skeletal muscle (and a little cardiac) excitation-contraction coupling ed balog applied physiology...
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Skeletal Muscle (and a little cardiac) Excitation-Contraction Coupling
Ed BalogApplied Physiology
555 14th St NW Rm 1303ed.balog@ap.gatech.edu
[Ca2+] 1 mM
Endo/SarcoplasmicReticulum
Ca2+ATP
ADP + Pi
Ca2+
ATP
AD
P +
Pi
Ca2+
Na2+[Ca2+] 100 nM
[Ca2+] 1-2 mM
IP3RRyR
Intracellular Calcium Signaling
Fertilization Stimulus-secretion
coupling Metabolism Muscle Contraction
Learning and Memory Gene regulation Cell death Immune cell activation
Berridge et al., Nature Rev. Mol. Cell Biol. 1:11, 2000.
Muscle Contraction Summary
Buffers
Mitochondria
Troponin& other Ca2+-
binding proteins
Ca2+
Ca2+ Movement in Muscle
SR
Cytoplasm
Flux that increases cytoplasmic Ca2+
Flux that decreases cytoplasmic Ca2+
Determinates of Contractile Force
1. Maximal Calcium Activated Force
2. Calcium Sensitivity
3. Calcium Delivered to Contractile Proteins
Excitation-Contraction (EC) Coupling
The process linking depolarization of the muscle cell surface membrane to the release of Ca2+ from the sarcoplasmic reticulum (SR).
EC coupling controls the [Ca2+] within the muscle cell; [Ca2+] controls force.
Skeletal Muscle Membrane System
Sarcolemma
TriadTransverse Tubule
SarcoplasmicReticulum
Hypotheses:1. Depolarization of the sarcoplasmic reticulum.
2. Chemical messenger from the transverse-tubules to the sarcoplasmic reticulum.
3. Ca2+-induced Ca2+ release (CICR).
4. Physical link between the transverse-tubules and the sarcoplasmic reticulum. This hypothesis also became to be known as depolarization-induced Ca2+ release (DICR)
Calcium-Induced Calcium Release?
Small amount of Ca2+entering the cell from the extracellular fluid triggers much larger SR Ca2+release.
Primary mechanism of EC coupling in the heart.
Skeletal muscle SR Ca2+ release can be triggered via CICR under experimental conditions.
But – Contraction can be elicited in the absence of extracellular Ca2+.
Physical Link?
Primary mechanism in skeletal muscle.
Electronmicrographs show electron-dense structures in triadic junction, called “feet” linking t-tubules and SR.
Dysgenic mouse muscle lacking “feet” also lack EC coupling.
Excitation-contraction coupling: a tale of two Ca2+ channels
aka “Ca2+ release channel” or “junctional foot protein”
Homotetramer; 2 million daltons.
aka “DHPR”, voltage sensor” or “L-type channel”
Heteromultimeric protein w/ 5 subunits.
Origin of charge movement & L-type Ca2+ current.
Ryanodine Receptor
Dihydropyridine Receptor
RyR tetramer
DHPR tetrad
T-tubule membrane
SR membrane
Lamb and Stephenson, Dept of Zoology, LaTrobe University
High K+ High Na+
Posterino, Proc Australian Physiol Pharmacol Soc 32: 28, 2001.
Mechanically Peeled Muscle Fiber
Methods to Study Excitation-Contraction Coupling
Fig. 5. Ca2+ sparks activated by membrane depolarization in skeletal muscle. The figure shows line scan images of sparks activated by small depolarizations (indicated at top) from a holding potential of −90 mV. For the pulses to −70 mV individual, randomly activated sparks are evident during the depolarization in the images and in the fluorescence records from individual, identified triads (below). The lowermost record in each column shows the average elevation of fluorescence from the entire image. The rightmost panel shows a depolarization to −60 mV during which the high frequency of Ca2+ sparks has resulted in a much larger elevation of fluorescence within the fiber, precluding the observation of individual sparks.
Klein and Schneider Prog Biophys Mol Biol 92:308, 2006
[3H]Ryanodine Binding
Ryanodine binds the open channel with high affinity and specificity.
Binding reflects the open state of the channel. Ryanodine
Single Channel Recording in a Planar Lipid Bilayer
LipidBilayer
Channels
Some of the Whole-Cell Measurable Events of Excitation-Contraction Coupling
DHPR (L-type Ca2+ Channel)
Member of voltage-gated ion channel superfamily, which also includes Na+ & K+ channels.
Pentamer; α1 subunit forms pore.
Cav1.1 : skeletal muscle isoform; 1873 aa in humans
Cav1.2: cardiac isoform; 2169 aa in humans
NC
βN
C
+++
+++
+++
+++
α1
N C
γ
α2
N
C
II-III loop and β-subunitvital for skeletal muscle ECC
δC
N
cytoplasm
t-tubulelumen
S
S
10 μA/μF
25 ms
DHPR electrical signals:L-type calcium current Cardiac current is larger
and is fully activated by a ventricular action potential. Ca2+ influx via the cardiac DHPR activates RyR2 via Ca2+ induced Ca2+ release (CICR).
Skeletal muscle current is smaller and is barely activated during a skeletal muscle action potential. The current is not required for contraction.
Cardiac
Skeletal Muscle
Wang et al Biophys J 77:2709, 1999Caputo & de Bolanos J Physiol 289: 175, 1979.
Skeletal Muscle Contraction Does Not Require the Entry of Extracellular Calcium
2.5 mM Ca2+ 0 Ca2+ 2.5 mM Ca2+
1. Voltage dependence of Ica and contraction differ.
2. Ica activation is too slow to contribute Ca entry during an action potential.
3. Skeletal muscle can contract in the absence of extracellular Ca.
Dulhunty and Gage J Physiol 399:63, 1988
Tetanus
Twitch
DHPR electrical signals: Charge movement
Arises from movement of charged amino acids across membrane electric field.
Similar to ion channel gating currents, but larger and slower.
Required for skeletal muscle contraction.
t-tubulemembrane
++
+
++
+ΔV
DHPRtetrad
+
Charge Movement & Ca2+ Release
Top: voltage dependence of skeletal muscle contraction.
A: Intracellular calcium transients recorded from a muscle fiber.
B: T-tubule charge movement records from the same fiber.
Below: Correlation between Charge movement and Ca2+ release rate.
Caputo & de Bolanos J Physiol 289: 175, 1979.
Ryanodine Receptor (RyR)
The open channel binds Ryanodine, an alkaloid derived from the South American plant Ryania speciosa.
Member of intracellular Ca2+ channel family, includes IP3 receptor. Largest known ion channel. Three isoforms: RyR1 (skeletal), RyR2 (cardiac), RyR3 (wide cellular
distribution, low abundance).
Sarcolemma
TriadTransverse Tubule
SarcoplasmicReticulum
Calcium Dependence of RyR1 and RyR2
High affinity calcium binding site – Activates channel when bound. Calcium selective (KCa/KMg ~100) .
Low affinity calcium binding site – Inhibits channel when bound. Relatively unselective for divalent cations (KCa/KMg ~1) .
A IAL
A
I
ALSR lumenal calcium binding site – Activates channel when bound.
Song et al Prog Biophys Mol Biol. 105:145, 2012.
RyR Macromolecular Complex
RyR Channel Modulators (Partial List)
ExogenousCaffeine
RyanodineRuthenium Red
Volatile AnestheticsDepolarizing Muscle
RelaxantsOxidizing/reducing agents
Local Anesthetics4-chloro-m-cresol
Dantrolene
EndogenousAdenine Nucleotides
CalmodulinMg2+
H+
Inorganic PhosphateDihydropyridine Receptor
FKBP12/12.6Reactive Oxygen Species
Nitric Oxide
Arrangement of DHPR and RyR1
4 DHPRs per coupled RyR
RyR1
α1
β
α2
γδ
SR Membrane
T-tubule Membrane
DHPRs
Junctophilin
Serysheva et al PNAS 99:10370, 2002.
RyR1
DHPR tetrads
ToadfishSwim Bladder
(very fast)
MammalianFast-twitch
Muscle
MammalianSlow-twitch
Muscle
DHPR:RyR Arrangement
and Ratio Varies with
Muscle Fiber Type
MammalianCardiac Muscle
RyR2
Individual DHPRs
How are uncoupled skeletal muscle ryanodine receptor
channels opened?
+ + +
+ + +
+ + +
+ + +
+ + +
Direct Coupling Ca2+-Induced Ca2+ Release
Comparison of Cardiac and Skeletal Muscle Excitation-Contraction Coupling
Cardiac Muscle:Calcium-InducedCalcium Release
Skeletal Muscle: Mechanical Coupling
DHPR mediates Ca2+ influx, Ca2+ binds to and activates the underlying RyR.
Charge movement within DHPR and subsequent conformational change activates RyR via direct physical interaction.
Ca2+
++ +++ ++ V
DHPR tetrad(α1s)
RyR1 Ca2+
Ca2+DHPR(α1c)
+
+V t-tubule
RyR2
SR
Two Forms of Ca2+ Entry in Skeletal Muscle
Store-Operated Calcium Entry (SOCE)
Requires depletion of the internal stores & has been best characterized in non-excitable cells.
Requires STIM1 and ORAI Significant SR Ca2+ depletion
required to reach activation threshold for SOCE only achieved during prolonged bouts of ECC.
SOCE is not responsible for refilling the SR during periods of fiber quiescence.
Excitation-Coupled Calcium Entry (ECCE)
Activated following prolonged membrane depolarization
Independent of the calcium stores.
Requires functioning L-type channel and RYR1, but molecular identity of the pore remains undefined although it is likely to involve the L-channel.
Store-Operated Ca2+ Channels
Lewis Nature 446: 284, 2007.
Calcium Transporters in Muscle
SERCA: Sarco/Endoplasmic Reticulum Calcium ATPasePMCA:Plasma Membrane Calcium ATPaseNCX:Na/Ca ExchangeMCU:Mitochondrial Uniporter
SERCA: Sarco/Endoplasmic Reticulum Calcium ATPase
• Encoded by 3 mammalian genes• ~1000 amino acids• 10 TM helices• Located in intracellular organelles: ER
and SR• 3 Cytoplasmic domains (A,N,P)• 2 Ca2+ transported per ATP hydrolyzed• Activity regulated by phospholamban and
sarcolipin
SERCA GenesSERCA1 – Expressed in fast-twitch skeletal muscle. Two splice variants.
SERCA1a in adult fast-twitch skeletal muscle.SERCA1b in embryonic skeletal muscle.
SERCA2 – Expressed in cardiac and slow-twitch skeletal muscle. Three splice variants.
SERCA2a in cardiac and slow-twitch skeletal muscle.SERCA2b low levels in most tissues (“house-keeper”)SERCA2c in embryonic heart cells and mesenchymal stem cells (give rise to muscle and bone)
SERCA3 – Expressed in smooth muscle. Five splice variants.In smooth muscle, blood, and neural cells. Co-expressed with SERCA2b (and others).
Why 3 genes with multiple splice variants?Tune enzyme activity (calcium affinity & maximal velocity) to cell type, ligand sites to modulate activity, binding sites for regulators, other unknown reasons.
PMCA:Plasma Membrane Ca2+-ATPase
• Encoded by 4 mammalian genes• ~1300 amino acids• 10 TM helices• Located in surface and t-tubule
membranes• 3 Cytoplasmic domains (A,N,P)• 1 Ca2+ transported out of cell per ATP
hydrolyzed• Activity regulated by calmodulin
Brini et al. FEBS J 280:5385, 2013
NCX:Sodium-Calcium Exchanger
• Encoded by 3 mammalian genes• ~950 amino acids• 9 TM helices• Located in surface and t-tubule
membranes• 1 Ca2+ transported out of cell per 3 Na+
into cell
• Driven by Na+ potential (Em-ENa)
• Reverses mode during depolarization Red = CalciumGreen = Sodium
MCU:Mitochondrial Calcium Uniporter
• A complex of proteins located in mitochondrial inner membrane.
• Facilitated diffusion of Ca into matrix.
• Driven by large electronegative potential (-180 mV).
Marchi & Pinton, in press J Physiol 2013
Ryanodine Receptor Diseases
RyR1
Malignant Hyperthermia
Central Core Disease
RyR2
Catecholaminergic Polymorphic Ventricular Tachycardia
Arrhythmogenic Right Ventricular Dysplasia
RyR3
None Identified, Yet
Malignant HyperthermiaAn autosomal dominant pharmacogenetic disease characterized
by an unusual metabolic reaction to volatile anesthetics and depolarizing muscle relaxants.
Symptoms include:HypercapniaCyanosisTachycardiaMuscle rigidityRhabdomyolysisHyperthermia
Incidence: 1 in 15,000 anaesthetized children 1 in 50,000 anaesthetized adults
With the introduction of dantrolene the mortality rate has been reduced from ~80% to current ~5%.
About 50% of cases linked to RyR1 mutations.
A1832GG2060CV2117LD2129ER2163CR2163H/PV2168MA2200VT2206M/RV2210FV2212AV2214IV2280II2321VR2336HN2342SE2344DV2346ME2348GA2350T
R367LR401C/G/HI403MQ474HY522S/CR530HR533C/HR533HR552WR614C/LS846LR1043CR1140CS1342GQ1589PP1592LS1728P/FM1729RP1787LM1814K
L13RC35RR 44C/HD60NQ155KR156KE160GR163C/LG165RD166N/GR177CY178CG215EV218IM226KD227VG248RR316LR328WG341R
R2355WF2364VP2366RA2367TG2375AA2428TD2431N/WG2434RR2435H/LA2437VR2452W/QI2453TR2454C/HR2458C/HP2496LR2508G/C/HY2510HE2545DV2550L
R2591WT2596IR2676WD2730H/GG2733DT2787SR2840WE2880KE3104KR3119HR3350HK3367RP3527SE3583QE3584QR3707LQ3756EV3840IR3903Q
I3916MD3986EG3990VS4050YT4081MN4119YΔ4124-4216R4136SI4138TV4234LE4283VT4637A/IG4638DR4645QΔ4647-4648L4650PH4651PP4668SF4684S
K4724QY4733EG4734ER4737W/GL4793PY4796CF4808IL4814FI4817FG4820WL4824PR4825C/PT4826IL4838VV4849IA4856GF4860VR4861C/HΔ4863-4969
Y4864CK4876RM4880TG4891RR4893W/QA4894T/VI4898TG4899R/EA4906VR4914G/TF4921SV4927FΔ4927-4928I4938MD4939EA4940TG4942VF4960YP4973L
0 1000 2000 3000 4000 5000
RyR1 MH/CCD Mutations
Amino Acid Sequence
Increased Sensitivity to RyR Activators Forms Basis for MH Screening
In Vitro Contracture TestCaffeine/Halothane Contracture Test
European Malignant Hyperthermia Group (EMHG)Low threshold = contracture of ≥0.2 g at a concentration of
≤2 mM caffeine or ≤ 2% halothane MH Susceptible(MHS): Low contraction threshold for both
Caffeine & HalothaneMH Equivocal(MHE): Low threshold for oneNormal (MHN): Normal threshold for both
North American Malignant Hyperthermia Group (NAMHG)
Low threshold = contracture of ≥0.3 g at a concentration of ≤2 mM caffeine or ≥0.3 g at ≤ 3% halothane
MH Susceptible(MHS): Low contraction threshold for either Caffeine or Halothane
Normal (MHN): Normal threshold for both
Gallant and Lentz Am J Physiol 262:C422, 1992
R615CHomozygous
R615CHeterozygous
Normal
MacLennan and Phillips Science 256:789, 1992.
How does abnormal Ca2+ regulation cause MH?
Is there a link between MH and exertional heat illness?
Porcine Stress Syndrome
26 soldier with exertional heat illness, all had positive in vitro contracture tests (Bendahan et al Anesth Anag 93: 683, 2001).
3 patients with positive IVC and RyR1 mutations; 2 had history of EHI (Brown et al Br J Anesth 88:508, 2002).
Effectiveness of dantrolene in treatment of heat illness questioned (see Hadad et al Sports Med 34: 501, 2004).
Y522S MH mutation knock-in mouse has an increased sensitivity to heat stress (Chelu et al FASEB J 20:329, 2006).
Cardiac Excitation Contraction Summary
Bers 2002 Nature 425:198.
Catecholaminergic Polymorphic Ventricular Tachycardia
Rare autosomal dominant (RyR2 mutations) or recessive (calsequestrin)
Symptoms:Rare before age 10.None at restVentricular arrhythmias of varying morphology upon exercise or catecholamine administration.SyncopeDeath
Mortality: 30-35% by age 30
CPVT Mutations
In Situ Confocal Imagingof a CPTV Heart
Chen et al Circ Arrhythmia Electrophys 5:841, 2012
CPVT Mechanisms
Calcium Leak from RyR2
Activation of Na/Ca exchange
Inappropriately timed depolarization
Arrhythmia
Sudden Death
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