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 E1E1-ATP-2CaE1P-ADP-2Ca E2P-2CaE2PE2 SERCA structure E1 E2

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,  -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 ATPPhenylephrine Larsen & Kummer, 2003

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

12. Decoding calcium signaling Competitive processes Kinetics –k on –K off Affinity –k d = k off /k on

13. Calcineurin/Calmodulin Kinase Calcineurin (Cn) –Ca/CaM dependent phosphatase –Ca k d = 0.2 uM, k off 0.001/s –High affinity, slow kinetics CaM Kinase II (CaMKII) –Ca/CaM dependent kinase –Ca k d = 1 uM, k off 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 Mg 2+ block –Calcium induced channel phosphorylation increases conductance –Long term potentiation Ca2+ influx via NMDA receptors Ca 2+ ->PKA-|I1->PP1-|AMPA Low frequency stimulation Low Calcium I1 activates PP1 Decreases AMPA High frequency stimulation High Calcium I1 is inhibited Reduces PP1 Activates CaMK Increases AMPA current

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

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

21. CaMKII autophosphorylation CaM Kinase II (CaMKII) –CaM dependent kinase –CaM k d = 2 nM, k off 0.3/s –High affinity, fast kinetics Phospho-CaMKII –CaM independent kinase –CaM k d = 0.1 pM, k off 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