Calcium regulation of skeletal muscle thin filament motility in vitro.
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.
Gordon
A M
AM
Department of Physiology and Biophysics, University of Washington, Seattle 98195, USA. amg@u.washington.edu
LaMadrid
M A
MA
Chen
Y
Y
Luo
Z
Z
Chase
P B
PB
D000199:Actins / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000494:pharmacology* / Q000502:physiology; D066298:In Vitro Techniques; D007700:Kinetics; D016018:Least-Squares Analysis; D008856:Microscopy, Fluorescence; D009068:Movement; D009119:Muscle Contraction / Q000502:physiology*; D018656:Muscle Fibers, Fast-Twitch / Q000187:drug effects / Q000502:physiology*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology*; D015879:Myosin Subfragments / Q000502:physiology; D009218:Myosins / Q000502:physiology; D009994:Osmolar Concentration; D011817:Rabbits; D014335:Tropomyosin / Q000502:physiology; D014336:Troponin / Q000502:physiology
D000199:Actins; D015879:Myosin Subfragments; D014335:Tropomyosin; D014336:Troponin; D009218:Myosins; D002118:Calcium