Freeze-Fracture Studies on the Cross-Bridge Angle Distribution at Various States and the Thin Filament Stiffness in Single Skinned Frog Muscle Fibers

Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb+) and conditional Neb knockout (Neb−) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca2+ activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb+ fibers and 68% in Neb− fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb− fibers than in Neb+ fibers, the increase in force per CB in the presence of Neb is ∼1.5 fold. The equilibrium constant of the CB detachment step (K2), its rate (k2), and the rate of the reverse force generation step (k−4) are larger in Neb+ fibers than in Neb− fibers. The rates of the force generation step (k4) and the reversal detachment step (k−2) change in the opposite direction. These effects can be explained by Le Chatelier’s principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin.

Download Full-text

Freeze-Fracture of physiologically identified neuromuscular junctions from single frog muscle fibers

Journal of Electron Microscopy Technique ◽

10.1002/jemt.1060040210 ◽

1986 ◽

Vol 4 (2) ◽

pp. 147-156 ◽

Cited By ~ 6

Author(s):

Chien-Ping Ko ◽

Jon W. Propst

Keyword(s):

Neuromuscular Junctions ◽

Muscle Fibers ◽

Freeze Fracture ◽

Frog Muscle

Download Full-text

BDM affects nucleotide binding and force generation steps of the cross-bridge cycle in rabbit psoas muscle fibers

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.2.c437 ◽

1994 ◽

Vol 266 (2) ◽

pp. C437-C447 ◽

Cited By ~ 29

Author(s):

Y. Zhao ◽

M. Kawai

Keyword(s):

Equilibrium Constants ◽

Muscle Fibers ◽

Psoas Muscle ◽

Isometric Tension ◽

Activation Mechanism ◽

Phosphate Binding ◽

Rabbit Psoas ◽

Cross Bridge ◽

The Cross ◽

Cross Bridges

The effect of 2,3-butanedione monoxime (BDM) on elementary steps of the cross-bridge cycle was studied with the sinusoidal analysis technique in skinned rabbit psoas muscle fibers. Our results showed that isometric tension and stiffness decreased progressively with an increase in the BDM concentration. The MgATP and MgADP binding constants increased 27 and 6 times, respectively, when BDM was increased from 0 to 18 mM, whereas the phosphate binding constant did not change significantly. The equilibrium constants of the ATP isomerization and detachment step were not sensitive to BDM, whereas the equilibrium constant of the attachment (power stroke) step decreased with BDM. Thus, in the presence of BDM, the number of attached cross bridges decreases; more cross bridges accumulate in the detached state, causing isometric tension and stiffness to decline. However, our detailed analysis shows that the decrease in the number of attached cross bridges is approximately 40%, which is not adequate to account for the 84% decrease in the isometric tension when 18 mM BDM was present. Therefore we suggest that a thin-filament activation mechanism is also affected by BDM.

Download Full-text