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Calcium

Calcium. [Ca2+]i very low ~50-100 nM Many calcium binding proteins = high buffering capacity Divalent cation forms ionic bridges Glutamic acid Aspartic acid Contribute to protein folding Quaternary Binding Substrate recognition. Sources of calcium. Intracellular

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Calcium

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  1. Calcium • [Ca2+]i very low ~50-100 nM • Many calcium binding proteins = high buffering capacity • Divalent cation forms ionic bridges • Glutamic acid • Aspartic acid • Contribute to protein folding • Quaternary Binding • Substrate recognition

  2. Sources of calcium • Intracellular • Endoplasmic (sarcoplasmic) reticulum • IP3 receptor • Sarco(endo)plasmic reticulum Ca ATPase (SERCA) • Extracellular • V-gated Ca channels • Ligand gated channels • Store operated calcium entry • Mitochondria • Mitochondrial calcium uniporter

  3. SERCA • ATP-driven calcium pump • E1-E2 model, P-type pumps E1 E1-ATP-2Ca E1P-ADP-2Ca SERCA structure E1 E2 E2 E2P E2P-2Ca

  4. IP3 • Endoplasmic reticulum IP3 channel • IP3 gated • Ca2+ activated

  5. Calcium Binding Domains • EF-Hand –calcium dependent protein binding • C2 –calcium dependent DAG binding • Gel (gelsolin)-calcium dependent actin binding

  6. Calcium effectors • Calpain • Calcium dependent protease • m-calpain, m-calpain • Troponin • Calcium dependent inhibitor of motility • Calmodulin • Calcium dependent cofactor • Synaptotagmin • Calcium dependent vesicle fusion • Myriad others

  7. Ca mediated protein modification • CaMK (I – IV) • Calmodulin mediated • Serine/threonine kinases • CaMK-III = eEF2 kinase • Post-synaptic density • Protein kinase C • Calcineurin • Calmodulin mediated • Serine/threonine phosphatase • Calpain (I-III) • Cysteine protease • Cytoskeletal remodeling

  8. Calcium dependent fusion • Neurotransmitter release • Complimentary v-SNARE t-SNARE complex • Complexin mediated docking, synaptogamin trigger • Membrane resealing • Injury repair • Extracellular Ca2+ • Spontaneous zipper model Sudhof & Rothman, 2009

  9. Calcium dependent membrane fusion

  10. Calcium dynamics • Spatially restricted • Time varying • Neural firing rate • Receptor dynamics Hepatocyte calcium oscillations Extracellular ATP Phenylephrine Larsen & Kummer, 2003

  11. Calcium sparks • Quantal Ca2+ release from ER • IP3, Ca, Voltage Position in cell (line scan) Time  Cheng et al., 1993

  12. Decoding calcium signaling • Competitive processes • Kinetics • kon • Koff • Affinity • kd = koff/kon

  13. Calcineurin/Calmodulin Kinase • Calcineurin (Cn) • Ca/CaM dependent phosphatase • Ca kd = 0.2 uM, koff 0.001/s • High affinity, slow kinetics • CaM Kinase II (CaMKII) • Ca/CaM dependent kinase • Ca kd = 1 uM, koff 0.3/s • Low affinity, fast kinetics • Small calcium signals activate Cn long time • Large calcium spikes activate CaMKII briefly

  14. Cn/CaMKII competition • Equilibrium/Steady state • Time course Resting [Ca]

  15. Cn/CaMKII in neural plasticity • CaMKII modulates cell motility • cdc42 phosphorylation • Increases actin filament polymerization • Dendrite remodeling • Synaptic strength (hours-days) • Axonal regrowth • Repair mechanism • Specific targeting

  16. Long term potentiation/depression • Glutamineric synapses have both AMPA and NMDA receptors • Long term potentiation: Tetanus increases subsequent EPSPs • Tetanic depolarization relieves Mg2+ block • Calcium induced channel phosphorylation increases conductance • Long term potentiation • Ca2+ influx via NMDA receptors • Ca2+->PKA-|I1->PP1-|AMPA High frequency stimulation High Calcium I1 is inhibited Reduces PP1 Activates CaMK Increases AMPA current Low frequency stimulation Low Calcium I1 activates PP1 Decreases AMPA

  17. Axonal outgrowth • Growth cone • Chemotaxis • Re-establish lost synapse Direction of initial growth Unsynapsed axon grows toward a chemoattractant Fast

  18. CaMKII dependent guidance • “Caged” Ca2+ NP-EDTA • Impose periodic, localized Ca2+ spikes • Guide growth cone development • CaMKII dependent Laser targeted Ca pulse Axon grows toward a chemoattractant & is diverted by intracellular calcium release

  19. Calcium dependent guidance • Low calcium media converts attraction to repulsion • Calcineurin dependent • Tune caged Ca content to produce repulsion Laser targeted Ca pulse with low NP-EGTA

  20. Cn/CaMKII competition CAM Chemoattractant molecule binds a receptor Ca2+ Triggering local calcium release Low concentrations of chemoattractant release little calcium and Cn activity dominates High concentrations of chemoattractant release lots of calcium and activate CaMKII Cn CaMKII Regulating the local phosphorylation of cdc42 cdc42 actin Promoting actin filament growth towards higher chemoattractant concentrations

  21. CaMKII autophosphorylation • CaM Kinase II (CaMKII) • CaM dependent kinase • CaM kd = 2 nM, koff 0.3/s • High affinity, fast kinetics • Phospho-CaMKII • CaM independent kinase • CaM kd = 0.1 pM, koff 10-6/s • Insanely high affinity, very slow kinetics • CaMKII autophosphorylation locks itself in an active conformation

  22. Rate decoding by CaMKII • Activity dependent muscle phenotype • “Slow” muscle • High oxidative capacity • Slow myosin kinetics • Frequent activation • “Fast” muscle • Low oxidative capacity • Fast myosin kinetics • Infrequent activation • Calcium dependent

  23. Rate decoding • Autophosphorylation is like integration • Dephosphorylation is like a high pass filter • eg: Deliver regular calcium pulses • Measure Ca independent activity • Elevated > 1 hr after exercise in muscle

  24. CaMKII phenotypic control • Acute modulation of contractility • Calcium release & re-uptake • Glucose transport • Mitochondrial biogenesis • Oxidative capacity • Contractile protein expression • Upregulation, increase content • Isoform specification, phenotype control

  25. Rate decoding: non-excitable cells • Calcium dependent metabolites • Hepatocytes • Phenylephrine dependent Ca2+ oscillations • Mitochondrial isocitrate dehydrogenase Calcium oscillations in different cells NADH content increases w/frequency Robb-Gaspers et al., 1998

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