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
– 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
SERCA
• ATP-driven calcium pump
• E1-E2 model, P-type pumps
E1
E1-ATP-2Ca
E2
E2P
E1P-ADP-2Ca
E2P-2Ca
SERCA structure
E1
E2
IP3
• Endoplasmic reticulum IP3 channel
– IP3 gated
– Ca2+ activated
Calcium Binding Domains
• EF-Hand –calcium dependent protein binding
• C2 –calcium dependent DAG binding
• Gel (gelsolin)-calcium dependent actin binding
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
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
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
Calcium dependent membrane fusion
Calcium dynamics
• Spatially restricted
• Time varying
– Neural firing rate
– Receptor dynamics
Hepatocyte calcium oscillations
Extracellular ATP
Phenylephrine
Larsen & Kummer, 2003
Calcium sparks
• Quantal Ca2+ release from ER
Position in cell
(line scan)
– IP3, Ca, Voltage
Time 
Cheng et al., 1993
Decoding calcium signaling
• Competitive processes
• Kinetics
– kon
– Koff
• Affinity
– kd = koff/kon
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
Cn/CaMKII competition
• Equilibrium/Steady state
• Time course
Resting [Ca]
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
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
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
Axonal outgrowth
• Growth cone
• Chemotaxis
• Re-establish lost synapse
Direction of initial growth
Unsynapsed axon grows toward a chemoattractant
Fast
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
Calcium dependent guidance
• Low calcium media converts attraction to
repulsion
Laser targeted Ca pulse
with low NP-EGTA
• Calcineurin dependent
• Tune caged Ca content to produce repulsion
Cn/CaMKII competition
Chemoattractant molecule
binds a receptor
CAM
Triggering local calcium
release
Ca2+
Low concentrations of
chemoattractant release
little calcium and Cn
activity dominates
Cn
CaMKII
cdc42
actin
High concentrations of
chemoattractant release
lots of calcium and activate
CaMKII
Regulating the local
phosphorylation of cdc42
Promoting actin filament growth
towards higher chemoattractant
concentrations
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
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
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
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
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