Excitation Contraction Coupling

Question 1

Active zones

Answer 1

• Dense spots over which synaptic vesicles are clustered;
• where fusion of synaptic vesicles & release of ACh occurs
• Oriented directly over secondary postsynaptic clefts between adjacent postjunctional folds

Question 2

postjunctional folds`

Answer 2

• Extensive invaginations on postsynaptic membrane directly under nerve terminal
• Increase surface area of muscle plasma membrane

Question 3

synaptic cleft

Answer 3

• ~ 50 nm, time delay in impulse transmission with ACh diffusion

Question 4

Nicotinic Acetylcholine Receptors

Answer 4

• located at crests of postjunctional folds in NNJ

Question 5

Acetylcholinesterase

Answer 5

(AChE)
High concentration associated with synaptic basal lamina (basement membrane)

*** Terminates synaptic transmission after AP

Hydrolyzes ACh →
choline & acetate

Question 6

Acetylcholine

Answer 6

• Motor neuron cell bodies in spinal cord produce NT vesicles
• fast axonal transport translocates vesiceles to nerve terminal through MT-mediated process
• Vesicles for ACh travel down axon empty- at nerve terminal ACh synthesis occurs and it is taken up by vesicles
• –> choline acetyltransferase
• –> ACh-H+ exchanger

Question 7

Choline Acetyltransferase

Answer 7

• synthesizes ACh from choline and acetyl coenzyme A

Question 8

ACh-H+ exchanger

Answer 8

• NT transport proteins
• ACh is taken up into synaptic vessicles through this exchanger, and H+ is released.
• driven by vesicular proton electrochemical gradient (positive voltage and low pH inside) - H+ proton pump uses ATP to make the gradient inside the synaptic vesicle acidic to enable the exchanger to work

Question 9

synaptobrevin

Answer 9

(v-SNARE)

• helps drive vesicle fusion - essential for NT release
• forms complex with SNAP-25 and syntaxin (t-SNAREs)

Question 10

synaptogamin

Answer 10

• Ca2+ receptor of synaptic vesicle
• detects rise in [Ca2+] in the nerve and triggers exocytosis docked vesicles

Question 11

t-SNAREs

Answer 11

Syntaxin and SNAP-25

• located in presynaptic membrane and play key role in fusion process

Synaptobrevin (located on synaptic vesicle): coils around the free ends of syntaxin and SNAP-25 - brings the vesicle closer to presynaptic membrane

Question 12

Synaptotagmin

Answer 12

• [Ca2+] sensor that is located on the synaptic vesicle
• When Ca2+ enters through voltage-gated Ca2+ channels near the active zone of the presynaptic membrane synaptogamin recognizes the rise of Ca2+ and triggers vesicle fusion and exocytosis of NT

Question 13

Neurotoxins that block fusion of synaptic vesicle

Answer 13

• Tetanus Toxin and Botulinum toxins B,D,F,G: endoproteinases that digest synaptobrevin
• Botulinum A/E: cleaves SNAP-25
• Botulinum toxin C1: cleaves syntaxin

–> all block the fusion process of synaptic vesicle with the nerve terminal membrane

Question 14

ACh Receptor

Answer 14

• ionotropic, Nicotinic AChR channel: nonselective cation channel at the muscle endplate
• premeable to cations: Na+, K+, Ca 2+
• not permeable to anions, except Cl-
• function to raise Vm above threshold
• When ACh binds it functions to open AChR channel at end of muscle plate:
• Na+ and K+ become equally permeable
• results in increasing the resting premeability of Na+ relative to K+
• Vm shifts to a vale between -80mv and Ena= +50 mV (end plate potential)

Question 15

End-plate potential (EPP)

Answer 15

*** a graded response to opening of AChR!

• a type of graded potential, an EPSP producted by transient opening of AChR
• due to increased Na+ conductanve driving Vm of end-plate region more positive

Decremental spread of current:
- Amplitude of change in membrane
potential diminishes at distances
from the end plate

Question 16

AcH and end-plate potentials

Answer 16

• presynaptive motor nerve axon AP –> depolarizing postsynaptic EPP
• EPP: ~40mV more positive than resting Vm
• NMJ can maintain high rate of AP transmission without significant loss of function, due to ability to synthesize ACh and repackage it

Question 17

Miniature end-plate potentials (MEPPs, minis)

Answer 17

• spontaneous fluctuations in Vm : similar to normal EPPs
• low-probability of ACh release in absence of nerve stimulation, due to opening only a few AChRs

Question 18

Summary of events at NMJ

Answer 18

1. AP in motor neuron is propogated to terminal buton
2. presence of AP triggers opening of voltage-gated Ca2+ channels and Ca2+ enters into terminal buton
3. Ca2+ triggers release of ACh from vesicles
4. ACh diffuses across NMJ and binds with AChR on motor end plate
5. binding brings about opening of cation channelas and large amounts of Na+ move into muscle cell compared to small amounts of K+ moving out
6. Result is an end-plate potential - local curent flows betwen the depolarized end plate and adjacent membrane
7. local curent flow opens voltagegated Na+ channels in adjacent membrane
8. Na+ reduces the potential to threshold, initiating an AP which is propogated through muscle fiber
9. ACh in NMJ is destroyed by acetylcholinesterase - terminating muscle cell’s response

Question 19

Thick Filaments

Answer 19

• 2 myosin heavy chains (MHC) have 2 important binding sites:
• Actin binding site - for cross-bridge formation
• myosin ATPase site - for binding and hydrolyzing ATP
• myosin light chains:
• Alkali light chain - essential for stabilizing the myosin head region
• regulatory light chain: regulates myosin ATPase activity

Question 20

Thin Filaments

Answer 20

• made of F-actin
• associated with 2 regulatory actin-binding proteins: troponin and tropomyosin
• the myosin binding site on actin is blocked by tropomyosin at rest. When Ca2+ binds trponin, tropomyosin slips away from its blocking position between actin and myosin. The exposure of the muosin-binding site on actin, allows a cross bridge to be formed and muscular contraction to occur

Question 21

Tropomyosin

Answer 21

• two alpha helices coiled around each other; regulates binding of myosin heads to myosin binding site on actin

Question 22

troponin

Answer 22

• troponin T: TNT, binds tropomyosin
• Troponin C: binds Ca2+
• Troponin I: binds to actin and inhibits contraction

Question 23

Titin

Answer 23

• largest knkown protein
• tethered from M line to Z line
• Appears to be involved in the elastic behavior of muscle by maintaining the resting length of muscle during relaxation

Question 24

Sarcolemma –> T-tubule

Answer 24

• A rise in [Ca2+]i is the intracellular signal that triggers and sustains muscle contraction (skeletal, cardiac, & smooth m.)
• APs originating at sarcolemma propgate to cell interior via T-tubules: T-tubules extend into muscle fiber and surround myofibrils at junctions of A and I bands
• Depolarization of the sarcolemmal membrane results in a rise in [Ca2+]i - due to release of Ca2+ from Sarcoplasmic Reticulum

Question 25

Triad

Answer 25

• T-tubule and its neighboring 2 cisternae (sarcoplasmic reticulum cisternae)
• Propogation of AP into T tubules depolarizes triad and results in Ca2+ release from lateral sacs of SR

Question 26

Dihydropyridine receptors and Ryanodine Receptor

Answer 26

• DHPR: L-type Ca2+ channels
• voltage gated channels located on T-tubule membrane
• conformational changes in the tetrad of DHP Ca2+ channels induces a conformational change in the Ca2+ release channel
• Ryanodine receptor located on SR membrane. Cluster at the portion of SR that faces T tubule. Releases stored Ca2+ from SR when bound with DHPR
• Summary: Depolarization of DHPR on T-tubule membrane results in mechanical activation of Ryanodine in the SR. Ca2+ stored in the SR rapidly leaves through the Ca2+ release channel.

Question 27

Power Stroke

Answer 27

• Ca2+ binds troponin, and troponin removes tropomyosin from the myosin binding site on actin.
• ATP binds to myosin head, causing the dissociation of the actin-myosin complex
• ATP is hydrolyzed (potential energy) causing myosin heads to return to resting conformation (cocked)
• cross-bridge forms and myosin head binds new position on actin
• P is released. Myosin heads change conformation, resulting in power stroke and filaments slide past each other
• ADP is released.
• ATP is needed for myosin head to dissociate (reason for rigor-mortis)

Question 28

Relaxation

Answer 28

• requires reuptake of Ca2+ from sarcoplasm back into SR
• If unregulated, cross-bridge cycle would continue until myocyte is depleted of ATP
• After an AP, Ca2+ must be removed from the cytoplasm for contraction to cease and for relaxation to occur
• When Ca2+ levels decrease, troponin and tropomyosin move back in place and cover myosin-binding site on actin
• not a passive process.

1. NA+Ca2+ exhcnager and ca2+: minor contribution to removing ca2+ from cytoplasm
2. Sarcoplasmic and Endoplasmic Reticulum Ca2+ ATPase (SERCA) = Ca2+ pump that re-uptakes Ca2+ into the SR - MOST IMPORTANT!!!

Question 29

Role of Ca2+ binding proteins in the SR

Answer 29

High [Ca2+] in the SR inhibits activity of SERCA (impacts gradient)

Ca2+-binding proteins in the SR lumen can delay inhibition of Ca2+ pump activity

Ca2+-binding proteins buffer increased [Ca2+] during Ca2+ re-uptake and can increase the Ca2+ capacity of the SR

• Calquesterin= Primary Ca2+-binding protein in skeletal muscle forms a complex with RYR, facilitates muscle relaxation by buffering Ca2+ AND unloads its Ca2+ in the vicinity of the Ca2+-release channel to facilitate EC coupling
• Calreticulin: Ca2+-binding protein in smooth muscle