1. The Excitation-contraction coupling in skeletal muscle Department of Animal Science & Technology National Taiwan University De-Shien Jong

2. Outline Excitation-contraction coupling of skeletal muscle Excitation-contraction coupling of skeletal muscle Charge movement & Calcium release Charge movement & Calcium release Poor man ’ s fura-2 Poor man ’ s fura-2 Model of Ca release Model of Ca release Effects of Ca on kinetics of charge movement Effects of Ca on kinetics of charge movement Model of charge movement Model of charge movement

3. Peachey, 1965 Schematic drawing of skeletal muscle

4. Excitation-Contraction coupling Skeletal muscle Skeletal muscle Action potential → Motor neuron → Neuromuscular junction → muscle surface → Transverse tubular system (T-system) → Triad → Dihydropyridine Receptors (DHPR) → Ryanodine Receptors (RyR) → Ca 2+ release from SR → bind to troponin → induce muscle contraction Action potential → Motor neuron → Neuromuscular junction → muscle surface → Transverse tubular system (T-system) → Triad → Dihydropyridine Receptors (DHPR) → Ryanodine Receptors (RyR) → Ca 2+ release from SR → bind to troponin → induce muscle contraction How does DHPR and RyR talk to each other? How does DHPR and RyR talk to each other?

5. Excitation-Contraction coupling Cardiac muscle Cardiac muscle Action potential → Triad (or Diad) → DHPR → Ca 2+ entry → Ca 2+ bind to RyR → Ca 2+ induce Ca 2+ Release → Ca 2+ bind to contractile protein → muscle contraction Action potential → Triad (or Diad) → DHPR → Ca 2+ entry → Ca 2+ bind to RyR → Ca 2+ induce Ca 2+ Release → Ca 2+ bind to contractile protein → muscle contraction DHPR is L-type voltage-gated Ca 2+ channel which has two isoforms : Skeletal type & Cardiac type DHPR is L-type voltage-gated Ca 2+ channel which has two isoforms : Skeletal type & Cardiac type

7. Tanabe et al. 1988

8. Tanabe et al. 1990

9. Ryanodine Receptor RyR has two isoforms in amphibian muscle :  &  RyR has two isoforms in amphibian muscle :  &  RyR has three isoforms in mammalian muscle : RyR1, RyR2, and RyR3 RyR has three isoforms in mammalian muscle : RyR1, RyR2, and RyR3 RyR1 mainly in skeletal muscle RyR1 mainly in skeletal muscle RyR2 in cardiac muscle RyR2 in cardiac muscle RyR3 in most other cells (i.e. brain) RyR3 in most other cells (i.e. brain)

10. Felder & Franzini-Armstrong, 2002 Ferguson et al. 1984

11. Franzini-Armstrong, 2004

12. Charge movement & Calcium release Dr. W. Knox Chandler Dr. Paul C. Pape Dr. Steve M. Baylor

13. Miledi et al. 1977, Caputo et al. 1984 Chandler et al. 1976

15. Effects of increased [Ca 2+ ] i Bind to contractile protein troponin Bind to contractile protein troponin Bind to various intrinsic Ca buffers Bind to various intrinsic Ca buffers Activate additional release of Ca from the SR (Ca induced Ca release) Activate additional release of Ca from the SR (Ca induced Ca release) Reduce additional Ca release from the SR (Ca inactivation of Ca release) Reduce additional Ca release from the SR (Ca inactivation of Ca release) Bind to Ca indicators Bind to Ca indicators

16. Irving et al. 1987

17. Poor man ’ s fura-2

18. Experimental methods A cut frog muscle fiber was mounted on a double Vaseline-gap chamber A cut frog muscle fiber was mounted on a double Vaseline-gap chamber Extracellular and intracellular solutions contain ion replacement to eliminate ionic currents Extracellular and intracellular solutions contain ion replacement to eliminate ionic currents Internal solution contained 20 mM EGTA plus 1.76 mM Ca 2+, which expecting to catch all the Ca released from the SR Internal solution contained 20 mM EGTA plus 1.76 mM Ca 2+, which expecting to catch all the Ca released from the SR

20. Fractional phenol red in the nonprotonated form f f = (r - r min ) / (r max - r min ) where r = A ind (570) / A ind (480) pH = pk + log (f / (1 – f)) 480 nm : isobestic wavelenth of phenol red 570 nm : a wavelength at which phenol red is sensitive to pH 690 nm : a wavelength not absorbed by phenol red

21. Estimation of Ca buffering power  of muscle fiber

25. Advantages of EGTA-phenol red method The EGTA-phenol red method estimates SR Ca release reliably (~ 96%) and Rapidly (<0.1 ms). The EGTA-phenol red method estimates SR Ca release reliably (~ 96%) and Rapidly (<0.1 ms). The change in pH does not alter physiological condition and the buffering power  is stable. The change in pH does not alter physiological condition and the buffering power  is stable. The dissociatin rate of Ca and EGTA, k -1, is small (~ 1 s -1 ) compared to that of Ca and fura- 2 (~10-30 s -1 ). The dissociatin rate of Ca and EGTA, k -1, is small (~ 1 s -1 ) compared to that of Ca and fura- 2 (~10-30 s -1 ).

26. k 1 [EGTA] -1 = 22  s So the  [Ca] signal estimated from  pH would be the same amplitude as the measurement with PDAA

27. Model of  [Ca] near a single SR Ca channel

28.  [Ca] =  / (4  D Ca r) * exp ( -r / Ca ) Where  Ca  D Ca  k 1 [EGTA] R } 1/2 = 81 nm

29. Time course of  [Ca] after a step change in  from a single point source in an isotropic infinite medium.

30. e-fold increase in d  [Ca T ]/dt every 3.73 mV

31. Effects of released Ca on charge movement

35. = I cm / (Q oo – Q cm )

44. Model of charge movement

45.  0 /  0 = exp[(v - v 0 )/k]

46.  n = f n  0,  n = f n  0  n = f n  0,  n = f -n  0 Case 1Case 2

47.  n = f ’ f n  0,  n = f ’ f -n  0 Case 3 Exp. Data

48. Calcium spark

49. Thank you