Transcript PPoint Lec 14

What is evidence that EC coupling works as described?
Model seems to be correct-at least for skeletal muscle 9
• if you remove parts of DHPR receptor
(molecularly)-you eliminate EC coupling-and if you
put them back restore EC coupling (Tanabe et al.
1990 Nature 346:567-569).
Takeshima et al, 1994
Nature 369:556-559
• mutant "dispedic"
mice
• EC coupling needing
for normal skeletal
development
Normal Dispedic
Ryanodine receptor HUGE -4 subunits of 6000 amino acids each
ryanodine is a plant poison that kills animals by binding to calcium
channels (RyR), prevents muscle contraction (can't move muscles to
breath)
common pesticide in 50s and 60s
malignant hyperthermia -condition characterized by uncontrolled heat
production
swine, dogs, humans, tuna??
can be caused by only one amino acid substitution out of 6000
Radermacher et al., J. Cell. Biol. 1994
127:411-423
More details about contraction…
-
+
Actin polymerizes from subunits called G actin to form actin filament
actin found in nearly all cells, not just muscle FIG 18-2 p. 754
Actin filaments have polarity which is very
important to their functionone can determine polarity by adding myosin heads
(stay tuned) to filaments-they will attach in
arrowhead configuration
-
+ end is where new g-actin subunits add on
(net effect)
-capping proteins can cause
filaments to grow in one
direction
18-3 p. 755, 18-13 p. 763
+
18-20,22 769-770
Myosin-the motor in muscle
tails intertwine and heads stick
out-form thick filaments
How do you know that myosin is the motor (vs. actin)?
Attach myosin to a slide and add ATP and actinmyosin heads will move actin filaments (but not vice-versa)
18-22, 771
Laser optical trap Fig. 18.23 p.772
-laser exerts force on actin through latex
bead, can precisely measure force myosin
puts on actin (3-4 pN) and distance myosin
head travels in single "stroke" (11 nm)
Note:
ATP needed for
unbinding of myosin
head from actin
Rief et al., 2000
P.N.A.S.
97:9482-9486
Models of sliding
filament still being
debated/refined
How does a rise in intracellular calcium trigger
contraction? 18-32, 780
Troponin C binds calcium, undergoes
conformational change which is
transmitted through Troponins I and T,
which are bound to Tropomyosin.
Tropomyosin undergoes conformational
change, moves on actin, exposing
binding site for myosin head on actin.
In reality (not a test tube) MUCH more complicated…
•multiple calcium binding proteins in muscle
(caldesmon, calsequestrin, parvalbumin)-all have different
binding constants for calcium
•relaxation, rather than contraction probably is rate-limiting
factor in contraction speed
•relaxation speeds in fish sped up by injecting parvalbumin cDNA
Capping proteins stabilize actin
filaments
-in the Z disk, alpha actinin +Cap Z
Titinmuscle has elasticity not
accounted for by actin,
myosin
titin very springy-stretches
from M-line to Z disk
largest protein that has its
coding sequence cloned
27000 amino acids, 3 million
molecular weight
can mix and match domains
of titin to adjust muscle
stiffness
Nebulin wraps around actin
with troponins and
tropomyosin
18.30, 778
Smooth muscle-loose array of actin
and myosin
-dense bodies within
cell probably function
similar to Z disk
-can contract and hold
contraction with very
little energy input
(contracted only uses
10% more ATP than
relaxed)
18-33 p. 781
-Has Tropomyosin but not troponins
-poorly developed SR-much of calcium that trigger contraction comes
from outside the cell
-caldesmon-a calcium binding protein-binds calcium with calcium is low,
then binds to thin filament and prevents myosin binding to actin (no
contraction)
-Caldesmon can be phosphorylated by protein kinase C -prevents it
from binding to actin-thus STIMULATES contraction
Cardiac Muscle
• some consider it striated
muscle because it is
striated, but important
differences
• single nucleus vs.
multinucleated
• no nebulin
• intercalated disk-site of
attachment of sarcomere
to plasma memebrane and
one myocyte (heart cell) to
another
Group Exercise #14
Complete this concept map-try it first by yourself,
then work with a partner(s).
DHPR receptor
thick filaments
myosin
thin filaments
contraction
actin
ATP
serca
troponins
sarcoplasmic reticulum
RyR receptor
calcium