FK506结合蛋白 12.6与心肌细胞钙动员

辛洪波 博士南昌大学转化医学研究院

中国，南昌

Ca2+ signaling (cycling) network

ADP ribosylcyclase

Sphingosinekinase

Ca2+ (1 M )

IP3R,RyRNAADPR

SCaMPER

Plasm a m em brane Ca2+ channelsCa2+ (1 m M )

+/ Ca

Ca2+ sensitive processes

ContractionProliferationFertilizationLearning and m em oryCrosstalking w ith other s ignaling pathw aysm em brane excitationSecretionM etabolismVesicle trafficking

M itochondrial enzym esATP synthesisStero id synthesis

Caspases: apoptosis

PLC

PLC

Sphingosine

NADNADP

Ptdins(4,5)P 2

RTK R VR R

Stimulus

Na+ /C a2+

Ca2+

Ca2+

PMCA SERCA

ER/SR

Ca2+

~100 M

Buffers/chaperones

Ca2+ buffers

Na+

Cytoplasm

Plasm aMem brane

2+

Cytochrome C

Na+

PTP

IP3cADPRNAADP

S1P

Ca2+ (1 M )IP3R,RyRNAADPR

SCaMPER

M itochondrion

Na+/ Ca2+

exchanger

C a 2+ buffers

P LC

G

Na+/C a2+

exchanger

C a2+

R

Intracellular Ca2+ release channels (ICRCs)

Inositol-1,4,5-trisphosphate receptors (IP3Rs)

IP3R1~5: IP3Ryanodine receptors (RyRs)RyR1: skeletal muscleRyR2: cardiomyocytes, brain and cells: cADPRRyR3: ubiquitous Nicotinic acid adenine dinucleotide phosphate receptor (NAADPR)Sphingolipid Ca2+ release-mediated protein of the ER (SCaMPER): S1P

FK506, FK506 binding proteins (FKBPs) and ryanodine receptors

(RyRs)

FK506 is a potent immnuosuppressive drug to prevent organ graft rejection and to cure immune diseases.

The mechanism is involved in inhibition of calcineurin by way of binding to FK506 binding proteins (FKBPs), a family of related intracellular FK506 receptors including FKBP12 and FKBP12.6.

FKBP12 and FKBP12.6 are associated with RyR1 in skeletal muscle and RyR2 in cardiomyocytes, respectively.

FKBP12 is tightly associated with RyR1 in skeletal muscle

RyR1

FKBP12?

1. FKBP12 is co-purified with RyR1 3. RyR1 is immunopreciptated by anti-FKBP12 antibody

2. Skeletal muscle FKBP12 cDNA was isolatedusing responding synthetic oligonucleotides of the amino acid sequence of the co-purified 12 kDa protein with RyR1

4. FKBP12 is co-localized with terminal Cisternae

25 50 100 25 50 100 5 10 g

FKBP12

Jayaraman T et al (1992). JBC. 267, 9474-77

Co-localization of FKBP12, calmodulin and RyR1

Wagenknecht T et al. J Biol Chem. 272:32463-71, 1997.Sharma MR et al. J Biol Chem. 273:18429-34, 1998.Sharma MR et al. J Biol Chem. 275:9485-91, 2000.

FKBP12.6 selectively binds to cardiac RyR2

Timerman et al. (1994) BBRC, 198:701-706;Lam E et al. (1995) JBC, 270, 26511-522

Time (min)

Xin HB et al (1995). BBRC. 214, 263-270Timerman et al (1996). JBC. 271, 20385-91

Skeletal musc le

U biquitous

FK B P 12.6

FK B P 12 RyR1

RyR2

RyR3

Heart, B rainSmooth musc leP anc reatic b-cells

Xin HB et al (1999). JBC. 274, 15315-19.

Gln31, Asn32 and Phe59 residues are responsible for selective binding of FKBP12.6 to RyR2

Xin HB et al (1999). JBC. 274, 15315-19.

The function of skeletal muscle RyR1 is modulated by both

FKBP12 and FKBP12.6 in vitro

Timerman et al (1996). JBC. 271, 20385-91; Barg S et al (1997). AJP. 272,C1726-33

FKBP12.6 fails to modulate cardiac RyR2 function in vitro

Generation of FKBP12.6 knockout mice

Xin HB et al (2002). Nature. 416, 334-338

Increased CICR gain in cardiomyocytes from FKBP12.6-/-

mice

Altered Ca2+ sparks in cardiomyocytes from FKBP12.6-/- mice

Sex-dependent cardiac hypertrophy in

FKBP12.6-/- mice

Cardiac hypertrophy in male FBP12.6-/- mice can be rescued by heart-specific

FKBP12.6 transgenic mice

pM H C

mF K B P 12.6

B GH

A . Cons truc t of mouse FK B P 12.6 transgene

Unpublished data

A

B

C D

0

20

40

60

80

100

100

10

1010

0

101

110

00

10

010

0

001

010

1000

100

000

F K 506F K BP 12F K BP 12.6

Conc entration ( M )

Ca

lcin

eu

rin

ac

tiv

ity

in

cru

de

e

xtr

ac

t in

he

art

(%

of

co

ntr

ol)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18p>0.05p<0.01

Male FemaleWT, n=7 K O, n=9K O, n=19WT, n=19

Cal

cine

uri

n ac

tivi

ty i

n he

art

(m

U/m

g pr

ote

in)

CN-A

M F M F

FKBP12.6+/+ FKBP12.6-/-

Determination of CN contents in crude Extracts in heart by IP

Alteration of calcineurin activity in heart from FKBP12.6-/- mice

FKBP12.6FKBP12

A. Comparison of FKBP12.6 expression in heart from cardiac-specific FKBP12.6 transgenic mice (TG) and wild type mice (WT)

FKBP12.6-TG

1 2 3 4 5 6

WT

B. Comparison of calcineurin expression in heart from cardiac-specific FKBP12.6 transgenic mice (TG) and wild type mice (WT)

1 2 3 4 5

WT TG

CN-A

C

Measurement of calcineurin activity in heart from cardiac-specific

FKBP12.6 transgenic mice

0.5

10 mV

-70 mV

-42 mV

0.5

3

20 mµ

100 ms

control

cADPR

control

cADPR

A

KO

WT

C ontro lc A DP R

0

0

10

10

20

20

30

30

40

40

50

50

60

70

WT WTK O K O

**

**

Nu

mb

er

of

Ca

Sp

ark

s(1

0 s

ec

)2

+ -1

1

2

0

1

2

3

4

5

WT WTK O K O

17

46

1716

***

F/F

0

FW

HM

(M

)

Ris

e T

ime

(m

s)

De

ca

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alf

Tim

e (

ms

)

75

50

25

0

WT (n=9) K O (n=10)

Nu

mb

er

of

Sp

ark

s

0 5 10T ime (min)

0

5

4

3

2

1WT (n=9)KO (n=10)

cADPR (100 nM)

B

FKBP12.6 plays an essential role in cADPR-induced Ca2+ release from SR

in cardiomyocytes

Zhang X et al (2009). Cardiovas Res 84:253-62

GST/FKBP12 + + + FK506(20 M) + cADPR(20 M) +

GST/FKBP12.6 + + + FK506(20 M) + cADPR(20 M) +

RyR1 RyR2

GST/FKBP12 + + + GST/FKBP12.6 + + +FK506(20 M) + + cADPR(20 M) + +

Cyclic ADPR selectively blocks the association of FKBP12.6 with RyR2

Dissociation of FKBP12.6 following RyR2 phosphorylation by PKA is not

essential in b-adrenergic stimulation-induced CICR

1

2

3

0

1

2

3

4

5

0

10

20

30

40

50

60

0

20

40

60

80

51

126 70

55

F/F

0R

ise t

ime

(m

s)

Half

tim

e d

eca

y (m

s)

FW

HM

(M

)µ

WT WT

WTWT

KO KO

KOKO

Ba2+

Ba +ISO2+

******

***

***

***

***

*** ***

C

Elevation of Ca2+ release stimulated with Iso is not resulted by

phosphorylation of phospholamban in myocytes of FKBP12.6-/- mice

Contro l2D122D12+I SO

1.0

1.5

0

1

2

3

0

10

20

30

40

50

F/F

0

FW

HM

(m

M)

Ris

ing

Tim

e (

ms)

*

n=23

n=56

n=21

**

*

n=54

n=88

n=

18

Nu

mb

er

of

Ca

Sp

ark

(1

0 S

ec

)2+

-1 n=9

Wildtype F K B P 12.6-/-

0

5

10

15

20

n=10

**

0

10

20

30

40

50

*

De

ca

y H

alf

Tim

e (

ms

)

Wildtype WildtypeF K B P 12.6-/- F K B P 12.6-/-

Wildtype WildtypeF K B P 12.6-/- F K B P 12.6-/-

D

Effects of beta-adrenergic stimulation with isoproterenol (Iso) on isolated cardiac papillary muscle

from mice

Alternations of plasma sex steroid hormones in FKBP12.6

deficient mice

FKBP12.6 -/-wt

0

1

2

3

4

0

5

10

15

20

25

Tes

tost

ero

ne

leve

l (n

g/m

l)

Est

rad

iol

leve

l (p

g/m

l)

Male Female Male Female

**

*

**

（ Unpublished data ）

HW

/BW

(m

g/g

) R

atio

0

1

2

3

4

5

6

7

wt

FKBP12.6 -/-

Castrated

FKBP12.6 -/-

**

**

Role of castration in cardiac hypertrophy of FKBP12.6

deficient male mice

Summary

我们发现 FKBP12.6 可选择性地与心脏 RyR2 结合，而这种结合是由FKBP12.6 的三个氨基酸 (Gln31, Asn32 and Phe59) 决定的。敲除 FKBP12.6 基因尽管可导致雌雄小鼠心肌细胞的钙释放异常，但仅成年雄性小鼠出现心肌肥大。给予雌性 FKBP12.6 基因敲除鼠 Tamoxifen 也可诱导心肌肥大。FKBP12.6 基因敲除导致的心肌肥大可被心脏特异性过表达 FKBP12.6 转基因鼠矫正。

FKBP12.6 在 cADPR 诱导的心肌细胞钙释放过程中起关键作用。

PKA 介导 RyR2 磷酸化所致的 FKBP12.6 与 RyR2 的解离并非 - 肾上腺能受体激活诱导的“钙诱导的钙释放（ CICR ）”的主要原因 .

Calcineurin 信号通路在 FKBP12.6 基因敲除所致雄性成年鼠的心肌肥大的作用还有待进一步阐明。