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Why Calcium?
Double positive charge provides increased affinity for negatively charged proteinsbut lower affinity than larger divalent cations such as Cu, Zn, or Mn. Thecoordination chemistry of Ca is higher and more flexible than for Mg.
The fact that Ca complexes with inorganic compounds and to proteins suggeststhat the maintenance of low [Ca] intracellularly would require less energy than forother cations. The maintenance of a large transmembrane gradient is critical fora second messanger ion.
This large transmembrane gradient provides the signal-to-noise ratio required forefficient signal transduction.
Resting intracellular [Ca] is ~100 nM versus mM extracellular [Ca] or aconcentration gradient of ~10,000.
VGCC
ROC
SOC
TRP
Stretch receptor
CNGnAChR
NMDA, AMPA rec
VR/temp rec
IP3R
RyR
VDAC
SERCA
SERCA
NCE
PMCA
mM [Ca]
100 nM [Ca]
mM [Ca]
mM [Ca]
2
Sarcoplasmic Reticulum (SR) / T Tubule System
3
4
Twitch
Summation
Tetanus
5
Role of Ca++ in contraction
6
SR SR
T-tubule T-tubuleA B
SRMuscle contraction
T-tubuleVDCC
RyRFKBP12/12.6
Ca release
C RyR Accessory proteins�FKBP12/12.6�Kinases�Phosphatases�Adaptor/anchoring/targeting proteins�Sorcin�S100s�Triadin�Junctin�Annexin VI�Calmodulin
VDCC
RyR1RyR2
vertebrate skeletal muscle contraction
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Excitation-contraction coupling
Ryanodine receptors
P r o pe r tie s o f C alci u m R e lease Ch an n els
R ya n o d ine R e cep to r s
R y R 1 R y R 2 R y R 3Siz e (a mi no a ci d s)
of m ono m er 5,0 3 7 4,9 7 0 4,8 7 0Siz e (d a l ton s ) o f
m o nome r ~ 565, 0 00 ~ 560, 0 00 ~ 560, 0 00S ed ime n tat ionc oef f icie nt of
tet rame r30 S 30 S 30 S
S to ic h iom e tr y o fF KB P /R yR 4 4 4
Si ng le c h a nne lc on d u c tan c ein C a 5 0 m M
~ 120 p S ~ 120 p S ~ 100 p S
Si ng le c h a nne lc on d u c tan c e
in Cs 2 5 0 m M~ 540 p S ~ 540 p S ?
E n d og e n o usm od u la to r s
µ M Ca a c t iv ate s y e s y e s y e sm M C a inh ib its y e s y e s y e sm M Mg in h ib its y e s y e s y e s
ki na s e s y e s y e s ?ph os phata ses y e s y e s ?
DH P R in tera c t io n y e s ? ?c a lm o du li n y e s y e s y e s
ad e n inenu cl e o t ide s y e s y e s y e s
M g A T P y e s y e s y e sN O y e s y e s ?
8
Pharmacology of Ryanodine Receptors
RyR1 RyR2 RyR3 Site of actionXanthines(caffeine) activates activates activates Ca activation sites
Ryanodine/ryanoids
subconductancestate
subconductancestate
subconductance state
carboxyterminus
ruthenium red inhibits inhibits inhibitsCa binding
site(s)/channelpore
Antraquinones(doxorubicin) activates activates activates ?
FK506 &rapamycin activates activates activates FKBP12/12.6Purinergicagonists/
antagonists(adenosine)
activates activates activates ATP binding si tes
Calmodulinantagonist
inhibits inhibits inhibits calmodulin bindingsite
Local anesthetics(tetracaine) inhibits inhibits inhibits ?Dantrolene inhibits inhibits ? ?
Phenol derivatives(4-chloro-m-
cresol)activates activates ? ?
NO generatingcompounds Activates/inhibits Activates/inhibits ? ?
RyR
SRMembrane
Cytosol
Lumen
NH2
CO2H5000
4000
3000
2000
1000 500
4500
3500
1500
2500FKBPPP2A
PP1
mAKAPRII-PKA PKA/CamKII
PO4 siteCalmodulin
Rya
nodi
ne
P
Ca-Inactivation
Ca-Activation
GIG
DHPR/RyR1
MH/CCD
MH/CCD
MH/CCD
M1 M2 M3 M4
RyR2
9
Voltage-gated ion channels
10
11
12
RyR2
SR
Lumen
Ca2+
Ca2+
P
P
P
β1AR β2AR
αβγ
AC
cAMP
Regulation of EC coupling by adrenergicsignaling --“fight or flight”
RyR2 macromolecular complex
RyR macromolecular complexes are held together by
leucine/isoleucine zippers
13
RyR2/calcium release channel phosphorylated by addition of cAMP alone
PKAphosphorylation activates RyR2
and inducessubconductance
states
14
Cytosolic[Ca]
RyR
2 op
en p
roba
bilit
y
ECC gainCa transientcontractility
DADs - SCD[Ca]
Time
Iso CHF
15
activatesinhibitsHeparininhibitsNoneRR
biphasicIP3R1 -biphasicIP3R2/3 - opens
Ca2+
ActivatesInhibitsCaffeine (5 mM)
Locks open/closes
NoneRyanodineNoneActivatesIP3 ++++++Neurons++++Smooth m.++++Skel muscle
RyRIP3R
Excitation-secretion coupling
Inositol 1,4,5-trisphosphatereceptors
16
IP3 Receptors
IP3R1 IP3R2 IP3R3Size (amino acids)
of monomer 2,749 2,691 2,685Size (daltons) of
monomer 313,000 ~300,000 ~300,000Stoichiometry of
FKBP/RyR 4 4 ?
Endogenousmodulators
IP3 activates activates activatesnM Ca activates yes yes yesµM Ca inhibits yes yes noATP <2 mM activates activates activatesATP >2 mM inhibits inhibits inhibits
tyrosine kinases activates ? ?phosphatases ? ? ?
Pharmacology of IP3 Receptors
IP3R1 IP3R2 IP3R3 Site of action(caffeine) inhibits inhibits inhibits Ca activation sitesheparin inhibits inhibits inhibits ?2-APB inhibits inhibits inhibits ?
xestospongin inhibits inhibits inhibits ?
IP3 signaling
17
Stable transfectant Jurkat T cells do not
express IP3R1
IP3R1
18
Activation of IP3-gated Ca2+ channel by fyn
19
Bcl-2
P
Bcl-2Bcl-2
Bcl-2
Rec
Growth Factor
Plasma Membrane
CytoplasmPIP2
PLC-γ
IP3ER Bad
P
Degradation
14-3-3
Bcl-xBad
Ca2+
IP3R
CalcineurinSERCA
Ca2+
Cytosolic TF
NuclearTF
Survival
Nucleus
Cell Death
DNADigestion
M M
Akt
Cytoskeletal Breakdown
Cytochrome CCaspases
CAD
Bad
Survival
+ X ?
Calcium signaling during apoptosis
VGCC
Stretch receptor
CNGnAChR
IP3R
RyR
VDAC
SERCA
SERCA
NCE
PMCA
mM [Ca]
100 nM [Ca]
mM [Ca]
mM [Ca]
T-tubule
Sarcoplasmicreticulum
Endoplasmicreticulum
mitochondria
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