Caldesmon enhances motility inhibition by myosin dephosphorylation.

Abstract

Some studies have suggested that smooth muscle caldesmon (hCaD) participates in force maintenance at low myosin light chain (LC20) phosphorylation. To elucidate the effects of hCaD on the molecular mechanics of dephosphorylating smooth muscle myosin, we injected myosin light chain phosphatase (MLCP) during in vitro motility measurements performed in the presence of hCaD (400 nM) or its absence. Because hCaD increases the binding force of unphosphorylated smooth muscle myosin to actin, we hypothesized that, upon LC20 dephosphorylation, the fraction (fmot) and the average velocity (vavg) of moving actin filaments would decrease at a greater rate in the presence of hCaD. We fitted sigmoidal curves to fmot and vavg and estimated the rate constant (k) and the midpoint (t0) of the decrease. k was larger in the presence of hCaD (3.6×10−1±6.4×10−2 s−1) than without (1.8×10−1±1.4×10−2 s−1), p = 0.039, n = 5. Additionally, t0 was smaller in the presence of hCaD (32±3 s) than without (44±2 s), p = 0.008, n = 5. These results can be explained either by an increased dephosphorylation rate in the presence of hCaD or by an increase of the reattachment of dephosphorylated myosin to actin. In line with previous experiments that demonstrated that hCaD inhibits the actomyosin ATPase, our sigmoidal fits also show a lower vavg in the presence of hCaD (0.45±0.04 µm/s) than without (0.63±0.06 µm/s), p=0.04, before the addition of MLCP. However, hCaD had no effect on k or t0 of vavg during MLCP injection. The lack of concordance between the fmot and vavg data may be due to the fact that the vavg data are noisier after dephosphorylation. Our results agree with previous data obtained at the whole muscle level that showed that hCaD increases the rate of relaxation of arterial smooth muscle and accelerates the decay in tension of skinned smooth muscle strips.