Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed

Malmqvist, Ulf; Trybus, Kathleen M.; Yagi, Shinobu; Carmichael, Jeffrey; Fay, Fredric S.

doi:10.1073/pnas.94.14.7655

Cited by 50 publications

(53 citation statements)

References 41 publications

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“…Unlike experiments on isolated myosin (14) or single cells (16), treatment of Triton-permeabilized rabbit bladder strips with TFP caused no detectable loss of the RLC, LC 17 , or calponin, as determined in whole homogenates (Fig. 1A) or MHC immunoprecipitates (Fig.…”

Section: The Effect Of Tfp On the Light Chain Content And Properties mentioning

confidence: 96%

“…2 However, the smooth muscle fibers in bladder bundles were better (more parallel) oriented and, unless otherwise stated, all mechanical experiments reported were performed using rabbit bladder detrusor muscle strips. Strips were stretched by 10% of their resting length and, after determining a response to high K ϩ , permeabilized in a pCa 6.5 solution containing Triton X-100 (0.05%, v/v) and A23187 (10 M), to deplete internal Ca 2ϩ stores, for 15-20 min before washing in relaxing solution and incubation in a rigor solution designed to facilitate light chain exchange and based on that used previously (16), it contained (mM): 1 TFP, 10 CDTA, 150 potassium methanesulfonate, and 20 PIPES (pH 6.5) for 15 min at 30°C (see Fig. 7A for illustration of this protocol).…”

Section: Tissue Preparation and Light Chain Exchangementioning

confidence: 99%

“…10 and the present study). We used TFP, a hydrophobic cation that facilitates extraction and/or exchange of light chains from isolated myosin (12,14,15) and single cells (16), to promote light chain exchange in permeabilized smooth muscle. Our results show a significant LC 17 isoform-specific effect on the unloaded shortening velocity of nonphosphorylated, slowly cycling cross-bridges, but no detectable effect on muscles in which the RLCs are thiophosphorylated.…”

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Myosin Essential Light Chain Isoforms Modulate the Velocity of Shortening Propelled by Nonphosphorylated Cross-bridges

Matthew

Khromov

Trybus

et al. 1998

Journal of Biological Chemistry

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The differential effects of essential light chain isoforms (LC 17a and LC 17b ) on the mechanical properties of smooth muscle were determined by exchanging recombinant for endogenous LC 17 in permeabilized smooth muscle treated with trifluoperazine (TFP). Co-precipitation with endogenous myosin heavy chain verified that 40 -60% of endogenous LC 17a could be exchanged for recombinant LC 17a or LC 17b . Upon addition of MgATP in Ca 2؉ -free solution, recombinant LC 17 exchange induced slow contractions unaccompanied by regulatory light chain (RLC) phosphorylation only in TFP-treated, but not in untreated, permeabilized smooth muscle; the shortening velocity and rate of force development were approximately 1.5 and 2 times faster, respectively, in response to LC 17a than LC 17b . Additional incubation with recombinant, thiophosphorylated RLC increased the shortening velocity, independent of the LC 17 isoform exchanged. The LC 17 -induced contractions of TFPtreated muscles were abolished by prior addition of nonphosphorylated RLC. We suggest that LC 17 stiffens the lever arm of myosin and, in the absence of regulation by RLC, permits cross-bridge cycling without requiring RLC phosphorylation. Our results are compatible with nonphosphorylated RLC acting as a repressor and with LC 17 isoforms modulating the MgADP affinity and, consequently, rate of cooperative cycling of nonphosphorylated cross-bridges.The markedly different rates of contraction and relaxation in fast, phasic and slow, tonic smooth muscles reflect differences not only at the level of the membrane potential, signal transduction, rates of myosin light chain phosphorylation and dephosphorylation, but also in the kinetic properties of the actomyosin motor. The muscle-specific differences in actomyosin kinetics have been ascribed to the existence of specific myosin isoforms, but their relative contributions to the mechanical properties of smooth muscles have not been unequivocally demonstrated (reviewed in Ref. 1). The expression of the two myosin heavy chain (MHC) 1 isoforms, SM-1 and SM-2 (2) that differ by, respectively, the presence or absence of a 34-amino acid extension of the carboxyl terminus (3), does not correlate with phasic or tonic kinetics of contraction. On the other hand, motility assays show different rates of movement propelled by MHC isoforms that are the products of an alternative splicing mechanism resulting in the presence or absence of a 7-amino acid insert near the nucleotide-binding region of the myosin head (4, 5). Disparate results have been obtained concerning the effects on contractile kinetics of the two LC 17 isoforms, acidic (LC 17a ) and basic (LC 17b ), that differ in 5 of the 9 COOHterminal amino acid residues and are products of a single gene generated by an alternative splicing mechanism (6). The faster kinetics of phasic, smooth muscle in situ (7-9) correlates with the expression of the LC 17a isoform (10, 11), albeit the proportion of myosin heavy chain containing the insert was also variant in these muscles, whereas ...

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Section: The Effect Of Tfp On the Light Chain Content And Properties mentioning

confidence: 96%

Section: Tissue Preparation and Light Chain Exchangementioning

confidence: 99%

mentioning

confidence: 99%

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Myosin Essential Light Chain Isoforms Modulate the Velocity of Shortening Propelled by Nonphosphorylated Cross-bridges

Matthew

Khromov

Trybus

et al. 1998

Journal of Biological Chemistry

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“…Some lines of evidence have been accumulated to indicate the in vitro biochemical properties of calponin (Haeberle et al ., 1994;Hodgkinson et al ., 1997;Malmqvist et al ., 1997). To elucidate the function of calponin physiologically, mice with a mutated basic calponin locus were generated and the mechanical properties of smooth muscle in intact tissue were compared between calponin knockout (KO) mice and wild type (WT) one Matthew et al ., 2000;Takahashi et al ., 2000).…”

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confidence: 99%

Altered Mechanical Properties in Smooth Muscle of Mice with a Mutated Calponin Locus

2002

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“…Exogenously added CaP is capable of inhibiting contractile activity of permeabilized smooth muscle (reviewed in Horowitz, Menice, LaPorte & Morgan, 1996b). Peptides from the CaP sequence have been reported to reverse an in situ effect of CaP to suppress vascular tone (Itoh, Suzuki, Watanabe, Mino, Naka & Tanaka, 1995;Horowitz, ClementChomienne, Walsh, Tao, Katsuyama & Morgan, 1996a) and extraction of CaP results in contraction of smooth muscle cells (Malmqvist, Trybus, Yagi, Carmichael & Fay, 1997). Thus, current evidence points to the possibility that CaP is a physiologically important regulator of contractility of differentiated smooth muscle.…”

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Cytoskeletal targeting of calponin in differentiated, contractile smooth muscle cells of the ferret

Parker

Takahashi

Tang

et al. 1998

The Journal of Physiology

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Biochemical and quantitative image analysis methods were used to investigate the anatomical basis for the previously described agonist‐induced redistribution of calponin. At 140 nm resolution, the quantitative distribution of calponin in resting cells was statistically indistinguishable from that of filament bundles containing α‐smooth muscle actin and myosin, but was significantly different from that of filaments containing β‐non‐muscle actin. Conversely, in stimulated cells, the distribution of calponin was not significantly different from that of β‐actin filaments in the subplasmalemmal cell cortex but was significantly different from the distribution of α‐actin‐ and myosin‐containing filamentous bundles. The distribution of calponin significantly differed from that of the intermediate filament proteins vimentin and desmin as well as that of the dense body protein α‐actinin either by ratio analysis of the subcellular distribution or by colocalization analysis. The imaging results, although limited to 140 nm spatial resolution, suggested the hypothesis that the agonist‐induced redistribution involves the binding of calponin to isoform‐specific actin filaments. This hypothesis was tested by quantifying the relative affinity of calponin for purified α‐ and β‐actin. Light scattering measurements showed that calponin induces bundle formation with β‐actin more readily than α‐actin, indicating that calponin may be preferentially sequestered by β‐actin under appropriate conditions. These results are consistent with a model whereby agonist activation decreases calponin's binding to filaments, but the tighter binding to β‐actin filaments results in a spatial redistribution of calponin to the submembranous cortex.

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Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed

Cited by 50 publications

References 41 publications

Myosin Essential Light Chain Isoforms Modulate the Velocity of Shortening Propelled by Nonphosphorylated Cross-bridges

Myosin Essential Light Chain Isoforms Modulate the Velocity of Shortening Propelled by Nonphosphorylated Cross-bridges

Altered Mechanical Properties in Smooth Muscle of Mice with a Mutated Calponin Locus

Cytoskeletal targeting of calponin in differentiated, contractile smooth muscle cells of the ferret

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