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Skeletal Muscle (and a little cardiac)
Excitation-Contraction Coupling
Ed Balog
Applied Physiology
555 14th St NW Rm 1303
[email protected]
[Ca2+]
1-2 mM
Ca2+
[Ca2+]
100 nM
RyR
Na2+
IP3R
[Ca2+]
1 mM
Endo/Sarcoplasmic
Reticulum
ADP + Pi
ATP Ca2+
Intracellular Calcium Signaling
 Fertilization
 Stimulus-secretion
coupling
 Metabolism
 Muscle Contraction
Berridge et al., Nature Rev. Mol. Cell Biol. 1:11, 2000.




Learning and Memory
Gene regulation
Cell death
Immune cell activation
Muscle
Contraction
Summary
Ca2+ Movement in Muscle
Flux that increases cytoplasmic Ca2+
Flux that decreases
cytoplasmic Ca2+
Cytoplasm
Mitochondria
Troponin
& other Ca2+binding proteins
SR
Ca2+
Buffers
Force
Determinates of Contractile Force
1. Maximal
Calcium
Activated
Force
2. Calcium
Sensitivity
Calcium
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
Triad
Transverse Tubule
Sarcoplasmic
Reticulum
Hypotheses:
1. Depolarization of the sarcoplasmic reticulum.
2. Chemical messenger from the transversetubules 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
DHPR tetrad
T-tubule membrane
RyR tetramer
Dihydropyridine Receptor
 aka “DHPR”, voltage
sensor” or “L-type channel”
 Heteromultimeric protein w/
5 subunits.
 Origin of charge movement
& L-type Ca2+ current.
Ryanodine Receptor
SR membrane
 aka “Ca2+ release
channel” or “junctional
foot protein”
 Homotetramer; 2 million
daltons.
Mechanically Peeled Muscle Fiber
Lamb and Stephenson, Dept of Zoology, LaTrobe University
High K+
High Na+
25
mg
2 sec
Posterino, Proc Australian Physiol
Pharmacol Soc 32: 28, 2001.
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
Channels
Lipid
Bilayer
Some of the Whole-Cell Measurable Events of ExcitationContraction Coupling
DHPR (L-type Ca2+ Channel)
α2
 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
α1
S
N
S
C
N
+
+
+
δ
+
+
+
γ
+
+
+
t-tubule
lumen
+
+
+
N
C
cytoplasm
C
C
N
N
β
II-III loop and β-subunit
C
vital for skeletal muscle ECC
DHPR electrical signals:
L-type calcium current
10 μA/μF
25 ms
 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).
Cardiac
Skeletal Muscle
 Skeletal muscle current is
smaller and is barely
activated during a skeletal
muscle action potential.
The current is not
required for contraction.
Skeletal Muscle Contraction Does Not Require the
Entry of Extracellular Calcium
Caputo & de Bolanos J Physiol 289: 175, 1979.
Wang et al Biophys J 77:2709, 1999
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.
Tetanus
Twitch
Dulhunty and Gage J Physiol 399:63, 1988
DHPR electrical signals: Charge movement
DHPR
tetrad
ΔV
t-tubule
membrane
+ +
+ +
20 ms
+ +
 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.
Charge Moved
(nC/uF)
+
20 nA
30
20
10
0
-100 -75 -50 -25
0
25
Test Potential (mV)
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)
Sarcolemma
Triad
Transverse Tubule
Sarcoplasmic
Reticulum
 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).
Channel Activity
Calcium Dependence of RyR1 and RyR2
RyR2
A
I
AL
RyR1
A
[Calcium]
High affinity calcium binding site –
Activates channel when bound.
Calcium selective (KCa/KMg ~100) .
I
Low affinity calcium binding site –
Inhibits channel when bound.
Relatively unselective for divalent
cations (KCa/KMg ~1) .
AL
SR lumenal calcium binding site –
Activates channel when bound.
RyR Macromolecular Complex
Song et al Prog Biophys Mol Biol. 105:145, 2012.
RyR Channel Modulators
(Partial List)
Endogenous
Adenine Nucleotides
Calmodulin
Mg2+
H+
Inorganic Phosphate
Dihydropyridine Receptor
FKBP12/12.6
Reactive Oxygen Species
Nitric Oxide
Exogenous
Caffeine
Ryanodine
Ruthenium Red
Volatile Anesthetics
Depolarizing Muscle
Relaxants
Oxidizing/reducing agents
Local Anesthetics
4-chloro-m-cresol
Dantrolene
Arrangement of DHPR and RyR1
DHPRs
γ
α1
α2
δ
T-tubule Membrane
β
Junctophilin
RyR1
SR Membrane
 4 DHPRs per coupled RyR
Serysheva et al PNAS 99:10370, 2002.
Toadfish
Swim Bladder
(very fast)
DHPR tetrads
Mammalian
Fast-twitch
Muscle
RyR1
Mammalian
Slow-twitch
Muscle
RyR2
Mammalian
Cardiac Muscle
Individual DHPRs
DHPR:RyR
Arrangement
and Ratio
Varies with
Muscle Fiber
Type
How are uncoupled skeletal
muscle ryanodine receptor
channels opened?
+++
+++
Ca2+-Induced Ca2+ Release
+++
Direct Coupling
+++
+++
Comparison of Cardiac and Skeletal
Muscle Excitation-Contraction Coupling
V
DHPR
(α1c)
++
++
+
DHPR
tetrad
(α1s)
Skeletal Muscle:
Mechanical Coupling
V
Ca2+
+
Cardiac Muscle:
Calcium-Induced
Calcium Release
t-tubule
++
SR
RyR1
Ca2+
Charge movement within DHPR
and subsequent conformational
change activates RyR via direct
physical interaction.
RyR2
Ca2+
DHPR mediates Ca2+ influx,
Ca2+ binds to and activates
the underlying RyR.
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 Lchannel.
Store-Operated Ca2+ Channels
Lewis Nature 446: 284, 2007.
Calcium Transporters in Muscle
SERCA:
Sarco/Endoplasmic Reticulum Calcium ATPase
PMCA:
Plasma Membrane Calcium ATPase
NCX:
Na/Ca Exchange
MCU:
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 Genes
SERCA1 – 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 = Calcium
Green = 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 Hyperthermia
An autosomal dominant pharmacogenetic disease characterized
by an unusual metabolic reaction to volatile anesthetics and
depolarizing muscle relaxants.
Symptoms include:
Hypercapnia
Cyanosis
Tachycardia
Muscle rigidity
Rhabdomyolysis
Hyperthermia
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.
RyR1 MH/CCD Mutations
L13R
C35R
R 44C/H
D60N
Q155K
R156K
E160G
R163C/L
G165R
D166N/G
R177C
Y178C
G215E
V218I
M226K
D227V
G248R
R316L
R328W
G341R
0
R367L
R401C/G/H
I403M
Q474H
Y522S/C
R530H
R533C/H
R533H
R552W
R614C/L
S846L
R1043C
R1140C
S1342G
Q1589P
P1592L
S1728P/F
M1729R
P1787L
M1814K
1000
A1832G
G2060C
V2117L
D2129E
R2163C
R2163H/P
V2168M
A2200V
T2206M/R
V2210F
V2212A
V2214I
V2280I
I2321V
R2336H
N2342S
E2344D
V2346M
E2348G
A2350T
R2355W
F2364V
P2366R
A2367T
G2375A
A2428T
D2431N/W
G2434R
R2435H/L
A2437V
R2452W/Q
I2453T
R2454C/H
R2458C/H
P2496L
R2508G/C/H
Y2510H
E2545D
V2550L
2000
R2591W
T2596I
R2676W
D2730H/G
G2733D
T2787S
R2840W
E2880K
E3104K
R3119H
R3350H
K3367R
P3527S
E3583Q
E3584Q
R3707L
Q3756E
V3840I
R3903Q
I3916M
D3986E
G3990V
S4050Y
T4081M
N4119Y
Δ4124-4216
R4136S
I4138T
V4234L
E4283V
T4637A/I
G4638D
R4645Q
Δ4647-4648
L4650P
H4651P
P4668S
F4684S
K4724Q
Y4733E
G4734E
R4737W/G
L4793P
Y4796C
F4808I
L4814F
I4817F
G4820W
L4824P
R4825C/P
T4826I
L4838V
V4849I
A4856G
F4860V
R4861C/H
Δ4863-4969
3000
Amino Acid Sequence
4000
Y4864C
K4876R
M4880T
G4891R
R4893W/Q
A4894T/V
I4898T
G4899R/E
A4906V
R4914G/T
F4921S
V4927F
Δ4927-4928
I4938M
D4939E
A4940T
G4942V
F4960Y
P4973L
5000
Increased Sensitivity to RyR Activators Forms Basis for
MH Screening
In Vitro Contracture Test
Caffeine/Halothane Contracture Test
R615C
Homozygous
European Malignant Hyperthermia Group (EMHG)
Low threshold = contracture of ≥0.2 g at a concentration of
≤2 mM caffeine or ≤ 2% halothane
R615C
MH Susceptible(MHS): Low contraction threshold for both
Caffeine & Halothane
Heterozygous
MH Equivocal(MHE): Low threshold for one
Normal (MHN): Normal threshold for both
Normal
Gallant and Lentz Am J Physiol 262:C422, 1992
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
How does abnormal Ca2+ regulation cause MH?
MacLennan and Phillips Science 256:789, 1992.
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 rest
Ventricular arrhythmias of varying morphology upon exercise
or catecholamine administration.
Syncope
Death
Mortality: 30-35% by age 30
CPVT Mutations
In Situ Confocal Imaging
of 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