regulation and measurement of intracellular calcium may 12, 2006
TRANSCRIPT
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