What is evidence that EC coupling works as described?

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 :

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 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, p. 763

18-20, 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 actin- myosin heads will move actin filaments (but not vice-versa) 18-22, 771

Laser optical trap Fig 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: Models of sliding filament still being debated/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 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

Titin- muscle 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 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 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

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