Effects of partial extraction of light chain 2 on the Ca²⁺ sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers

Various functional roles for myosin light chain 2 (LC₂) have been suggested on the basis of numerous and predominantly in vitro biochemical studies. Using skinned fibers from rabbit psoas muscle, the present study examines the influence of partial removal of LC₂ on isometric tension, stiffness, and maximum velocity of shortening at various levels of activation by Ca²⁺. Isometric tension, stiffness, and velocity of shortening were measured at pCa values between 6.6 and 4.5 (a) in a control fiber segment, (b) in the same fiber segment after partial removal of LC₂, and (c) after recombination with LC₂. The extraction solution contained 20 mM EDTA, 20 or 50 mM KCl, and either imidazole or PO₄^2- as a pH buffer (pH 7.0). The amount of LC₂ extracted varied with the temperature, duration of extraction, and whether or not troponin C (0.5 mg/ml) was added to the extraction solution. Extraction of 20-40% LC₂ resulted in increased active tensions in the range of pCa's between 6.6 and 5.7, but had no effect upon maximum tension. The tension-pCa relationship was left-shifted to lower [Ca²⁺] by as much as 0.2 pCa units after LC₂ extraction. At low concentrations of Ca²⁺, an increase in stiffness proportional to the increase in tension was observed. Readdition of LC₂ to these fiber segments resulted in a return of tension and stiffness to near control values. Stiffness during maximal activation was unaffected by partial extraction of LC₂. LC₂ extraction was shown to uniformly decrease (by 25-30%), the velocity of shortening during the high velocity phase but it did not significantly affect the low velocity phase of shortening. This effect was reversed by readdition of purified LC₂ to the fiber segments. On the basis of these findings we conclude that LC₂ may modulate the number of cross-bridges formed during Ca²⁺ activation and also the rate of cross-bridge detachment during shortening. These results are consistent with the idea that LC₂ may modulate contraction via an influence upon the conformation of the S1-S2 hinge region of myosin.