Excitation Contraction Coupling Dr. Atif Mahmood May 14, 2010

EXCITATION- CONTRACTION COUPLING (SEQUENCE OF EVENTS)

EXCITATION- CONTRACTION COUPLING

EXCITATION- CONTRACTION COUPLING (cont’d)

EXCITATION- CONTRACTION COUPLING (cont’d)

Muscle Contraction and Relaxation Four actions involved in this process Excitation = nerve action potentials lead to action potentials in muscle fiber Excitation-contraction coupling = action potentials on the sarcolemma activate myofilaments Contraction = shortening of muscle fiber Relaxation = return to resting length Images will be used to demonstrate the steps of each of these actions

Nerve Activation of Individual Muscle Cells (cont.)

Excitation/contraction coupling Action potential along T-tubule causes release of calcium from cisternae of TRIAD Cross-bridge cycle

Begin cycle with myosin already bound to actin

1. Myosin heads form cross bridges Myosin head is tightly bound to actin in rigor state Nothing bound to nucleotide binding site

2. ATP binds to myosin Myosin changes conformation, releases actin

3. ATP hydrolysis ATP is broken down into: ADP + Pi (inorganic phosphate) Both ADP and Pi remain bound to myosin

4. Myosin head changes conformation Myosin head rotates and binds to new actin molecule Myosin is in high energy configuration

5. Power stroke Release of Pi from myosin releases head from high energy state Head pushes on actin filament and causes sliding Myosin head splits ATP and bends toward H zone. This is Power stroke.

6. Release of ADP Myosin head is again tightly bound to actin in rigor state Ready to repeat cycle

THE CROSS-BRIDGE CYCLE Relaxed state Crossbridge energised Crossbridge attachment A + M l ADP l Pi Ca2+ present A – M l ATP AlMlADPlPi Crossbridge detachment Tension develops ADP + Pi ATP AlM A, Actin; M, Myosin

Cross Bridge Cycle

Rigor mortis Myosin cannot release actin until a new ATP molecule binds Run out of ATP at death, cross-bridges never release

Need steady supply of ATP! Many contractile cycles occur asynchronously during a single muscle contraction Need steady supply of ATP!

Regulation of Contraction Tropomyosin blocks myosin binding in absence of Ca2+ Low intracellular Ca2+ when muscle is relaxed

Ca+2 binds to troponin during contraction Troponin-Ca+2 pulls tropomyosin, unblocking myosin-binding sites Myosin-actin cross-bridge cycle can now occur

How does Ca2+ get into cell? Action potential releases intracellular Ca2+ from sarcoplasmic reticulum (SR) SR is modified endoplasmic reticulum Membrane contains Ca2+ pumps to actively transport Ca2+ into SR Maintains high Ca2+ in SR, low Ca2+ in cytoplasm

The action potential triggers contraction How does the AP trigger contraction? This question has the beginning (AP) and the end (contraction) but it misses lots of things in the middle! We should ask: how does the AP cause release of Ca from the SR, so leading to an increase in [Ca]i? how does an increase in [Ca]i cause contraction?

Structures involved in EC coupling A band (myosin) I band (actin) Z disc Contractile proteins in striated muscle are organised into sarcomeres T-tubules and sarcoplasmic reticulum are organised so that Ca release is directed toward the regulatory (Ca binding) proteins The association of a t-tubule with SR on either side is often called a ‘triad’ (tri meaning three) Z disc M line Z disc sarcoplasmic reticulum t-tubules Triad junctional feet

Structures involved in EC coupling - Skeletal Muscle - T-tubule sarcolemma out in sarcoplasmic reticulum voltage sensor? junction foot

Ca2+ Controls Contraction Ca2+ Channels and Pumps Release of Ca2+ from the SR triggers contraction Reuptake of Ca2+ into SR relaxes muscle So how is calcium released in response to nerve impulses? Answer has come from studies of antagonist molecules that block Ca2+ channel activity 19

Dihydropyridine Receptor In t-tubules of heart and skeletal muscle Nifedipine and other DHP-like molecules bind to the "DHP receptor" in t-tubules In heart, DHP receptor is a voltage-gated Ca2+ channel In skeletal muscle, DHP receptor is apparently a voltage-sensing protein and probably undergoes voltage-dependent conformational changes 20

The "foot structure" in terminal cisternae of SR Ryanodine Receptor The "foot structure" in terminal cisternae of SR Foot structure is a Ca2+ channel of unusual design Conformation change or Ca2+ -channel activity of DHP receptor apparently gates the ryanodine receptor, opening and closing Ca2+ channels Many details are yet to be elucidated! 21

Skeletal muscle The AP: moves down the t-tubule voltage change detected by DHP （双氢吡啶） receptors DHP receptor is essentially a voltage-gated Ca channel is communicated to the ryanodine receptor which opens to allow Ca out of SR activates contraction out in voltage sensor (DHP receptor) junctional foot (ryanodine receptor) sarcoplasmic reticulum sarcolemma T-tubule

Cardiac muscle The AP: moves down the t-tubule voltage change detected by DHP receptors (Ca channels) which opens to allow small amount of (trigger) Ca into the fibre Ca binds to ryanodine receptors which open to release a large amount of (activator) Ca (CACR) Thus, calcium, not voltage, appears to trigger Ca release in Cardiac muscle! out in voltage sensor & Ca channel (DHP receptor) junctional foot (ryanodine receptor) sarcoplasmic reticulum sarcolemma T-tubule

The Answers! Skeletal Cardiac The trigger for SR release appears to be calcium (Calcium Activated Calcium Release - CACR) The t-tubule membrane has a Ca2+ channel (DHP receptor) The ryanodine receptor is the SR Ca release channel The ryanodine receptor is Ca- gated & Ca release is proportional to Ca2+ entry Skeletal The trigger for SR release appears to be voltage (Voltage Activated Calcium Release- VACR) The t-tubule membrane has a voltage sensor (DHP receptor) The ryanodine receptor is the SR Ca release channel Ca2+ release is proportional to membrane voltage

Transverse tubules connect plasma membrane of muscle cell to SR

Ca2+ release during Excitation-Contraction coupling Action potential on motor endplate travels down T tubules Ryanodyne R Ca-release ch.

Voltage -gated Ca2+ channels open, Ca2+ flows out SR into cytoplasm Ca2+ channels close when action potential ends. Active transport pumps continually return Ca2+ to SR Ca ATPase (SERCA)

Excitation-Contraction Coupling Depolarization of motor end plate (excitation) is coupled to muscular contraction Nerve impulse travels along sarcolemma and down T-tubules to cause a release of Ca2+ from SR Ca2+ binds to troponin and causes position change in tropomyosin, exposing active sites on actin Permits strong binding state between actin and myosin and contraction occurs ATP is hydrolyzed and energy goes to myosin head which releases from actin

Summary: Excitation-Contraction Coupling

Sliding Filament Model I: Actin myofilaments sliding over myosin to shorten sarcomeres Actin and myosin do not change length Shortening sarcomeres responsible for skeletal muscle contraction During relaxation, sarcomeres lengthen

Sliding filament model II:

Sarcomere Shortening

Sliding Filament Theory for Muscle Contraction Muscle contraction occurs when actin and myosin, the major proteins of the thin and thick filaments, respectively, slide past each other in an ATP-driven enzymatic reaction. Huxley AF and Niedergerke R, Nature 173, 971-973 (1954) Structural changes in muscle during contraction; interference Microscopy of living muscle fibers. Huxley HE and Hanson J, Nature 173, 973-976 (1954) Changes in cross-strations of muscle during contraction and stretch and their structural interpretation.

Structure of Actin and Myosin

Myosin structure:

Thick filament structure:

Structure of the M-line:

Structure of thin filament:

Cross-bridge formation:

Sliding Filament Theory for Muscle Contraction Coupling of chemical reactions with vectorial motion. ATP (Crossbridge) ADP + Pi + Energy Cross-bridge hypothesis of muscle contraction: Sliding of thin and thick filaments is caused by the cross-bridges that extend from the myosin filament, attach to actin, pull the thin filaments toward the center of the sarcomere and detach. This cyclic interaction is coupled with the hydrolysis of ATP.

Initiation of Contraction, Ca2+ release:

The micrograph shows myosin bound to actin EM evidence for sliding filament theory The micrograph shows myosin bound to actin Contraction of one sarcomere

Mechanism of muscle contraction