Ca2+ regulation of rabbit skeletal muscle thin filament sliding: role of cross-bridge number.
We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to approximately 60% or the fraction of Ca(2+)-activatable regulatory units was reduced to approximately 33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca(2+). These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.
Liang
Bo
B
Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195, USA.
Chen
Ying
Y
Wang
Chien-Kao
CK
Luo
Zhaoxiong
Z
Regnier
Michael
M
Gordon
Albert M
AM
Chase
P Bryant
PB
D008841:Actin Cytoskeleton / Q000737:chemistry*; D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D008856:Microscopy, Fluorescence; D009068:Movement; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D009218:Myosins / Q000737:chemistry; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014336:Troponin / Q000737:chemistry
D000199:Actins; D011994:Recombinant Proteins; D014336:Troponin; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins; D002118:Calcium