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 E1P-ADP-2Ca

E2P-2CaE2PE2

SERCA structureE1 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, -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 oscillationsExtracellular ATP Phenylephrine

Larsen & Kummer, 2003

Calcium sparks

• Quantal Ca2+ release from ER– IP3, Ca, Voltage

Cheng et al., 1993

Time

Po

siti

on

in c

ell

(lin

e sc

an)

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

9 8 7 6 5 4 30

0.2

0.4

0.6

0.8

1.0

Cn

pCa

CaMKIICn/CaMKIIA

ctivity

Cn/CaMKII competition

• Equilibrium/Steady state

• Time course

Resting [Ca]

0 10 100 10000

0.2

0.4

0.6

0.8

1.0 CnCaMKII

Act

ivit

y

Time (s)

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 stimulationLow CalciumI1 activates PP1Decreases AMPA

High frequency stimulationHigh CalciumI1 is inhibitedReduces PP1

Activates CaMKIncreases AMPA current

Axonal outgrowth

• Growth cone

• Chemotaxis

• Re-establish lost synapse

Direction of initial growth

FastUnsynapsed axon grows toward a chemoattractant

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

• Calcineurin dependent

• Tune caged Ca content to produce repulsion

Laser targeted Ca pulsewith low NP-EGTA

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

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