Publications by authors named "Christine R Cremo"

28 Publications

  • Page 1 of 1

Evidence for S2 flexibility by direct visualization of quantum dot-labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro.

J Gen Physiol 2021 Mar;153(3)

Department of Pharmacology, School of Medicine, University of Nevada, Reno, Reno, NV.

Myosins in muscle assemble into filaments by interactions between the C-terminal light meromyosin (LMM) subdomains of the coiled-coil rod domain. The two head domains are connected to LMM by the subfragment-2 (S2) subdomain of the rod. Our mixed kinetic model predicts that the flexibility and length of S2 that can be pulled away from the filament affects the maximum distance working heads can move a filament unimpeded by actin-attached heads. It also suggests that it should be possible to observe a head remain stationary relative to the filament backbone while bound to actin (dwell), followed immediately by a measurable jump upon detachment to regain the backbone trajectory. We tested these predictions by observing filaments moving along actin at varying ATP using TIRF microscopy. We simultaneously tracked two different color quantum dots (QDs), one attached to a regulatory light chain on the lever arm and the other attached to an LMM in the filament backbone. We identified events (dwells followed by jumps) by comparing the trajectories of the QDs. The average dwell times were consistent with known kinetics of the actomyosin system, and the distribution of the waiting time between observed events was consistent with a Poisson process and the expected ATPase rate. Geometric constraints suggest a maximum of ∼26 nm of S2 can be unzipped from the filament, presumably involving disruption in the coiled-coil S2, a result consistent with observations by others of S2 protruding from the filament in muscle. We propose that sufficient force is available from the working heads in the filament to overcome the stiffness imposed by filament-S2 interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1085/jgp.202012751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809879PMC
March 2021

Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain.

Cytoskeleton (Hoboken) 2019 02 20;76(2):192-199. Epub 2019 Mar 20.

Department of Pharmacology, University of Nevada, Reno, Nevada.

The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys-108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin-activated steady-state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C-terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC-Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cm.21516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520152PMC
February 2019

A mixed-kinetic model describes unloaded velocities of smooth, skeletal, and cardiac muscle myosin filaments in vitro.

Sci Adv 2017 12 13;3(12):eaao2267. Epub 2017 Dec 13.

Department of Pharmacology, University of Nevada, Reno School of Medicine, 1664 North Virginia Street, Reno, NV 89557, USA.

In vitro motility assays, where purified myosin and actin move relative to one another, are used to better understand the mechanochemistry of the actomyosin adenosine triphosphatase (ATPase) cycle. We examined the relationship between the relative velocity () of actin and myosin and the number of available myosin heads () or [ATP] for smooth (SMM), skeletal (SKM), and cardiac (CMM) muscle myosin filaments moving over actin as well as from actin filaments moving over a bed of monomeric SKM. These data do not fit well to a widely accepted model that predicts that is limited by myosin detachment from actin (/), where equals step size and equals time a myosin head remains attached to actin. To account for these data, we have developed a mixed-kinetic model where is influenced by both attachment and detachment kinetics. The relative contributions at a given vary with the probability that a head will remain attached to actin long enough to reach the end of its flexible S2 tether. Detachment kinetics are affected by /, where is related to the tether length. We show that is relatively long for SMM, SKM, and CMM filaments (59 ± 3 nm, 22 ± 9 nm, and 22 ± 2 nm, respectively). In contrast, is shorter (8 ± 3 nm) when myosin monomers are attached to a surface. This suggests that the behavior of the S2 domain may be an important mechanical feature of myosin filaments that influences unloaded shortening velocities of muscle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aao2267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733112PMC
December 2017

Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates.

Cell Biochem Funct 2016 Oct 16;34(7):469-474. Epub 2016 Aug 16.

Department of Pharmacology, University of Nevada Reno School of Medicine, Reno, Nevada, USA.

Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher k for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas K values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (k /K ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.

Significance Of The Study: Phosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5076371PMC
http://dx.doi.org/10.1002/cbf.3209DOI Listing
October 2016

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle.

J Gen Physiol 2015 Oct;146(4):267-80

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557

Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot-labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto-myosin and MLCK-myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting "stuck" on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1085/jgp.201511483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586593PMC
October 2015

Velocities of unloaded muscle filaments are not limited by drag forces imposed by myosin cross-bridges.

Proc Natl Acad Sci U S A 2015 Sep 20;112(36):11235-40. Epub 2015 Aug 20.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557

It is not known which kinetic step in the acto-myosin ATPase cycle limits contraction speed in unloaded muscles (V0). Huxley's 1957 model [Huxley AF (1957) Prog Biophys Biophys Chem 7:255-318] predicts that V0 is limited by the rate that myosin detaches from actin. However, this does not explain why, as observed by Bárány [Bárány M (1967) J Gen Physiol 50(6, Suppl):197-218], V0 is linearly correlated with the maximal actin-activated ATPase rate (vmax), which is limited by the rate that myosin attaches strongly to actin. We have observed smooth muscle myosin filaments of different length and head number (N) moving over surface-attached F-actin in vitro. Fitting filament velocities (V) vs. N to a detachment-limited model using the myosin step size d=8 nm gave an ADP release rate 8.5-fold faster and ton (myosin's attached time) and r (duty ratio) ∼10-fold lower than previously reported. In contrast, these data were accurately fit to an attachment-limited model, V=N·v·d, over the range of N found in all muscle types. At nonphysiologically high N, V=L/ton rather than d/ton, where L is related to the length of myosin's subfragment 2. The attachment-limited model also fit well to the [ATP] dependence of V for myosin-rod cofilaments at three fixed N. Previously published V0 vs. vmax values for 24 different muscles were accurately fit to the attachment-limited model using widely accepted values for r and N, giving d=11.1 nm. Therefore, in contrast with Huxley's model, we conclude that V0 is limited by the actin-myosin attachment rate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1510241112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568697PMC
September 2015

The kinetics underlying the velocity of smooth muscle myosin filament sliding on actin filaments in vitro.

J Biol Chem 2014 Jul;289(30):21055-70

Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ~0.63 μm long and contain ~176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment- limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment- limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.564740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4110310PMC
July 2014

Sucrose increases the activation energy barrier for actin-myosin strong binding.

Arch Biochem Biophys 2014 Jun 25;552-553:74-82. Epub 2013 Dec 25.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV, United States. Electronic address:

To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880mM) slowed the rate of actin-myosin (A-M) strong binding by 71±8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21±3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85±9% with a smaller inhibitory effect on ATP-induced A-M dissociation, kT (39±2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25±2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A-M strong binding by 37±3%, indicating that sucrose inhibits the rate of A-M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A-M rigor binding (71%) are comparable in magnitude to sucrose's effects on both V (79±33% decrease) and maximal actin-activated ATPase, kcat, (81±16% decrease), indicating that the rate of A-M strong bond formation significantly influences both kcat and V.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.abb.2013.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4043939PMC
June 2014

Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

Biochemistry 2013 Nov 11;52(47):8489-500. Epub 2013 Nov 11.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine , Reno, Nevada 99557, United States.

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (<0.1-0.2 s⁻¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi401001xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3886827PMC
November 2013

The myosin duty ratio tunes the calcium sensitivity and cooperative activation of the thin filament.

Biochemistry 2013 Sep 3;52(37):6437-44. Epub 2013 Sep 3.

Department of Electrical and Biomedical Engineering, University of Nevada, Reno , Reno, Nevada 89557, United States.

In striated muscle, calcium binding to the thin filament (TF) regulatory complex activates actin-myosin ATPase activity, and actin-myosin kinetics in turn regulates TF activation. However, a quantitative description of the effects of actin-myosin kinetics on the calcium sensitivity (pCa50) and cooperativity (nH) of TF activation is lacking. With the assumption that TF structural transitions and TF-myosin binding transitions are inextricably coupled, we advanced the principles established by Kad et al. [Kad, N., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 16990-16995] and Sich et al. [Sich, N. M., et al. (2011) J. Biol. Chem. 285, 39150-39159] to develop a simple model of TF regulation, which predicts that pCa50 varies linearly with duty ratio and that nH is maximal near physiological duty ratios. Using in vitro motility to determine the calcium sensitivity of TF sliding velocities, we measured pCa50 and nH at different myosin densities and in the presence of ATPase inhibitors. The observed effects of myosin density and actin-myosin duty ratio on pCa50 and nH are consistent with our model predictions. In striated muscle, pCa50 must match cytosolic calcium concentrations and a maximal nH optimizes calcium responsiveness. Our results indicate that pCa50 and nH can be predictably tuned through TF-myosin ATPase kinetics and that drugs and disease states that alter ATPase kinetics can, through their effects on calcium sensitivity, alter the efficiency of muscle contraction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi400262hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7207222PMC
September 2013

Actin Sliding Velocities are Influenced by the Driving Forces of Actin-Myosin Binding.

Cell Mol Bioeng 2013 Mar;6(1):26-37

University of Nevada Reno School of Medicine, Department of Biochemistry and Molecular Biology, Reno, NV 89557.

Unloaded shortening speeds, , of muscle are thought to be limited by actin-bound myosin heads that resist shortening, or = ·· where is the rate at which myosin detaches from actin and is myosin's step size. The -term describes the efficiency of force transmission between myosin heads, and has been shown to become less than one at low myosin densities in a motility assay. Molecules such as inorganic phosphate, P, and blebbistatin inhibit both and actin-myosin strong binding kinetics suggesting a link between and attachment kinetics. To determine whether these small molecules slow by increasing resistance to actin sliding or by decreasing the efficiency of force transmission, , we determine how inhibition of by Pi and blebbistatin changes the force exerted on actin filaments during an in vitro sliding assay, measured from changes in the rate, , at which actin filaments break. Upon addition of 30 mM P to a low (30 μM) [ATP] motility buffer decreased from 1.8 to 1.3 μm·sec and from 0.029 to 0.018 sec. Upon addition of 50 μM blebbistatin to a low [ATP] motility buffer, decreased from 1.0 to 0.7 μm·sec and from 0.059 to 0.022 sec. These results imply that blebbistatin and P slow by decreasing force transmission, , not by increasing resistive forces, implying that actin-myosin attachment kinetics influence .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12195-013-0274-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3627502PMC
March 2013

Modification of interface between regulatory and essential light chains hampers phosphorylation-dependent activation of smooth muscle myosin.

J Biol Chem 2012 Jun 1;287(26):22068-79. Epub 2012 May 1.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557, USA.

We examined the regulatory importance of interactions between regulatory light chain (RLC), essential light chain (ELC), and adjacent heavy chain (HC) in the regulatory domain of smooth muscle heavy meromyosin. After mutating the HC, RLC, and/or ELC to disrupt their predicted interactions (using scallop myosin coordinates), we measured basal ATPase, V(max), and K(ATPase) of actin-activated ATPase, actin-sliding velocities, rigor binding to actin, and kinetics of ATP binding and ADP release. If unphosphorylated, all mutants were similar to wild type showing turned-off behaviors. In contrast, if phosphorylated, mutation of RLC residues smM129Q and smG130C in the F-G helix linker, which interact with the ELC (Ca(2+) binding in scallop), was sufficient to abolish motility and diminish ATPase activity, without altering other parameters. ELC mutations within this interacting ELC loop (smR20M and smK25A) were normal, but smM129Q/G130C-R20M or -K25A showed a partially recovered phenotype suggesting that interaction between the RLC and ELC is important. A molecular dynamics study suggested that breaking the RLC/ELC interface leads to increased flexibility at the interface and ELC-binding site of the HC. We hypothesize that this leads to hampered activation by allowing a pre-existing equilibrium between activated and inhibited structural distributions (Vileno, B., Chamoun, J., Liang, H., Brewer, P., Haldeman, B. D., Facemyer, K. C., Salzameda, B., Song, L., Li, H. C., Cremo, C. R., and Fajer, P. G. (2011) Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 108, 8218-8223) to be biased strongly toward the inhibited distribution even when the RLC is phosphorylated. We propose that an important structural function of RLC phosphorylation is to promote or assist in the maintenance of an intact RLC/ELC interface. If the RLC/ELC interface is broken, the off-state structures are no longer destabilized by phosphorylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.343491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381165PMC
June 2012

Biochemistry of smooth muscle myosin light chain kinase.

Arch Biochem Biophys 2011 Jun 3;510(2):135-46. Epub 2011 May 3.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, 89557, USA.

The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca(2+)-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.abb.2011.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382066PMC
June 2011

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation.

Proc Natl Acad Sci U S A 2011 May 2;108(20):8218-23. Epub 2011 May 2.

National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310, USA.

Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the short-tailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the off-state, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1014137108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100986PMC
May 2011

Role of the tail in the regulated state of myosin 2.

J Mol Biol 2011 May 23;408(5):863-78. Epub 2011 Mar 23.

Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2011.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776433PMC
May 2011

Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells.

Proc Natl Acad Sci U S A 2011 Jan 4;108(4):1421-6. Epub 2011 Jan 4.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.

The 10S self-inhibited monomeric conformation of myosin II has been characterized extensively in vitro. Based upon its structural and functional characteristics, it has been proposed to be an assembly-competent myosin pool in equilibrium with filaments in cells. It is known that myosin filaments can assemble and disassemble in nonmuscle cells, and in some smooth muscle cells, but whether or not the disassembled pool contains functional 10S myosin has not been determined. Here we address this question using human airway smooth muscle cells (hASMCs). Using two antibodies against different epitopes on smooth muscle myosin II (SMM), two distinct pools of SMM, diffuse, and stress-fiber-associated, were visualized by immunocytochemical staining. The two SMM pools were functional in that they could be interconverted in two ways: (i) by exposure to 10S- versus filament-promoting buffer conditions, and (ii) by exposure to a peptide that shifts the filament-10S equilibrium toward filaments in vitro by a known mechanism that requires the presence of the 10S conformation. The effect of the peptide was not due to a trivial increase in SMM phosphorylation, and its specificity was demonstrated by use of a scrambled peptide, which had no effect. Based upon these data, we conclude that hASMCs contain a significant pool of functional SMM in the 10S conformation that can assemble into filaments upon changing cellular conditions. This study provides unique direct evidence for the presence of a significant pool of functional myosin in the 10S conformation in cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1011784108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3029703PMC
January 2011

Effects of actin-myosin kinetics on the calcium sensitivity of regulated thin filaments.

J Biol Chem 2010 Dec 2;285(50):39150-9. Epub 2010 Oct 2.

Department of Biochemistry, University of Nevada School of Medicine, Reno, Nevada 89557, USA.

Activation of thin filaments in striated muscle occurs when tropomyosin exposes myosin binding sites on actin either through calcium-troponin (Ca-Tn) binding or by actin-myosin (A-M) strong binding. However, the extent to which these binding events contributes to thin filament activation remains unclear. Here we propose a simple analytical model in which strong A-M binding and Ca-Tn binding independently activates the rate of A-M weak-to-strong binding. The model predicts how the level of activation varies with pCa as well as A-M attachment, N·k(att), and detachment, k(det), kinetics. To test the model, we use an in vitro motility assay to measure the myosin-based sliding velocities of thin filaments at different pCa, N·k(att), and k(det) values. We observe that the combined effects of varying pCa, N·k(att), and k(det) are accurately fit by the analytical model. The model and supporting data imply that changes in attachment and detachment kinetics predictably affect the calcium sensitivity of striated muscle mechanics, providing a novel A-M kinetic-based interpretation for perturbations (e.g. disease-related mutations) that alter calcium sensitivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.142232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998111PMC
December 2010

Kinetic and motor functions mediated by distinct regions of the regulatory light chain of smooth muscle myosin.

Biochim Biophys Acta 2009 Nov 25;1794(11):1599-605. Epub 2009 Jul 25.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.

To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP(95-98)/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg(2+)-ATPase (V(max) and K(ATPase)) and in vitro actin-sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the up-state (0.007-0.030 s(-1)) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (K(ATPase)) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the V(max) was 64% and the actin-sliding velocity was approximately 50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbapap.2009.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757001PMC
November 2009

Characterization of tightly associated smooth muscle myosin-myosin light-chain kinase-calmodulin complexes.

J Mol Biol 2009 Jul 25;390(5):879-92. Epub 2009 May 25.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, 89557, USA.

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73+/-9), the ratio was approximately 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at approximately 1 nM free [Ca(2+)]. There were two MLCK pools that bound unphosphorylated SMM with K(d) approximately 10 and 0.2 microM and phosphorylated SMM with K(d) approximately 20 and 0.2 microM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca(2+) and phosphorylation of SMM occurred at a pCa(50) of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2009.05.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742357PMC
July 2009

The kinesin-1 motor protein is regulated by a direct interaction of its head and tail.

Proc Natl Acad Sci U S A 2008 Jul 25;105(26):8938-43. Epub 2008 Jun 25.

Department of Cell and Molecular Biology, Northwestern University, Chicago, IL 60611, USA.

Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1's microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-A cryoEM reconstruction of the cross-linked head-tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0803575105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2449343PMC
July 2008

H(2)O(2)-induced kinetic and chemical modifications of smooth muscle myosin: correlation to effects of H(2)O(2) on airway smooth muscle.

J Biol Chem 2007 Feb 22;282(7):4336-4344. Epub 2006 Nov 22.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557. Electronic address:

The effect of H(2)O(2) on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of P(i) release were measured by single-turnover kinetics. H(2)O(2) treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, approximately 50% of the molecules lost the ability to bind to ATP. H(2)O(2) treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H(2)O(2)-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H(2)O(2)-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mM ATP, H(2)O(2) caused a approximately 50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H(2)O(2) directly affects the force generating complex. Dithiothreitol treatment reversed the H(2)O(2) inhibition of the maximal force by approximately 50%. These data, when compared with the in vitro kinetic data, are consistent with a H(2)O(2)-induced loss of functional myosin heads in the muscle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M609499200DOI Listing
February 2007

The N-terminal lobes of both regulatory light chains interact with the tail domain in the 10 S-inhibited conformation of smooth muscle myosin.

J Biol Chem 2006 Dec 29;281(50):38801-11. Epub 2006 Sep 29.

Department of Biochemistry and Molecular Biology, School of Medicine, University of Nevada, 1664 N. Virginia Street, Reno, NV 89557, USA.

In the presence of ATP, unphosphorylated smooth muscle myosin can form a catalytically inactive monomer that sediments at 10 Svedbergs (10 S). The tail of 10 S bends into thirds and interacts with the regulatory domain. ADP-P(i) is "trapped" at the active site, and consequently the ATPase activity is extremely low. We are interested in the structural basis for maintenance of this off state. Our prior photocross-linking work with 10 S showed that tail residues 1554-1583 are proximal to position 108 in the C-terminal lobe of one of the two regulatory light chains ( Olney, J. J., Sellers, J. R., and Cremo, C. R. (1996) J. Biol. Chem. 271, 20375-20384 ). These data suggested that the tail interacts with only one of the two regulatory light chains. Here we present data, using a photocross-linker on position 59 on the N-terminal lobe of the regulatory light chain (RLC), demonstrating that both regulatory light chains of a single molecule can cross-link to the light meromyosin portion of the tail. Mass spectrometric data show four specific cross-linked regions spanning residues 1428-1571 in the light meromyosin portion of the tail, consistent with cross-linking two RLC to one light meromyosin. In addition, we find that position 59 can cross-link internally to residues 42-45 within the same RLC subunit. The internal cross-link only forms in 10 S and not in unphosphorylated heavy meromyosin (lacking the light meromyosin), suggesting a structural rearrangement within the RLC attributed to the interaction of the tail with the head.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M606555200DOI Listing
December 2006

Regulatory and catalytic domain dynamics of smooth muscle myosin filaments.

Biochemistry 2006 May;45(19):6212-21

Biology Department, Institute of Molecular Biophysics and the National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32306, USA.

Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, tau(r), were 24 +/- 6 micros and 441 +/- 79 micros for the catalytic and regulatory domains, respectively. The corresponding amplitudes of motion were 42 +/- 4 degrees and 24 +/- 9 degrees as determined from steady-state phosphorescence anisotropy. These results suggest that the two domains have independent mobility due to a hinge between the two domains. Although a similar hinge was observed for skeletal myosin (Adhikari and Fajer (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 9643-9647. Brown et al. (2001) Biochemistry 40, 8283-8291), the latter displayed higher regulatory domain mobility, tau(r)= 40 +/- 3 micros, suggesting a smooth muscle specific mechanism of constraining regulatory domain dynamics. In the myosin monomers the correlation times for both domains were the same (approximately 4 micros) for both smooth and skeletal myosin, suggesting that the motional difference between the two isoforms in the filaments was not due to intrinsic variation of hinge stiffness. Heavy chain/regulatory light chain chimeras of smooth and skeletal myosin pinpointed the origin of the restriction to the heavy chain and established correlation between the regulatory domain dynamics with the ability of myosin to switch off but not to switch on the ATPase and the actin sliding velocity. Phosphorylation of smooth muscle myosin filaments caused a small increase in the amplitude of motion of the regulatory domain (from 24 +/- 4 degrees to 36 +/- 7 degrees ) but did not significantly affect the rotational correlation time of the regulatory domain (441 to 408 micros) or the catalytic domain (24 to 17 micros). These data are not consistent with a stable interaction between the two catalytic domains in unphosphorylated smooth muscle myosin filaments in the absence of nucleotides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi060037hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090715PMC
May 2006

Novel sensors of the regulatory switch on the regulatory light chain of smooth muscle Myosin.

J Biol Chem 2004 Sep 15;279(38):39905-14. Epub 2004 Jul 15.

Department of Biochemistry, University of Nevada, Reno, Nevada 89557, USA.

Smooth muscle myosin can be switched on by phosphorylation of Ser-19 of the regulatory light chain. Our previous photocross-linking results suggested that an element of the structural mechanism for the regulatory switch was a phosphorylation-induced motion of the regulatory light chain N terminus (Wahlstrom, J. L., Randall, M. A., Jr., Lawson, J. D., Lyons, D. E., Siems, W. F., Crouch, G. J., Barr, R., Facemyer, K. C., and Cremo, C. R. (2003) J. Biol. Chem. 278, 5123-5131). Here we used three different approaches to test this notion, which are reactivity of cysteine thiols, pyrene and acrylodan spectral analysis, and pyrene fluorescence quenching. All methods detected significant differences between the unphosphorylated and phosphorylated regulatory light chain N termini in heavy meromyosin, a double-headed subfragment with an intact regulatory switch. These differences were not observed for subfragment-1, a single-headed, unregulated subfragment. In the presence of either ATP or ADP, phosphorylation increased the solvent exposure and decreased the polarity of the environment about position 23 of the regulatory light chain of heavy meromyosin. These phosphorylation-induced structural changes were not as evident in the absence of nucleotides. Nucleotide binding to unphosphorylated heavy meromyosin caused a decrease in exposure and an increase in polarity of the N terminus, whereas the effects of nucleotide on phosphorylated heavy meromyosin were the opposite. We showed a direct correlation between the kinetics of nucleotide binding/turnover and the conformational change reported by acrylodan at position 23 of the regulatory light chain. Acrylodan-A23C also reports the heads up (extended) to flexed (folded) transition in unphosphorylated heavy meromyosin. This is the first demonstration of direct coupling of nucleotide binding to conformational changes in the N terminus of the regulatory light chain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M407062200DOI Listing
September 2004

Fluorescent nucleotides: synthesis and characterization.

Methods Enzymol 2003 ;360:128-77

Department of Biochemistry, University of Nevada, Reno 89557, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0076-6879(03)60109-6DOI Listing
May 2003

Both heads of tissue-derived smooth muscle heavy meromyosin bind to actin in the presence of ADP.

J Biol Chem 2003 Feb 2;278(7):4410-5. Epub 2002 Dec 2.

Department of Biochemistry, University of Nevada, Reno, Nevada 89557, USA.

The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240-23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M211016200DOI Listing
February 2003

Structural model of the regulatory domain of smooth muscle heavy meromyosin.

J Biol Chem 2003 Feb 21;278(7):5123-31. Epub 2002 Nov 21.

Department of Biochemistry, University of Nevada, Reno, Nevada 89557, USA.

The goal of this study was to provide structural information about the regulatory domains of double-headed smooth muscle heavy meromyosin, including the N terminus of the regulatory light chain, in both the phosphorylated and unphosphorylated states. We extended our previous photo-cross-linking studies (Wu, X., Clack, B. A., Zhi, G., Stull, J. T., and Cremo, C. R. (1999) J. Biol. Chem. 274, 20328-20335) to determine regions of the regulatory light chain that are cross-linked by a cross-linker attached to Cys(108) on the partner regulatory light chain. For this purpose, we have synthesized two new biotinylated sulfhydryl reactive photo-cross-linking reagents, benzophenone, 4-(N-iodoacetamido)-4'-(N-biotinylamido) and benzophenone, 4-(N-maleimido)-4'-(N-biotinylamido). Cross-linked peptides were purified by avidin affinity chromatography and characterized by Edman sequencing and mass spectrometry. Labeled Cys(108) from one regulatory light chain cross-linked to (71)GMMSEAPGPIN(81), a loop in the N-terminal half of the regulatory light chain, and to (4)RAKAKTTKKRPQR(16), a region for which there is no atomic resolution data. Both cross-links were to the partner regulatory light chain and occurred in unphosphorylated but not phosphorylated heavy meromyosin. Using these data, data from our previous study, and atomic coordinates from various myosin isoforms, we have constructed a structural model of the regulatory domain in an unphosphorylated double-headed molecule that predicts the general location of the N terminus. The implications for the structural basis of the phosphorylation-mediated regulatory mechanism are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M206963200DOI Listing
February 2003

A novel mechanism by which hydrogen peroxide decreases calcium sensitivity in airway smooth muscle.

Am J Physiol Lung Cell Mol Physiol 2003 Feb 18;284(2):L324-32. Epub 2002 Oct 18.

Department of Anesthesiology and Physiology, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, MN 55905, USA.

The purpose of this study was to test the hypothesis that H(2)O(2) decreases the amount of force produced by a given intracellular Ca(2+) concentration (i.e., the Ca(2+) sensitivity) in airway smooth muscle (ASM) in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. A new preparation was developed and validated in which canine ASM strips were first exposed to H(2)O(2) and then permeabilized with 10% Triton X-100 to assess the persistent effects of H(2)O(2) on Ca(2+) sensitivity. Experiments in which H(2)O(2) was administered before permeabilization revealed a novel mechanism that contributed to reduced Ca(2+) sensitivity independently of changes in rMLC phosphorylation, in addition to an rMLC phosphorylation-dependent mechanism. The mechanism depended on factors not available in the permeabilized ASM strip or in the buffer to which the strip was exposed, since there was no effect when H(2)O(2) was added to permeabilized strips. H(2)O(2) treatment of a maximally thiophosphorylated purified myosin subfragment (heavy meromyosin) significantly reduced actomyosin ATPase activity, suggesting one mechanism by which the phosphorylation-independent reduction in Ca(2+) sensitivity may occur.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajplung.00159.2002DOI Listing
February 2003