Calcium regulation of skeletal muscle thin filament motility in vitro

Biophys J. 1997 Mar;72(3):1295-307. doi: 10.1016/S0006-3495(97)78776-9.

Abstract

Using an in vitro motility assay, we have investigated Ca2+ regulation of individual, regulated thin filaments reconstituted from rabbit fast skeletal actin, troponin, and tropomyosin. Rhodamine-phalloidin labeling was used to visualize the filaments by epifluorescence, and assays were conducted at 30 degrees C and at ionic strengths near the physiological range. Regulated thin filaments exhibited well-regulated behavior when tropomyosin and troponin were added to the motility solutions because there was no directed motion in the absence of Ca2+. Unlike F-actin, the speed increased in a graded manner with increasing [Ca2+], whereas the number of regulated thin filaments moving was more steeply regulated. With increased ionic strength, Ca2+ sensitivity of both the number of filaments moving and their speed was shifted toward higher [Ca2+] and was steepest at the highest ionic strength studied (0.14 M gamma/2). Methylcellulose concentration (0.4% versus 0.7%) had no effect on the Ca2+ dependence of speed or number of filaments moving. These conclusions hold for five different methods used to analyze the data, indicating that the conclusions are robust. The force-pCa relationship (pCa = -log10[Ca2+]) for rabbit psoas skinned fibers taken under similar conditions of temperature and solution composition (0.14 M gamma/2) paralleled the speed-pCa relationship for the regulated filaments in the in vitro motility assay. Comparison of motility results with the force-pCa relationship in fibers suggests that relatively few cross-bridges are needed to make filaments move, but many have to be cycling to make the regulated filament move at maximum speed.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / physiology
  • Animals
  • Calcium / pharmacology*
  • Calcium / physiology
  • In Vitro Techniques
  • Kinetics
  • Least-Squares Analysis
  • Microscopy, Fluorescence
  • Movement
  • Muscle Contraction / physiology*
  • Muscle Fibers, Fast-Twitch / drug effects
  • Muscle Fibers, Fast-Twitch / physiology*
  • Muscle, Skeletal / drug effects
  • Muscle, Skeletal / physiology*
  • Myosin Subfragments / physiology
  • Myosins / physiology
  • Osmolar Concentration
  • Rabbits
  • Tropomyosin / physiology
  • Troponin / physiology

Substances

  • Actins
  • Myosin Subfragments
  • Tropomyosin
  • Troponin
  • Myosins
  • Calcium