Observing the pCa-Force Relationship with a 3-Bead Laser Trap Assay

Abstract

Muscular force is highly dependent on the amount of free Ca++ in the myoplasm because actomyosin binding is carefully regulated by troponin (Tn) and tropomyosin (Tm). To gain molecular insight into the Ca++ dependence of force, we directly observed the effect of Ca++ on the force-generating capacity of a mini-ensemble of myosin (∼6 heads) interacting with a single reconstituted thin filament (actin+Tn+Tm) in a three bead laser trap assay at 100uM ATP. Maximum force decreased in a Ca++-dependent manner (6.75, 4.75, 3.25, and 1.85pN at pCa 5, 6, 6.5 and 7 respectively). Furthermore, the average forces were significantly different (p 1.5pN where only 0.1% of the events achieved this force level at pCa7. In addition, the total percent of time the myosin molecules were strongly bound to the thin filament were 41, 21, 16, and 14% at pCa 5, 6, 6.5 and 7 respectively. Overall, these data provide unique insight into the molecular events that underlie the Ca++-dependence of force generation in a regulated system.