Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. Funb from F-actin alone (0.12 ± 0.01pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01pN). To further verify that this enhancement of Funb was due to cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25 mM (0.21 ± 0.02pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01pN). This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.
Tuesday, June 11, 2013
Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin
Horia Nicolae Roman, Nedjma B. Zitouni, Linda Kachmar, Gijs Ijpma, Lennart Hilbert, Oleg Matusovskiy, Andrea Benedetti, Apolinary Sobieszek, Anne-Marie Lauzon
Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. Funb from F-actin alone (0.12 ± 0.01pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01pN). To further verify that this enhancement of Funb was due to cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25 mM (0.21 ± 0.02pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01pN). This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.
Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. Funb from F-actin alone (0.12 ± 0.01pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01pN). To further verify that this enhancement of Funb was due to cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25 mM (0.21 ± 0.02pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01pN). This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.
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