1

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

7

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