Calponin Cross-Links Unphosphorylated Myosin to Actin and Enhances its Binding Force

Abstract

Smooth muscle is unique in its capability to maintain force for long periods of time at low ATP (energy) consumption. This property, called the latch-state, is hypothesized to occur due to the dephosphorylation of myosin while attached to actin. Alternative theories have proposed that dephosphorylated-detached myosin can also re-attach to actin and contribute to force maintenance. The goal of this study was to investigate the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. To confirm the calponin regulation of our actin filaments at physiological concentrations, we used the in-vitro motility assay to measure the rate (1/2max) of actin propulsion by myosin. Our results showed a significant decrease in 1/2max (from 0.54±0.01 μm/s (mean±SE) in the absence to 0.43±0.01 μm/s in the presence of calponin). When calponin was phosphorylated, this inhibitory effect was suppressed (0.50±0.01 μm/s). To measure the force of binding (Funb) of unphosphorylated myosin to actin, we used the laser trap assay. Funb was normalized by the number of myosin molecules estimated per actin filament length. Funb of unregulated actin (0.12±0.01 pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02 pN). When calponin was phosphorylated, this enhancement was lost (0.12±0.01 pN). To verify whether this enhancement of Funb is due to cross-linking of myosinto actin by calponin, we repeated the measurements at high [KCl], as the calponin affinity for myosin decreases at high ionic strength. Indeed, the Funb, in presence of calponin, obtained at a [KCl] of 25mM (0.21±0.02 pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01 pN). These data demonstrate that calponin enhances the force of binding of unphosphorylated myosin to actin by cross-linking them together.