9Model seems to be correct-at least for skeletal muscle• if you remove parts of DHPR receptor

(molecularly)-you eliminate EC coupling-and if youput them back restore EC coupling (Tanabe et al.1990 Nature 346:567-569).

Takeshima et al, 1994Nature 369:556-559• mutant "dispedic"

mice• EC coupling needing

for normal skeletaldevelopment

Normal Dispedic

What is evidence that EC coupling works as described?

Ryanodine receptor HUGE -4 subunits of 6000 amino acids eachryanodine 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 60smalignant hyperthermia -condition characterized by uncontrolled heat productionswine, 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

Actin polymerizes from subunits called G actin to form actin filamentactin found in nearly all cells, not just muscle FIG 18-2 p. 754

More details about contraction…

-

+

Actin filaments have polarity which is very important to their function-

one 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 muscletails intertwine and heads stickout-form thick filaments

How do you know that myosin is the motor (vs. actin)?Attach myosin to a slide and add ATP and actin-myosin 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 myosinhead from actin

Rief et al., 2000P.N.A.S.97:9482-9486

Models of slidingfilament still beingdebated/refined

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.

How does a rise in intracellular calcium trigger contraction? 18-32, 780

In reality (not a test tube) MUCH more complicated…

•multiple calcium binding proteins in muscle(caldesmon, calsequestrin, parvalbumin)-all have differentbinding constants for calcium

•relaxation, rather than contraction probably is rate-limitingfactor in contraction speed

•relaxation speeds in fish sped up by injecting parvalbumin cDNA

Capping proteins stabilize actinfilaments-in the Z disk, alpha actinin +Cap Z

Titin-muscle has elasticity not accounted for by actin, myosintitin very springy-stretches from M-line to Z disklargest protein that has its coding sequence cloned27000 amino acids, 3 million molecular weightcan mix and match domains of titin to adjust muscle stiffnessNebulin wraps around actin with troponins and tropomyosin18.30, 778

Smooth muscle--loose array of actinand myosin-dense bodies within cell probably functionsimilar to Z disk-can contract and holdcontraction with verylittle energy input(contracted only uses10% 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 isstriated, but importantdifferences

• single nucleus vs.multinucleated

• no nebulin • intercalated disk-site of

attachment of sarcomereto plasma memebrane andone myocyte (heart cell) toanother

Group Exercise #14

Complete this concept map-try it first by yourself,then work with a partner(s).

myosin

sarcoplasmic reticulum

thick filaments

actin

DHPR receptor

RyR receptor

ATP

serca

thin filaments

contraction

calcium

troponins