molecular biology of ryanodinereceptors prof. christopher h. … · 2015. 12. 25. · ryanodine...

Molecular Biology of Ryanodine ReceptorsProf. Christopher H. George

The screen versions of these slides have full details of copyright and acknowledgements 1

1

Molecular Biology

of Ryanodine Receptors

Christopher H. George

Department of Cardiology,

School of Medicine, Cardiff University,

Cardiff, UK

2

I. Overview

Molecular cloning

Channel structure

II. RyR channel regulation

RyR:RyR interactions

Divalent cations

Phosphorylation

Protein-protein interactions

III. Genetic basis of RyR dysfunction

Skeletal muscle

Cardiac muscle

IV. Investigating the molecular basis of RyR-linked disease

3

I. Overview



4

Ryanodine receptors - an overview

Identification Electron-microscopy visualised as ‘foot’ structure

Purification 3H-ryanodine affinity

Size ~5000 aa

Structure Homo-tetramer / hetero-tetramer

Isoforms Three RyR1 skeletal muscle

RyR2 cardiac muscle and brain

RyR3 skeletal and smooth muscle

Distribution Widespread, particularly prominent in excitable tissue

Chromosomal location (gene size)

RyR1 : 19q13.1 (153.8kb)

RyR2 : 1q42.1-q43 (790.9kb)

RyR3 : 15q14-q15 (555.1kb)

Phenotype resulting from genetic ablation

RyR1 : Perinatal lethal

RyR2 : Embryonic lethal

RyR3 : Minor muscular and neurological dysfunction

5hRyR2 (4967aa; 14904bp)

Molecular cloning of human RyR214904bp

4967aa

6George et al. (2005) Cell. Biochem. Biophys 42; 197-222

More than just a Ca2+ channel: complexity of RyR function



7

RyR domain architecture

28nm

17nm

Cytoplasmic view Side-on view

Serysheva et al., (2005), J. Mol. Biol., 345, 427-431

Ludtke et al., (2005), Structure, 13, 1203-1211

Regulatory

‘scaffold’

Pore

8

II. RyR channel regulation

9Liu et al., (2004), J. Mol. Biol, 338: 533-545

Intrinsic lattice formation of RyR tetramers

Yin and Lai, (2000), Nat. Cell. Biol., 2: 669-671

Inter-RyR2 contact sites(divergent region 2)



10Marx et al., (2001) Circ Res 88: 1151-1158

“Coupled gating”: synchronous opening of multiple channels

O

O2

O

O2

O

O2

O3

11

Pharmacological Biochemical Proteins

Ryanodine Ca2+ / Mg2+ FK-506 binding proteins (FKBP)

Caffeine ATP L-type Ca2+ channels

4-chloro-m-cresol cADP ribose Calmodulin

Ruthenium red Phosphorylation Calcineurin (PP2B)

Tetracaine S-Nitrosylation Protein kinase A, C and G

FK506 / rapamycin Redox status Triadin / Junctin

Digoxin Protein phosphatases 1 and 2a

Imperatoxin Calsequestrin

Bastadins Sorcin

Regulators of RyR channel activity

12

Purified RyR: biophysical properties

Laver et al., (1997), J. Membr. Biol., 156: 213-229

Channel regulation by divalent cations

Ca2+

10 µmol/L

Ca2+ / Mg 2+



13Marx et al., (2000), Cell, 101: 365-376

Regulation by phosphorylation

C

O

C

O

C

O‘Substate’

Conductiv ity of the channel (pA)

14Bers, (2004), J. Mol. Cell Cardiol, 37: 417-429

Regulation by protein-protein interaction:the macromolecular complex

15

Building a picture of RyR domainlocalisation

RyR

RyR + inserted epitope

Images from Liu et al., (2004),J. Mol. Biol, 338: 533-545

RyR + accessory protein



16

• Common dimerizati on domain found in some DNA regulatory proteins

• Each half of a leucine zipper consists of a short alpha-helix

with a LEU residue at every seventh position

(commonly at the d position of the heptad repeat)

• 3.6 residues/tur n in an alpha-helix , this positions one LEU

at every second turn, just short of being exactly under each other

in the coil - leucines form the hydrophobic core of a coiled coil

Sticky patches: leucine zippers (LZ) and coiled-coil domains

17

Coiled-coil domains : stitching monomers together

18Marx et al., (2001), J. Cell. Biol., 154: 699-708

The role of LZ motifs in RyR protein-protein interaction



19

Cellular ‘superCellular ‘superCellular ‘superCellular ‘super----networks’ enable localised s ignal conetworks’ enable localised s ignal conetworks’ enable localised s ignal conetworks’ enable localised s ignal co----ordinationordinationordinationordination

Dodge-Kafka and Kapiloff, (2006), Eur. J. Cell. Biol,. 85: 593-602

LIF/gp130receptor

α-ARAdenylylcyclase

Gαq β γ γ β Gαs

β-AR

cAMP

PKA

Rap1

MEKK

MEK5

ERK5

P

5’ AMP

PDE4D3

Epac

Nesprin-1α

RyR

P

P

PmAKAP

Ca2+

CaNAβ

NFAT

NFATP

NFAT

Nuclearpore

Hypertrophic gene expression

CC

20Marks, A.R, et al., (2000), Cell 101: 365-376

B) Dissociation of PP1 / PP2A

Phosphorylation of RyR2 at Ser 2809

A) Hyper-adrenergic state: ↑ PKA activity

Dissociation of FKBP12.6

Abnormal Ca2+ release

Disruption of the RyR macromolecular complex: the molecular basis of heart failure?

Marks, A.R, 2003 J. Clin. Invest. 111: 597-600

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GTP

AC

cAMP

S2809

PKA

FKFK

CaCa Ca

Ca

CaCa Ca CaCa

Ca Ca

CaCa Ca

Ca

CaCa Ca CaCa Ca

Ca

CaCa

CaCa

FK

FK

FK

FK

!! Ca2+ !!

P (+ loss of PP1/P2A)

FKBP12.6 d issociation: a ‘unifying’ mechanism of pathological CaFKBP12.6 d issociation: a ‘unifying’ mechanism of pathological CaFKBP12.6 d issociation: a ‘unifying’ mechanism of pathological CaFKBP12.6 d issociation: a ‘unifying’ mechanism of pathological Ca2+2+2+2+ leak?leak?leak?leak?

ααααs

Adr

Adr

AdrAdr

Adr

Mechanism implicated in:Heart failure

Ventricular arrhythmiaAtrial fibrillation

Stress-induced arrhythmia

?

?

CaMKII?

?

? ?



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III. Genetic basis of RyR dysfunction

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Mutations in EC coupling: arrhythmogenic culprits

Bers (2002) Nature 415: 198-205

Cav1.2: Arrhythmia

RyR2: Arrhythmia

PLB: Cardiopathology

CSQ: Arrhythmia

Troponin: Cardiopathology

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The genetic basis of stressThe genetic basis of stressThe genetic basis of stressThe genetic basis of stress----induced VT : RyRinduced VT : RyRinduced VT : RyRinduced VT : RyR2 2 2 2 mutationsmutationsmutationsmutations

bVT

pVT

Clinically affected

gene carrier

Silent gene carrier

Non gene carrier

Unexplained sudden

cardiac death

Not tested

bVT

Priori et al., (2002),Circulation 106: 69-74

Affected male, affected female

(status determined by DNA analysis)

Non affected male, non affected female

(status determined by DNA analysis)Obligated carrier

Sudden death under age of 35

(age indicated in parenthesis),

no DNA sample available

Status unknown

Laitinen et al., (2001), Circulation 103: 485-490

SD



25

0

20

40

60

80

100

0

20

40

60

80

100

2000 2002 2003 2004 2005 2006 *

0

10

20

30

40

RyR2- mutation carriers

Mutations identified

Novel mutations (% )

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Laitinen et al., (2001) Circulation 103 485-490

R420W

Y2392C

Bauce et al., (2002) JACC 40 341-349

Laitinen et al., (2003) Eur. J. Hum. Genet. 11 888-891

N2386I

R176Q

L433PT2504M

Tiso et al., (2001) Hum. Mol. Genet. 10 189-194

Priori et al., (2002) Circulation 106 69-74

Bagattin et al., (2004) Clin. Chem. 50 1148-1155

Aizawa et al., (2005) Int. J. Cardiol. 99 343-345

Hasdemir et al., (2004) J. Cardiovasc. Electrophysiol. 15 729

Choi et al., (2004) Circulation 110 2119-2124

P466A

C3800F S4124T

A4556T

Ins EY @4657

Tester et al., (2005) Heart Rhythm 2 1099-1105

Tester et al., (2005) Mayo Clin. Proc .80 596-600

A77V

D’Amati et al., (2005) Hum. Pathol 36 761-767

Postma et al., (2005) J. Med. Genet. 42 863-870

Creighton et al., (2006) J. Mol. Diagnostics . 8 62-67

N4097S

E4146K

T4158P

Tester et al., (2004) Mayo Clin. Proc.79 1380-1384

R169Q

Hseuh et al., (2006) Int. J. Cardiol. 108 276-278

CPVT III

CPVT IV

R2401H/L

V2475F

F2331S

Ryanodine receptor mutations

4967aa2000 3000 4000

P2328S

Q4201R

V4653F

S2246LR2474S

N4104K/I

R4497C

Priori et al., (2001) Circulation 103 196-200

V2306I

P4902L/S

R4959Q

E2311D

G3946S

L3778F

V4771I

N4895D

I4867M

A4860G

E4950K

M4504IA2387P/TV488

0A

A4607P

L2534V

F4499C

I4848VA4510T

G4671RA2403T

E1724K A2254V

A2394G

F4020LE4076K

H4108Q/NH4762P

G4662S

10000

P164S

R414L/C

I419F

CPVT I

CPVT II

27

Mutation clustering is not linked to genotype-phenotype correlation

Domain Locus

(amino acids)

Mutations

(% of total)

De novo

mutations(% ) a

Inherited

mutations(% ) a

Age

onset (yr) b

Male:female bias (% )

I 77-466 17.4 ND 42 16.7 ± 9.5 55:45

II 2246-2534 27.5 31 31 16.9 ± 11.1 48:52

III 3778-4201 23.2 25 25 21.0 ± 21.8 46:54

IV 4497-4959 31.9 14 41 22.1 ± 15.314 41 22.1 ± 15.3 44:56



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CPVT I

CPVT II

CPVT III

CPVT IV

Amino acid changes exhibit locus specificity

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Mutation loci: roles in Ca2+ sensitivity and intra-RyR2 interaction

EC coupling

Domain interaction Domain interaction

Agonist sensitivity

Ca2+ -regulation

FKBP12.6 interaction

Domain interaction

Ca2+ sensitivity Ca2+ regulation

Ca2+ sensor

Channel modulation

Agonist sensitivity

FKBP12.6 interaction FKBP12.6 interaction

Ca2+ binding

CaM-like domain

CaM- regulation

ER retention

Snapin interaction

Ca2+ regulation

Pore-forming ‘P-loop’

Ca2+-binding

EC coupling

Domain interaction

Oligomerisation

496740003000200010000

77 466 2246 2534 3778 4201 4497 4959

CPVT-I CPVT-II CPVT-III CPVT-IV

Oxidoreductase-like domain

EC coupling

30

IV. Investigating the molecular basis

of RyR-linked diseases



31

Loke and MacLennan (1998), Am. J. Med.,

104: 470-486

The basis of skeletal muscle pathologies

32

Defective skeletal RyR regulation : Malignant HyperthermiaMolecular basis of malignant hyperthermia

33Expression (mammalian, X. Laevis, insect cells)

*

The use of recombinant systems

Genomic DNAcDNA library

PromoterResistance marker

PolyA tail

** = mutation



34

Experimental systems for investigating RyRExperimental systems for investigating RyRExperimental systems for investigating RyRExperimental systems for investigating RyR2 2 2 2 function: HLfunction: HLfunction: HLfunction: HL----1 1 1 1 cellscellscellscells

35

HL-1 cardiomyocytes: a functional syncitium

36

Fluorescent proteins to monitor intracellular expressionFluorescent proteins to monitor intracellular expressionFluorescent proteins to monitor intracellular expressionFluorescent proteins to monitor intracellular expression

Aequorea victoria Green fluorescent protein (GFP)

Discosoma sp. Red fluorescent protein (DsRed)



37

Visualising RyR2 in cardiomyocytes

hRyR2

38

Mechanistic defects in RyR2 mutants

Increased luminal Ca2+ sensitiv ity(SOICR)

Decreased Ca2+-dependent inactiv ation

Decreased Mg2+-dependent inhibition

‘Hyperphosphorylation’ / altered FKBP12.6 interaction

FKBP12.6-independent

Abnormal Ca2+ release

Increased cytoplasmic Ca2+ sensitiv ity(SPCR)

Abnormaliti es of cellular ‘sensing’

39

Ca2+ release

dysfunction

Molecular aspects of RyR2-linked arrhythmia

Abnormal

‘sensing’ of cellular

environment

‘Trigger’

Transduction

‘Transduction’-

How is abnormal RyR2 cellular ‘sensing’

converted into defective Ca2+ release ?



40Kobayashi et al., (2004) Biochem J .380: 561-569

Domain ‘unzipping’ and channel instability

41

Telling proteins apart……..

Merge

eGFP DsRed

42

Norm

alized

absorb

ance/e

missio

n

Fluorescence resonance energy transfer (FRET):

utilising eGFP and DsRed

Em: 583nm

DsRed

Energ

y

eGFP

Em: 507nm

Ex: 488nm

<100Å

Em: 507nm

FRET Ex: 558nm



43

Identification of the interacting (IIdentification of the interacting (IIdentification of the interacting (IIdentification of the interacting (I ----) domain: a molecular ‘hinge’) domain: a molecular ‘hinge’) domain: a molecular ‘hinge’) domain: a molecular ‘hinge’

George et al., (2004), Mol. Biol. Cell, 15: 2627-2738

dsRed

eGFP

I-domain

Liu et al., (2002) J. Biol.

Chem 277: 46712-46719

FRET

dsRed

eGFP

I-domain

3722-4610 aaL3778F S4124TC3800F E4146KG3946S T4158PF4020L Q4201R

E4076K R4497CN4097S F4499CN4104K M4504I

N4104I A4510TH4108Q A4556TH4108N A4607P

FRETCytosol

Lumen

44

The link between channel opening and Ca2+

release: mutations destabilise RyR2

George et al., (2006) Circ. Res. 98: 88-97

45

Summary

• RyR are massive, complex multifunc tional Ca2+ release channels

• RyR decode cellular environment to elicit appropri ate Ca2+ release –

act as ‘signal integrators’

• Size and complexity hinders characterisati on of structure and function

• Channel dysfunction causes skeletal muscle and cardiac muscle diseases,

and arises from defective ‘sensing’ of the cellular environment

• To date, characterisati on of the molecular basis of RyR dysfuncti on

implicates defects in diverse mechanisms



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molecular biology of ryanodinereceptors prof. christopher h. … · 2015. 12. 25. · ryanodine...

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