Flash photolysis studies of relaxation and cross-bridge detachment: higher sensitivity of tonic than phasic smooth muscle to MgADP

Fuglasang, Annette; Khromov, Alexander S.; Török, Katalin; Somlyo, Avril V.; Somlyo, Andrew P.

doi:10.1007/bf00141563

Cited by 93 publications

(121 citation statements)

References 48 publications

(4 reference statements)

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“…An alternate view proposed by Butler and co-workers (57) suggested that the cycling rate of a given myosin head, regardless of its phosphorylation state, depends on the fraction of phosphorylated heads in the ensemble and is thus modulated as the extent of phosphorylation changes during a contraction. Finally, the Somlyos and their co-workers (58) proposed that latch results from the high ADP affinity of minus-insert myosin leading to a longer attached lifetime, which activates the thin filament and allows cooperative binding of both phosphorylated and dephosphorylated myosin to actin. This strong binding would maintain force for sustained periods of time.…”

Section: Discussionmentioning

confidence: 99%

The Unique Properties of Tonic Smooth Muscle Emerge from Intrinsic as Well as Intermolecular Behaviors of Myosin Molecules

Baker

Brosseau

Fagnant

et al. 2003

Journal of Biological Chemistry

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To better understand the molecular basis for some of the unique mechanical properties of tonic smooth muscle, we use a laser trap to assay the mechanochemistry of single smooth muscle heavy meromyosin molecules lacking a seven-amino acid insert in the nucleotide binding loop (minus insert). We measured a second-order ATP-induced actin dissociation rate, k T , of 2. , and an ADP affinity, K D , of 3.2 M, which is more than 100-fold greater than that measured for skeletal muscle myosin. By performing in vitro motility studies under nearly identical conditions, we show that the relatively slow actin velocity generated by minus-insert heavy meromyosin is significantly influenced, but not limited, by k ؊D . Our results support a model in which two separate intermediate steps in the actin-myosin catalyzed ATP hydrolysis reaction are energetically coupled through mechanical interactions, and we discuss this model in the context of the ability of tonic muscle to maintain high forces at low energetic cost (latch).Muscle shortening and force generation result from actinmyosin binding events that are coupled to the actin-myosin catalyzed ATP hydrolysis reaction illustrated in Fig. 1. Upon binding to an actin filament (A) and releasing inorganic phosphate (P i ), myosin undergoes a large and discrete rotation of its lever-like light chain domain, which is capable of generating both motion and force (1-5). With the subsequent release of ADP (at the rate k ϪD ) an additional rotation of the light chain domain of myosin has been observed in smooth muscle myosin (6, 7), but unlike the work generating rotation associated with actin binding/P i release, the rotation associated with ADP release is thought to be a strain-sensing biochemical step (8 -10). Following the release of ADP, ATP binding induces the detachment of myosin from the actin filament (at the rate k T ), after which ATP is hydrolyzed.Muscles differ significantly in their shortening speeds and force-generating capacities. For instance, smooth muscle produces a greater average force per myosin and slower speeds of shortening than skeletal muscle (11). To a large extent these mechanical differences are caused by kinetic differences among the different myosin isoforms that exist within different muscle types (12, 13). For example, phasic and tonic smooth muscle (found in the intestine and aorta respectively) express two myosin heavy chain isoforms that differ by a seven-amino acid insert in a surface loop spanning their nucleotide binding pocket (14 -16). Phasic smooth muscle contains primarily the plus-insert myosin, whereas tonic smooth muscle contains primarily the minus-insert myosin. In addition, two essential light chain isoforms are coordinately expressed with the heavy chain isoforms. The acidic isoform (LC 17a ) is coexpressed with the plus-insert heavy chain whereas the basic isoform (LC 17b ) coexpresses with the minus-insert heavy chain (17-19). Based on in vitro motility studies, the presence or absence of the sevenamino acid insert in the heavy chain is t...

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Section: Discussionmentioning

confidence: 99%

The Unique Properties of Tonic Smooth Muscle Emerge from Intrinsic as Well as Intermolecular Behaviors of Myosin Molecules

Baker

Brosseau

Fagnant

et al. 2003

Journal of Biological Chemistry

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“…In tonic smooth muscles, such as rabbit femoral artery and aorta and porcine aorta, expression of LC 17b ranges between 43 and 58% of total LC 17 content (10,11,31). Therefore, the level of exchange achieved in the present study was consistent with the LC 17a /LC 17b ratio that correlates with the lower ATPase activity and slower V us and rates of force development by tonic smooth muscle (10,11,31).…”

Section: Discussionmentioning

confidence: 99%

“…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)(8)(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 in vitro studies have produced conflicting results. Increasing the LC 17a content of isolated aortic myosin has been reported to increase ATPase activity (12), but motility assays failed to detect a difference in the velocity of actin movement as a function of LC 17 isoform, although they showed faster movement propelled by myosin containing the 7-amino acid insert in the motor domain (4, 13).…”

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confidence: 99%

“…To that end, we exchanged the basic isoform, LC 17b , into bladder and amnion smooth muscles, both of which have fast, phasic properties and express only the LC 17a isoform (Ref. 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.…”

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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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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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“…This high force-low phosphorylation state (catch-like state, also known as latch) occurs in smooth muscle after a decline in Ca 2þ concentration that initiates dephosphorylation of RLC (Dillion et al 1981). The high affinity of smooth muscle myosin for ADP (Vyas et al 1992;Fuglsang et al 1993;Nishiye et al 1993;Butler & Siegman 1998;Cremo & Geeves 1998;Gollub et al 1999), cycling of dephosphorylated cross-bridges (Vyas et al 1992;Butler & Siegman 1998;Somlyo et al 1988) and the effects of strain and RLC thiophosphorylation on product release (Khromov et al 2004) contribute to this catch-like state and will be one focus of this review along with a survey of some of the structure-linked changes evident in smooth muscle myosin.…”

Section: Introductionmentioning

confidence: 99%

Smooth muscle myosin: regulation and properties

Somlyo

Khromov

Webb³

et al. 2004

Phil. Trans. R. Soc. Lond. B

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The relationship of the biochemical states to the mechanical events in contraction of smooth muscle crossbridges is reviewed. These studies use direct measurements of the kinetics of P i and ADP release. The rate of release of P i from thiophosphorylated cycling cross-bridges held isometric was biphasic with turnovers of 1.8 s À1 and 0.3 s À1 , reflecting properties and forces directly acting on cross-bridges through mechanisms such as positive strain and inhibition by high-affinity MgADP binding. Fluorescent transients reporting release of an ADP analogue 3 0 -deac-edaADP were significantly faster in phasic than in tonic smooth muscles. Thiophosphorylation of myosin regulatory light chains (RLCs) increased and positive strain decreased the release rate around twofold. The rates of ADP release from rigor cross-bridges and the steady-state P i release from cycling isometric cross-bridges are similar, indicating that the ADP-release step or an isomerization preceding it may limit the ATPase rate. Thus ADP release in phasic and tonic smooth muscles is a regulated step with strain-and dephosphorylation-dependence. High affinity of cross-bridges for ADP and slow ADP release prolong the fraction of the duty cycle occupied by strongly bound AMÁADP state(s) and contribute to the high economy of force that is characteristic of smooth muscle. RLC thiophosphorylation led to structural changes in smooth muscle cross-bridges consistent with our findings that thiophosphorylation and strain modulate product release.

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Flash photolysis studies of relaxation and cross-bridge detachment: higher sensitivity of tonic than phasic smooth muscle to MgADP

Cited by 93 publications

References 48 publications

The Unique Properties of Tonic Smooth Muscle Emerge from Intrinsic as Well as Intermolecular Behaviors of Myosin Molecules

The Unique Properties of Tonic Smooth Muscle Emerge from Intrinsic as Well as Intermolecular Behaviors of Myosin Molecules

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

Smooth muscle myosin: regulation and properties

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