Download Topics Covered MEMBRANE FUNCTION

PEP 8302 Module 2
Topics Covered
Membrane Structure and Function.
- Membrane structure and components.
- Membrane biophysical properties.
- Mechanical loading effects on membrane properties.
- Role of cholesterol in membrane protein function.
PEP 8302 Module 2
MEMBRANE FUNCTION
• To serve as a physical barrier between the
external and internal cellular environment
• To serve as a three-dimensional scaffold to
support a variety of signaling proteins
embedded in the three dimensional matrix of
the membrane
PEP 8302 Module 2
1
Mammalian Plasma Membrane
Cholesterol
Receptor
Phospholipid
Bilayer
G
Ion Channel
Kinase
(RYR-1, DHP, SERCA)
Lipid Raft
(CRMD)
Fluid Mosaic Model
http://www.youtube.com/watch?v=owEgqrq51zY
- Cholesterol is the major structural lipid of the mammalian membrane
- some proteins require lipid raft localization to function correctly
PEP 8302 Module 2
Biomechanical Properties of the
Living Cell Membrane
-rigidity, elasticity, fluidity, tensile strength
(all physical parameters)
- MEMBRANE ORDER (MO)
- cholesterol enrichment of large vessel endothelial cell
membranes results in both an increase in MO and an
increase in susceptibility to mechanically-induced
membrane wounding (Clarke et al., 1995; Endothelium
4:127-139).
- reversed by the acute (i.e. 2 min) addition of a synthetic
membrane disordering agent (i.e. decreases MO).
PEP 8302 Module 2
Biomechanical Changes In Myofiber Membranes
Associated with Mechanical Loading/Exercise
- exercise-induced hypertrophy includes the assembly of large amounts
of new membrane components required to service the new contractile
components.
- myofiber membranes are more resistant to damage than those in
unexercised muscle as evidenced by reduced levels of circulating
myofiber damage markers after a period of regular exercise.
- appears to be a “training effect” not only on contractile level but also
the membrane level.
- mechanical loading induces membrane wounding and release of the
muscle growth factor, fibroblast growth factor (FGF).
- this may explain the requirement for increasing resistive loads to
maintain muscle hypertrophic response during training regime.
PEP 8302 Module 2
2
Mechanically-Induced, Myofiber Wound-Mediated
FGF Release
- FGF has no signal transduction peptide sequence released via a nonsecretory pathway.
- release of FGF from myofiber cytoplasm via transient disruptions
or “membrane wounds” of the sarcolemma
- myofiber wounding increases as mechanical load increases.
- FGF release increases as mechanical load increases
- both acidic and basic isoforms of FGF released in this manner
- this phenomenon first shown indirectly in rat and mouse skeletal
muscle after exercise in vivo. Similar results obtained in rat
myocardium.
- phenomenon exacerbated in Duchenne’s muscular dystrophy
as mechanical strength of the myofiber is reduced. (?)
PEP 8302 Module 2
Myofiber Wounding In Eccentrically Loaded Mdx Mouse Muscle
FDx
FGF
Linear correlation between wounding and FGF release
PEP 8302 Module 2
Biomechanical Changes In Myofiber Membranes
Associated with Mechanical Unloading/Microgravity
Exposure
- myofiber is more susceptible to reloading-induced mechanical damage
than control muscle
- rat hindlimb suspension (HLS) muscles and space flown rat muscle
(Krippendorf and Riley, 1993; Riley et al., 1996).
- human subjects after 14 days of bedrest (Clarke et al., 1998).
- damage is associated with mechanical disruption of the myofiber
membranes as evidenced by increased circulating damage markers
and membrane lesions at the EM level.
PEP 8302 Module 2
3
Creatine Kinase (MM) as A Marker of
Myofiber Wounding During Bedrest
CK (mm) specific
to skeletal muscle,
released upon
myofiber damage
as a consequence
of mechanical load
Clarke et al., (1998)
JAP 85: 593-600.
PEP 8302 Module 2
In Vivo Correlation Between Myofiber Wounding
and Myofiber CrossCross-Sectional Area
Linear Correlation Between
Myofiber Wounding
And
Myofiber CSA
Atrophy
Hypertrophy
Clarke et al., (1998)
JAP 85: 593-600.
PEP 8302 Module 2
Creatine Kinase (MM) as A Marker of
Myofiber Wounding During Bedrest
Unexpected observation
that unloaded muscle
membranes are
susceptible to
mechanical damage than
normal membranes
Clarke et al., (1998)
JAP 85: 593-600.
PEP 8302 Module 2
4
Effect of Mechanical Unloading On
Muscle Membrane Cholesterol Content
(Atrophic)
Human Bedrest
(Hypertrophic)
Rat Hindlimb Suspension
PEP 8302 Module 2
Electro-mechanical
activation of the SR
membrane Ryanodine
Channel (RyR1) via the
dihydropyridine receptor
(DHP) results in Ca2+
efflux initiating
contraction. SR. Ca2+ reuptake into the SR brings
about relaxation.
Ca2+
RyR1
DHP
CaATPase
PEP 8302 Module 2
Membrane Localization of Cholesterol In Skeletal Muscle
Clarke et al., (2000) J. App. Physiol.
Physiol. 89: 731731-741
PEP 8302 Module 2
5
Membrane Localization of Cholesterol In Skeletal Muscle
Clarke et al., (2000) J. App. Physiol.
Physiol. 89: 731731-741
PEP 8302 Module 2
Cholesterol Immunostaining In Skeletal Muscle
Control
Atrophied
Rat Soleus Muscle
(14 days of HLS)
Human MVL
(Young vs Old)
Young (35 yrs)
Old (70 yrs)
PEP 8302 Module 2
Cholesterol Enrichment and
Membrane Function
•increased cholesterol content affects skeletal muscle
membrane protein function
–increased calcium flux through DHP-sensitive calcium channels
–inhibition of sarcoplasmic reticulum (Ca2+)-ATPase activity in
cardiac myocte membranes
–decreased muscle sensitivity to circulating insulin and IGF-1
•increased levels of free sarcoplasmic calcium ??
• increased cytosolic calcium is a catabolic stimulus
PEP 8302 Module 2
6
Relationship Between Myofiber Cholesterol
and Myofiber Free Calcium Levels
Calcium Green -1 Staining
Cholesterol vs. Calcium
PEP 8302 Module 2
Effect of Modifying Membrane Cholesterol Content on
SERCA-1 and SERCA-2 Ca2+ATPase Activity
SERCA-1
SERCA-2
PEP 8302 Module 2
Effect of Cholesterol Modulation on Ca2+ ATPase
Activity Rates in Rat Muscle Membranes
(N=4 per group, p < 0.05 between all groups)
Crude Membrane
Fraction of Rat
Soleus Muscle
CaATPase Rate
Assay.
PEP 8302 Module 2
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Effect of Modulating Membrane Cholesterol Content
On RyR1 Activity in a Lipid Bilayer Model
(1) Addition of MβC-CHOL
(2) Remove MβC-CHOL, add MβC alone
(3) Remove MβC, add MβC-CHOL again
PEP 8302 Module 2
The Effect of Dynamic Foot Pressure (DFP) on Myofiber Cross
Sectional Area in Rat Soleus Muscle With and Without HLS.
HLS Induces
approx. 50%
reduction in CSA.
DFP applied
intermittently for
4 hr per day during
HLS.
DFP completely
abolishes myofiber
atrophy in the
soleus of HLS rats.
PEP 8302 Module 2
Insulin Signaling in Skeletal Muscle
PEP 8302 Module 2
8
Vesicle Fusion
PEP 8302 Module 2
GLUT-4 Mobilization in Response to
Insulin Signaling
• GLUT-4 (glucose transporter)
• http://www.bristol.ac.uk/biochemistry/tavare/r
esearch/glut4.html
PEP 8302 Module 2
Diabetes and Skeletal Muscle
• Alteration in sarcolemma function
– Same number of sarcolemmasarcolemma-bound insulin receptors as nonnondiabetic muscle
– Same number of intracellular GLUTGLUT-4 (glucose channels) as
nonnon-diabetics
– Less GLUTGLUT-4 located in the sarcolemma of diabetic than
nonnon-diabetic muscle
– The result of less GLUTGLUT-4 in the sarcolemma of those with
T2DM is that the muscle is less able to uptake glucose than
those without T2DM
– The mechanism behind this reduction of GLUTGLUT-4 in the
sarcolemma is unknown
PEP 8302 Module 2
9
Immuno-localization of Cholesterol in Rat Soleus Muscle
Of Ambulatory Controls, HLS and HLS-DFP Animals
PEP 8302 Module 2
Immuno-localization of Inducible GLUT-4 in Rat Soleus Muscle Of
Ambulatory Controls, HLS and HLS-DFP Animals
PEP 8302 Module 2
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