ca2+ Uniporter –Na + /Ca 2+ exchanger Cytoplasm –Calmodulins –Parvalbumin –Other Ca 2+ -binding proteins

18. Calcium Channels Voltage Dependent –Slow, fast Intracellular Ca2+ channel

19. Receptor-operated Ca 2+ channels

20. Ca 2+ Channels Receptor-operated Ca 2+ channels –Nicotinic cholinergic receptor –NMDA receptor ion channel –Purinergic receptor P2X

21. Voltage-gated Ca 2+ channels

22. Gene Superfamily of Voltage- Gated Ion Channels Voltage-gated Na + channels Voltage-gated K + channels Voltage-gated Ca 2+ channels

23. Distinct classes of Ca 2+ curents L-type –High activation voltage –Large conductance –Long lasting –Blocked by dihydropyridine, phenylalkylamine, benzothiazepine

24. Other high-voltage-activated Ca 2+ channels N-type –Neuronal P/Q-type R-type Not blocked by DHPs, blocked by polypeptide toxins

25. Low-voltage activated Ca 2+ current T-type –Tiny conductance –Transient current –

26. Nomenclature Ion conducted Main regularor Alpha-1 subunit gene family Cav1.1

27. EA Ertel Neuron 25:533, 2000.

28. Molecules and curents Cav1.1 Cav1.2 Cav1.3 Cav1.4 Cav2.1 Cav2.2 Cav2.3 Cav3.1 Cav3.2 Cav3.3 L-type P/Q-type N-type R-type T-type

29. Blockers Cav1.1 Cav1.2 Cav1.3 Cav1.4 Cav2.1 Cav2.2 Cav2.3 Cav3.1 Cav3.2 Cav3.3 DHP Not known  -Agatoxin IVA Conotoxin None

32. Fluorescence

33. Jablonski Diagram

37. Fluorescence Resonance Energy Transfer

38. What is FRET It is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.

39. Conditions for FRET Proximity (10-100Å) Absorption spectrum of acceptor overlaps emission spectrum of donor Donor and acceptor transition dipole orientation is parallel

41. Excitation and emission spectra of different ”GFP”

42. Cameleons

43. Miyawaki A. 1997, Nature 388:6645