Regulation and measurement of intracellular calcium

May 12, 2006

The four units of the Ca signaling network. Stimuli act by generating Ca-mobilizing signals that act on various ON mechanisms to trigger an increase in the intracellular concentration of Ca. The increased level of Ca stimulates various Ca-sensitive processes to trigger many different cellular pathways. The response is terminated by OFF mechanisms that restore Ca to its resting level.

Nature reviews (MCB) 2000, 1, 11- 21

Nat. Rev. MCB (2003) 4, 552

The regulation of intracellular calcium compartmentalization

The four units of the Ca signaling network. Stimuli act by generating Ca-mobilizing signals that act on various ON mechanisms to trigger an increase in the intracellular concentration of Ca. The increased level of Ca stimulates various Ca-sensitive processes to trigger many different cellular pathways. The response is terminated by OFF mechanisms that restore Ca to its resting level.

Nature reviews (MCB) 2000, 1, 11- 21

Ca ATPase2Ca/1ATP

Ca ATPase (Ca pump)2Ca/1ATP

Structure of the catalytic alpha subunit of the muscle Ca-ATPase

K

Na Ca

K

H

NaGlucose

NaCaH

Ca

Glucose

H

Cl

Cl

HCO3

HCO3

lysosome

ER

mitochondria

Na = 145 mMCa = 2 mM

K = 150 mMCa = 0.1 μM

The free energy change of moving solute X, from one side of a membrane to theotherside with concentration of C1 and C2, respectively, is Delta G = RT ln C2/C1 = 2.3 RT log C2/C1

C1 < C2, delta G is positive, need energy to occur, so called “active transport”C1 > C2, delta G is negative, spontaneously occur, “passive diffusion” or “passiveTransport”

For example, 10 fold concentration gradient across the membraneDelta G = 2.3 RT log 0.1/1 = -1,359 cal/mol (energy released in the process)

If X is a charged compound, both the chemical concentration and the electric Potential have to be consider.Delta G = 2.3 RT log C2/C1 + ZF delta V

Z: the number of chargeF: the Faraday constantDelta V: the difference of electric

potential across the membrane

ATP

Na

K

Na

Ca

Ouabaindigitoxigenin

3Na/1CaElectrogenicReversible

- - - - - - -

+ + + + +

Na

Ca

Na = 145 mM

Na = 5 mM

Nat. Rev. MCB (2003) 4, 552

The regulation of intracellular calcium compartmentalization

The four units of the Ca signaling network. Stimuli act by generating Ca-mobilizing signals that act on various ON mechanisms to trigger an increase in the intracellular concentration of Ca. The increased level of Ca stimulates various Ca-sensitive processes to trigger many different cellular pathways. The response is terminated by OFF mechanisms that restore Ca to its resting level.

Nature reviews (MCB) 2000, 1, 11- 21

Nat. Rev. MCB (2003) 4, 552

The regulation of intracellular calcium compartmentalization

Ion channelsVoltage-gated ion channelsLigand-gated ion channels

Annu. Rev. Cell Dev. Biol. (2000) 16: 521-555

Skeletal muscle

Annu. Rev. Cell Dev. Biol. (2000) 16: 521-555

Cardiac muscle

Membrane 去極化超過 threshold Channel 才 open

Structure and function of the voltage-gated ion channels.

Nat. Rev. MCB (2003) 4, 552

The regulation of intracellular calcium compartmentalization

Acetylcholine receptor

Structure of the acetylcholine receptor ion channel.

An action potential is generated about every 4 ms.Action potentials move down the axonat speeds up to 100 meters per second.Their arrival at a synapse causes releaseof neurotransmitters that bind toreceptors in the postsynaptic cells,generally depolarizing the membrane(making the potential less negative) and tending to induce an action potential on it.

The threshold potential for generation of an action potential in a postsynaptic cell.

ATPKNa

K

Na

K

Na CaAch

Ca Secretion contraction

Memb.potential

Ligand-gated ion channelVoltage-gated ion channel

EK = - 91 mVENa = 64 mV

[K] = 140 mM[Na] = 145 mM

Nat. Rev. MCB (2003) 4, 552

The regulation of intracellular calcium compartmentalization

Activate PKC

Release calcium from ER

Hormone-activated phospholipase C and IP3

IP3 receptor was cloned in 1989.

Ryanodine receptorIP3 receptor

Molecular and Cellular Biology (1999) 190, 185-190

IP3 receptor

Molecular and Cellular Biology (1999) 190, 185-190

Ryanodine receptor

Voltage gated Ca channel

Sequential activation of gated ion channels at a neuromuscular junction.1 voltage-gated Ca channel2 ligand-gated nicotinic receptors3 voltage-gated Na channel to generated action potential4 voltage-gated Ca channel and Ca induced Ca release channel (ryanodine receptor)

Role of voltage-gated and ligand-gated ion channels in neural transmission

Annu. Rev. Cell Dev. Biol. (2000) 16: 521-555

Troponin and tropomyosin block the interaction between myosin and actin.Troponin C: binds CaTroponin I: bind actinTroponin T: bind tropomyosinTropomyosin: double helix polypeptide

Cardiac muscleCa entry (L channel)Ca induced Ca release from SR via ryanodine receptor

Skeletal muscle L channel directly activates Ca release from SR, extracellular Ca is not required.

Smooth musclemyosin light chain phosphorylation catalyzed by Ca/CaM MLC kinase.

+ Ca

- Ca

Myosin binding site exposed.

neuron

myocyte

Na

Na

L (skeletal muscle)

(cardiac muscle)

L

nonexcitable cell

CaR G E

IP3

Cadepletion

(SOC)

(VOC)Ca

Na

(ROC)

Na

IP3

Ca

cAMP

Ryanodine receptor

Ca

Ca

Ca

RGE RGE

(IP3 receptor)

(IP3 receptor)

(1983)(1996)

?

Nat. Rev. MCB (2003) 4, 552

The regulation of intracellular calcium compartmentalization

Capacitative Ca entry

Nature reviews (MCB) 2000, 1, 11- 21

Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature (`1992) 355, 353-356.Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca-permeable channel. Nature (1992) 355, 356-358.

Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature (1993) 364, 809-814.Depletion of InsP3 stores activates a Ca and K current by means of a phosphatase and a diffusible messenger. Nature (1993) 364, 814-818

Ca influx factor (CIF)

Activation of store-operated Ca current in xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell (1999) 98, 475-485.Store-operated Ca entry: evidence for a secretion-like coupling model. Cell (1999) 98, 487-499

Cell (1999) 98, 487

Cell (1999) 99, 5

TINS (2002) 23:63-70

Nat. Rev. MCB (2003) 4, 552

The regulation of intracellular calcium compartmentalization

Science’s STKE 2004, January 13

Science’s STKE 2004, January 13

Biochim. Biophys. Acta (2004) 1742:119-131

The four units of the Ca signaling network. Stimuli act by generating Ca-mobilizing signals that act on various ON mechanisms to trigger an increase in the intracellular concentration of Ca. The increased level of Ca stimulates various Ca-sensitive processes to trigger many different cellular pathways. The response is terminated by OFF mechanisms that restore Ca to its resting level.

Nature reviews (MCB) 2000, 1, 11- 21

Elements of the Ca signaling toolkit.

Nature reviews (MCB) 2000, 1, 11- 21

Catalytic domain

T286CaM-inhibitory

associationC

CaM kinase

Catalytic subunit

InhibitorycAMP cAMP

PKA

Catalytic domain

inhibitory Lipid/CaPKC

N

Calcium-calmodulin complex mediates many cellular responses.

N CCatalytic domain

Inhibitory domain

Calmodulin binding

Association domain

0%100%

100%

20 – 80%

20 – 80%

thr286

Thr305, 306

CaMKII

Ca/CaM

Ca increase

Ca decrease

Ca decrease

ATP

- CaM

(trapped)

(capped)

Calcium frequency decoding mechanism by CaM-K II autophosphorylation

Biochem. J. (2002) 364, 593-611

Calcium regulationProteins involved in Ca signaling (mobilization)

“off” Ca pump (plasma membrane and endoplasmic reticulum)Na/Ca exchanger

“on” Ca entry (VOC, ROC and SOC)Ca release (IP3 receptor and ryanodine receptor)

Calcium sensitive cellular functionsecretioncontractionactivating calmodulin and CaMKII

Role of mitochondria in calcium regulationCalcium measurement

[Ca]i

Ca

CaATP

IP3

Saponine or digitonin is used to permeabilized cells.

Ca electrode

IP3 was shown to be the second messenger to induce calcium release.Nature (1983) 306, 67-68

Fura-2 acidFura-2/acetoxymethyl (AM) (membrane permeable)

Ratioing method fura-2 + Ca ---- > fura-2-Ca (Cf) (Cb)Kd = Ca x Cf/CbCb = Cf x Ca/Kd F = SCS = I Φεl while I, incident intensity, Φ, quantum yield, ε, extinction coefficient, and l, length of light path.C = concentration of fura-2F1 (fluorescence at 340 nm) = Sf1Cf + Sb1CbF2 (fluorescence at 380 nm) = Sf2Cf + Sb2CbR = F1/F2 = (Sf1Cf + Sb1Cf x Ca/Kd) / (Sf2Cf + Sb2Cf x Ca/Kd)Ca = Kd x [(R – (Sf1/Sf2)/(Sb1/Sb2) – R] x (Sf2/Sb2)While Ca = 0, no Cb, R = Sf1/Sf2 designated RminWhile Ca = saturating conc., no Cf, R = Sb1/Sb2 designated RmaxCa = Kd x [(R – Rmin)/(Rmax – R)] x (Sf2/Sb2)Sf2/Sb2 = F of fura-2 at zero Ca (380 nm) / F of fura-2 at saturating Ca (380 nm)

= Sf2 Cf / Sb2 Cb

Ca = Kd x [(R – Rmin)/(Rmax – R)] x (Sf2/Sb2)

Peak at 340 nm, Ca under saturating conc.Ca-bound

Peak at 365nm, Ca = 0unbound

340 380360 (isosbestic point)

Excitation 340 and 380 nmEmission 505 nm

JBC (1985) 260, 3440-3450

Calcium sensors

Ratiometric dyes

Non-ratiometric dyes

Nat. Rev. MCB (2003) 4, 579

Protein-based calcium sensors

Nat. Rev. MCB (2003) 4, 579

aequorin

cameleon

camgaroo

pericam

coelenterazine

Tyr145 146

Nat. Rev. MCB (2003) 4, 579

Nat. Rev. MCB (2003) 4, 579

Nat. Rev. MCB (2003) 4, 579