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Intracellular Ca 2+ Signaling

Intracellular Ca 2+ Signaling. 2004. Why Calcium?. Na + and Cl - are sea water Excluded to maintain low osmotic pressure [K + ] i kept high for electrical neutrality [Ca 2+ ] i maintained very low Prevents precipitation of organic anions Mg 2+ helps solubilize organic anions.

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Intracellular Ca 2+ Signaling

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  1. Intracellular Ca2+ Signaling 2004

  2. Why Calcium? • Na+ and Cl- are sea water • Excluded to maintain low osmotic pressure • [K+]i kept high for electrical neutrality • [Ca2+]i maintained very low • Prevents precipitation of organic anions • Mg2+ helps solubilize organic anions

  3. How cells keep [Ca]i low • All eukaryotic cells have PM Ca2+-ATPase • Excitable cells also have Na+/Ca2+ exchanger (NCX) • ER Ca2+-ATPase (against a high grad) • Mitochondrial high capacity (low affinity) pump • When [Ca]i very high (dangerous) levels (>10-5 M) • Inner mitochondrial membrane • Uses the electrochemical gradient generated during electron-transfer of oxidative-phosphorylation

  4. Calcium Concentrations • [Ca2+]o / [Ca2+]i >104 • [Ca2+]o ~10-3 M • [Ca2+]ER ~10-3 M • [Ca2+]i <10-7 M at rest

  5. Ca2+ - a versatile signal

  6. Ca2+ - a versatile signal • Synaptic vesicle release (ms) • Excitation-contraction coupling (ms) • Smooth muscle relaxation (ms-sec) • Excitation-transcription coupling (min-h) • Gene transcription (h) • Fertilization (h)

  7. How Ca2+ is measured • Ratiometric dyes • Cameleons

  8. How cells ↑ [Ca]i • Voltage-gated Ca2+ Channels • Membrane potential drives Ca2+ down its chemical gradient • Different channels in different cells • Different properties for different purposes

  9. Ca2+ shut-off pathways • Voltage-gated Ca2+ channels inactivate • IP3 rapidly dephosphorylated • Ca2+ rapidly pumped out

  10. Ca2+ oscillations Biochemical Society Transactions (2003) Volume 31, part 5

  11. Intracellular Ca2+ Targets • EF hand-containing proteins • Calmodulin • C2 domain proteins • PKC • Synaptogamin

  12. Troponin C Calmodulin Calcineurin Calpain Recoverin S100 Intracellular Ca2+ TargetsEF hand proteins

  13. Calmodulin (CaM) • 16.8 kDa Ca2+-binding protein • Highly conserved in all eukaryotes • Up to 1% of protein • Binds 4 mol Ca2+/mol • Binding affinity ~ .25-1x106 M • Cooperativity with Hill constant ~ 2

  14. Classical mode of CaM regulationRelief of autoinhibition Cell, Vol. 108, 739–742, March 22, 2002

  15. CaMKII Autoinhibition and Frequency Decoder

  16. New modes of CaM interaction Cell, Vol. 108, 739–742, March 22, 2002

  17. New modes of CaM interactionNew modes of CaM regulation Cell, Vol. 108, 739–742, March 22, 2002

  18. Intracellular Ca2+ TargetsC2 Domains • Protein kinase C • Synaptotagmin • Phospholipase A2

  19. C2 domains - PKC Chem. Rev. 2001, 101, 2353-2364

  20. Synaptotagmin and neurotransmitter release

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