Excitation Contraction Coupling (E-C coupling)

Question 1

What is the structure of Na+/Ca++ exchanger

Answer 1

• Monomeric protein = 1 subunit
• 9 TM-spanning regions
• Ca++ regulatory site between the 5th and 6th segment
• IC Ca++ binding is necessary for its activity

Question 2

Binding of what ion is necessary for activity of Na+/Ca++ exchanger?

Answer 2

intracellular Ca++

Question 3

Genes encoding Na/Ca exchanger?

Answer 3

• NCX1 - heart, skeletal muscle
• NCX2 - smooth muscle
• NCX3 - skeletal muscle

Question 4

NCX is an ________ with two different modes, what are those modes?

Answer 4

NCX is an electrogenic transporter with two different modes, what are those modes?

1. Forward mode:
   
   • 1 Ca++ out and
   • 3 Na+ in
   • (depolarizing)
2. Reverse mode
   
   • 3 Na+ out
   • 1Ca++ in
   • repolarizing

Question 5

What is the contribution of NCX to an action potential?

Answer 5

• Active at phase 0
  
  • Reverse mode
    
    • Try to remove excess Na+ that is entering through Na+ channels
    • repolarizing
• Active at phase 1/2
  
  • Forward mode
    
    • Try to remove excess Ca++ coming in through L-type Ca++ channels
    • Net depol
      
      • contribute to sustained depolarization

Question 6

Structure of Na/K ATPase

Answer 6

• Member of active cation transport proteins
• Pumps 3Na+ out for 2K+ in
• Heterotrimeric protein comprised of an alpha, beta and gamma subunits

Question 7

What are the important subunits of the Na/K ATPase?

Answer 7

• Alpha-subunit
  
  • Forms the conformational alterations
  • 3 domains on the cytoplasmic side
• Beta-subunit
  
  • chaperone protein ( brings alpha subunit to the membrane)
• Gamma subunit
  
  • increases affinity for ATP

ALL THREE ARE REQUIRED FOR NORMAL FUNCTION

Question 8

5 steps for Na+/K+ ATPase

Answer 8

1. ATP binds - allows three intracellular Na+ ions to bind
2. ATP is hydrolyzed and the pump is phosphorylated (at P-domain of alpha subunit)
   
   • ADP is released
3. Conformational change exposes Na+ to outside
   
   • phosphorylated form has low affinity for Na+ - Na+ is released
4. Pump binds to 2 extracellular K+ ions
   
   • causes dephosphorylation reverting back
   • K+ exposed to intracellular side
5. Dephosphorylated form has higher affinity for Na+ ions than K+ ions so the K+ ions are released.
   
   • ATP binds
   • process restarts

Question 9

What is excitation-contraction coupling?

Answer 9

the relationship between the action potential (electrical), the intracellular Ca2+ concentration (chemical) and myocyte contraction (mechanical)

Question 10

What are the 10 steps of E-C coupling?

Answer 10

1. AP enters from adjacent cell
2. Voltage-gated Ca++ channels open (L-type)
   
   • Ca++ enters cell
3. Ca++ induces Ca++ release through ryanodine receptor channels (RyR)
4. Local release causes Ca++ spark
5. Summed Ca++ sparks create a Ca++ signal
6. Ca++ ions bind to troponin to initiate contraction
7. Relaxation occurs when Ca++ unbinds from troponin
8. Ca++ is pumped back into the sarcoplasmic reticulum via SERCA for storage
9. Ca++ is exchanged with Na+ (forward NCX) - 1Ca++ out, 3 Na+ in
10. Na+ gradient is maintained by Na+/K+ ATPase
    
    • 3Na+ out; 2K+ in

Question 11

What causes the Ca++ spark?

Answer 11

Local releases of Ca++ from the SR where L-type Ca++ channels meet the ryanodine receptor channels

Ca++ released from a cluster of RyR2

Question 12

Define:

Ca++ sparklet

Answer 12

Sparklet: Ca++ flux through the L-type Ca++ channel

Question 13

define ca++ blink

Answer 13

Blink: Ca++ release from individual RyR2

Question 14

What inactivates L-type Ca++ channels and RyR2?

Answer 14

Negative feedback from Ca++ released from SR

Question 15

What stops Ca++ release from the sarcoplasmic reticulum?

Answer 15

1. Inactivation of RyR2 and L-type Ca++ channels
2. Loss of Ca++ stores from SR (diffusional driving force)

Question 16

What are the molecules involved in the contractile apparatus?

• _______
• _______ runs along the groove of F-actin
• _______ blocks the myosin-binding sites on actin molecules (prevent contraction)
• Tropomyosin held in position by ______

Answer 16

What are the molecules involved in the contractile apparatus?

• Myosin and Actin (G and F)
• tropomyosin runs along the groove of F-actin
  
  • blocks the myosin-binding sites on actin molecules (prevent contraction)
• Tropomyosin held in position by troponin complex (TnI, TnC, TnT)

Question 17

What are the three subunits of troponin complex and their functions?

Answer 17

1. TnI - binds to actin and inhibits myosin ATPase
   
   • ​​binds to TnC and TnT and to actin
   • prevents binding of myosin and the activation of myosin ATPase
2. TnC - binds to Ca++**​​
   
   • Ca++ sensor for cardiac muscle
   • binding to Ca++ increases interaction of TnI with TnC - relieves interaction with actin
3. TnT - links troponin complex to tropomyosin
   
   • ​​holds the Tn complex together with actin and tropomyosin
   • Its C-terminal interacts with TnI and TnC while its N-terminal binds to tropomyosin
   • Interaction with TnI-TnC-Tm increases in the absence of Ca++ (inhibiting myosin ATPase)

Question 18

What are the 6 steps of cross-bridge cycling?

Answer 18

1. Tight binding in the rigor State
   
   • the crossbridge is at 45 degree angle relative to the filament
2. ATP binds to its binding site on the myosin
   
   • Myosin dissociates from actin
3. ATPase activity of myosin hydrolyzes the ATP; ADP and Pi remain bound to myosin
4. Myosin head swings over and binds weakly to a new actin molecule
   
   • crossbridge at 90 degree angle relative to the filament
5. Release of Pi initiates the power stroke
   
   • myosin head rotates pushing actin filament past it
6. At the end of the power stroke the myosin head releases ADP and assumes the tightly bound rigor state

Question 19

What needs to happen in the troponin complex in order to allow myocyte contraction?

Answer 19

• Ca++ binding to TnC induces a conformation change in tropomyosin and its location on actin
  
  • when calcium is bound to TnC it increases the interaction of TnI with TnC - relieving interaction with actin
• This movement along actin exposes the myosin binding site - thus allowing for the myosin head to interact with actin
  
  • Cross-bridge formation

Question 20

What is SERCA?

Answer 20

Sarco(endo)plasmic Reticulum Ca++ ATPase

• P-type ATPase
  
  • can autophosphorylate and switch between conformations Eq and E2
• Uptake of Ca++ into SR occurs against a concentration gradient

Question 21

What are the 6 steps of SERCA?

Answer 21

1. ATP binds
2. 2 Ca++ ions bind
3. SERCA is phosphorylated (E1~P)
4. E1~P is high energy intermediate
   
   • conformational change to E2-P (low energy state)
5. 2 Ca++ released into the SR lumen
6. EP-2 dephosphorylation and return to original conformation - ready to bind Ca++ on the cytosolic site

Question 22

What regulates the activity of SERCA2?

Answer 22

Phospholamban (PLN) - can be phosphorylated to allow Calcium flow into SR

• Phosphorylation of PLN alleviates SERCA2 inhibition

Question 23

Point mutation in PLN causes:

Answer 23

Point mutation in Phospholamban causes

• Dominant inherited cardiomyopathy
  
  • Phospholamban cant be phosphorylated = Ca++ uptake into SR is reduced

Question 24

Stop mutation in PLN causes

Answer 24

Stop mutation in phospholamban causes:

• Inherited cardiomyopathy
  
  • Unstable PLN
    
    • sometimes interacting with SERCA2 but sometimes not - unpredictable uptake of Ca++ into SR

Question 25

Deletion (null-mutation) of PLN causes?

Answer 25

Uncontrolled Ca++ uptake

Question 26

Pseudo-phosphorylated PLN mutant causes:

Answer 26

Uncontrolled Ca++ uptake as SERCA2 thinks it is always phosphorylated = no inhibition

Question 27

What is the structure of Phospholamban?

Answer 27

• 52 amino acids
• Three domains
  
  • Ia - cytosolic
    
    • three sites of phosphorylation (S10, S16, S17)
  • Ib - links cytosolic to transmembrane domain
  • Il - transmembrane
    
    • interacts closely with SERCA