Motility processes in Acantharia. II. A Ca2+ dependent system of contractile 2–4 nm filaments isolated from demembranated myonemes

Myonemes of the acantharians are contractile ribbon-like organelles. As previously shown, their motility is based on the coiling mechanism of double-twisted 2–4 nm nonactin filaments [14]. Myonemes have been isolated and manipulated in vitro. After demembranation, the contraction takes place when the Ca²⁺ concentration is above 10^?7 M, whereas relaxation occurs below this threshold concentration. The response to Ca²⁺ ions is an on/off mechanism. Both contraction and relaxation can be induced repeatedly without fatigue phenomena. Other divalent cations such as Sr²⁺, Ba²⁺, Ma²⁺, CO²⁺, and La³⁺ can replace Ca²⁺ in inducing contraction of the demembranated myonemes although with less efficiency. Contraction and relaxation are ATP-independent and calmodulin is not involved in this in vitro motility process. The myoneme is a strongly resistant structure which is capable of contracting and relaxing under various extreme conditions which indicates very stable proteins and resistant functions.     

Ca2+ -induced tropomyosin movement in scallop striated muscle thin filaments

Striated muscle contraction in most animals is regulated at least in part by the troponin-tropomyosin (Tn-Tm) switch on the thin (actin-containing) filaments. The only group that has been suggested to lack actin-linked regulation is the mollusks, where contraction is regulated through the myosin heads on the thick filaments. However, molluscan gene sequence data suggest the presence of troponin (Tn) components, consistent with actin-linked regulation, and some biochemical and immunological data also support this idea. The presence of actin-linked (in addition to myosin-linked) regulation in mollusks would simplify our general picture of muscle regulation by extending actin-linked regulation to this phylum as well. We have investigated this question structurally by determining the effect of Ca²⁺ on the position of Tm in native thin filaments from scallop striated adductor muscle. Three-dimensional reconstructions of negatively stained filaments were determined by electron microscopy and single-particle image analysis. At low Ca²⁺, Tm appeared to occupy the “blocking” position, on the outer domain of actin, identified in earlier studies of regulated thin filaments in the low-Ca²⁺ state. In this position, Tm would sterically block myosin binding, switching off filament activity. At high Ca²⁺, Tm appeared to move toward a position on the inner domain, similar to that induced by Ca²⁺ in regulated thin filaments. This Ca²⁺-induced movement of Tm is consistent with the hypothesis that scallop thin filaments are Ca²⁺ regulated.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Structural basis for the regulation of muscle contraction by troponin and tropomyosin

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca²⁺ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca²⁺ binding to TnC (the Ca²⁺ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the “inhibitory” subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca²⁺ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.     

Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca²⁺ on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca²⁺ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca²⁺ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca²⁺-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca²⁺, troponin operates as an inhibitor, while in activated muscles at high Ca²⁺, it acts as a promoter to initiate contraction.     

Determinants of relaxation rate in skinned frog skeletal muscle fibers

The influences of sarcomere uniformity andCa²⁺ concentration on the kineticsof relaxation were examined in skinned frog skeletal muscle fibersinduced to relax by rapid sequestration ofCa²⁺ by the photolysis of theCa²⁺ chelator, diazo-2, at10°C. Compared with an intact fiber, diazo-2-induced relaxationexhibited a faster and shorter initial slow phase and a fast phase witha longer tail. Stabilization of the sarcomeres by repeated releases andrestretches during force development increased the duration of the slowphase and slowed its kinetics. When force of contraction was decreasedby lowering the Ca²⁺concentration, the overall kinetics of relaxation was accelerated, withthe slow phase being the most sensitive toCa²⁺ concentration. Twitchlikecontractions were induced by photorelease ofCa²⁺ from a cagedCa²⁺ (DM-Nitrophen), withsubsequent Ca²⁺ sequestration byintact sarcoplasmic reticulum orCa²⁺ rebinding to cagedCa²⁺. These twitchlike responsesexhibited relaxation kinetics that were about twofold slower than thoseobserved in intact fibers. Results suggest that the slow phase ofrelaxation is influenced by the degree of sarcomere homogeneity andrate of Ca²⁺ dissociation fromthin filaments. The fast phase of relaxation is in part determined bythe level of Ca²⁺ activation.

     

Regulation analysis of contractile ATPase flux in skeletal muscle

The [Ca²⁺] regulation of contractile ATPase flux, J _p, in skeletal muscle was analysed by computation of the Response R ^Jp _Ca2+ for a 10 Hz range of electrical stimulation frequencies. Results of our analysis of the kinetic controls in ATP free energy metabolism in a network model of contracting muscle (J.A.L. Jeneson, H.V. Westerhoff and M.J. Kushmerick (2000) Am. J. Physiol. 279, C813–C832) formed the basis for the computations of R ^Jp _Ca2+. We found that neural regulation of sustained force generation via simple [Ca²⁺]_cyto frequency encoding in the network was robust for frequencies up to 2 Hz. Above 2 Hz, however, this regulation design broke down because of a shift in contractile ATPase flux control from the Ca²⁺-sensitive contractile filaments to mitochondria with low Ca²⁺ sensitivity. The role of glyco(geno)lytic ATP production at high contraction workloads is discussed in the context of this result     

Calcium wave for cytoplasmic streaming of Physarum polycephalum

The plasmodium Physarum polycepharum exhibits periodic cycles of cytoplasmic streaming in association with those of contraction and relaxation movement. In the present study, we injected Calcium Green dextran as a fluorescent Ca²⁺ indicator into the thin‐spread living plasmodium. We found changes in the [Ca²⁺]i (intracellular concentration of Ca²⁺), which propagated in a wave‐like form in its cytoplasm. The Ca²⁺ waves were also detected when we used Fura dextran which detected [Ca²⁺]i by the ratio of two wavelengths. We prepared the plasmodial fragment from the thin‐spread and found that the cycles of the contraction–relaxation movement was so synchronized that the measurement of its area provided an indication of the movement. We observed that [Ca²⁺]i also synchronized in the entire fragment and that the relaxation ensued upon the reduction in [Ca²⁺]i. We suggest that the Ca²⁺ wave generated periodically is one of the major factors playing a crucial role in the relaxation of P. polycepharum.     

Calcium ion and troponin: Professor S. Ebashi's epoch-making achievement

The processes by which Professor Setsuro Ebashi accomplished his great work are described. Independently of Marsh, Ebashi discovered the relaxing factor in homogenized muscle and showed that it has a lipid-containing particulate fraction with ATPase activity, later identified as the sarcoplasmic reticulum. He then solved the mechanism of relaxation of the relaxing factor through the following findings. A minute amount of calcium ion (Ca²⁺) is necessary for the physiological contractile reaction. The relaxing factor strongly accumulates Ca²⁺ in the presence of ATP and sufficiently removes Ca²⁺ from the contractile system to bring about relaxation. Ebashi found that the contractile reaction of myosin and actin is regulated by Ca²⁺ only in the presence of a tropomyosin-like protein factor, which he later showed to be a complex of tropomyosin and a new protein, troponin. He proved that troponin is the Ca²⁺-receptive protein and proposed the correct scheme for the molecular mechanism of regulation of contraction and relaxation.     

Analysis of the mechanism of dinoflagellate flagella contraction-relaxation cycle

Summary— Dinoflagellates possess two flagella. One of them, the longitudinal flagellum, retracts from time to time in some species, such as Ceratium and Peridinium. Additional structures which run along the axoneme seem to be responsible for this particular behaviour. The retraction which is rapid (less than 60 ms) may be subdivided into several steps: i) the undulating movement stops; ii) the flagellum appears then as a jagged line during 20 ms; iii) finally a rapid retraction (20 ms) takes place, the flagellum being folded 20 times inside the cylindrical flagellar pocket. The measurements on video-records suggest that the R-fibre shortens to 30% of its original length. The contraction and relaxation mechanism of nanofilaments is proposed to be through coiling and uncoiling dependent on Ca²⁺ concentration.     

Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca²⁺) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca²⁺ plays an important role in adult cardiac muscle cells for excitation–metabolism coupling, little is known about whether Ca²⁺ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca²⁺ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca²⁺ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca²⁺ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca²⁺-mediated ROS generation in cardiac myocytes. These results suggest that Ca²⁺, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.     

Ca2+ Uptake and Its Regulation in the Cyanobacterium Nostoc MAC

The uptake of Ca²⁺ and its regulation in the cyanobacterium Nostoc MAC were investigated. Cation uptake pattern was found to be biphasic, consisting of (a) rapid binding of cations to the negatively charged cell surface and (b) its metabolism dependent on intracellular import at least up to 60 min with the saturation at 2 mM Ca²⁺ (K _m , 1.5 mM, Vmax 42.1 nmol Ca²⁺ mg⁻¹ protein min⁻¹ ). The cellular Ca²⁺ uptake was light and ATP dependent, and the addition of 3(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or exogenous ATP proved the vital role of PS II-generated energy to drive the process. The significant inhibition of Ca²⁺ uptake by different metabolic inhibitors and uncouplers like p-chloromercuribenzoate (pCMB), carbonylcyanide-p-nitrofluoromethoxylphenyl hydrazone (FCCP), N′N-dicyclohexylcarbodiimide (DCCD) and azide revealed that -SH group(s), proton gradient across the cell membrane, and ATP hydrolysis were involved in the transmembrane movement of Ca²⁺ in Nostoc MAC cells. Verapamil showed antagonism, abscisic acid (ABA) agonism, while trifluoroperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) exerted no effect on Ca²⁺ uptake.     

A Ca2+‐binding protein with numerous roles and uses: parvalbumin in molecular biology and physiology

Parvalbumins (PVs) are acidic, intracellular Ca²⁺‐binding proteins of low molecular weight. They are associated with several Ca²⁺‐mediated cellular activities and physiological processes. It has been suggested that PV might function as a “Ca²⁺ shuttle” transporting Ca²⁺ from troponin‐C (TnC) to the sarcoplasmic reticulum (SR) Ca²⁺ pump during muscle relaxation. Thus, PV may contribute to the performance of rapid, phasic movements by accelerating the contraction–relaxation cycle of fast‐twitch muscle fibers. Interestingly, PVs promote the generation of power stroke in fish by speeding up the rate of relaxation and thus provide impetus to attain maximal sustainable speeds. However, immunological monitoring of diverse tissues demonstrated that PVs are also present in non‐muscle cells. The axoplasmic transport and various intracellular secretory mechanisms including the endocrine secretions seem to be controlled by the Ca²⁺ regulation machinery. Any defect in the Ca²⁺ handling apparatus may cause several clinical problems; for instance, PV deficiency alters the neuronal activity, a key mechanism leading to epileptic seizures. Moreover, atypical relaxation of the heart results in diastolic dysfunction, which is a major cause of heart failure predominantly among the aged people. PV may offer a unique potential to correct defective relaxation in energetically compromised failing hearts through PV gene transfer. Consequently, PV gene transfer may present a new therapeutic approach to correct cellular disturbances in Ca²⁺ signaling pathways of diseased organs. Hence, PVs appear to be amazingly useful candidate proteins regulating a variety of cellular functions through action on Ca²⁺ flux management.     

Fluctuations in the Concentration of Extracellular ATP Synchronized with Intracellular Ca2+ Oscillatory Rhythm in Cultured Cardiac Myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca²⁺ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca²⁺ oscillation). Both the contraction and Ca²⁺ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP‐purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca²⁺ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca²⁺ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca²⁺ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca²⁺ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca²⁺ oscillation. In addition, power spectrum and cross‐correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca²⁺ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca²⁺ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca²⁺, contributing to the intercellular synchronization of Ca²⁺ oscillation among cultured cardiac myocytes.     

ATP und Ca2+ induzierte actomyosinartige Kontraktion glycerinisierter Makronuklei

Summary The addition of 2 mM-3 mM ATP to macronuclei ofParamecium bursaria suspended in a glycerol buffer medium causes a decrease in their volume up to 23% within 3 minutes. The infiltration medium must not only contain Ca²⁺, but must also be of low ionic strength for ATP to be effective. A slow, careful exchange of the glycerol medium for the contraction solution is also necessary. Ca²⁺ present alone in the standard contraction buffer can likewise induce a limited volume decrease; in the presence of low concentrations of Ca²⁺, Mg²⁺ shows no detectable effect on glycerinated nuclei. When the nuclear volume has been reduced by ATP in the presence of Ca²⁺, the addition of EGTA induces a reexpansion of the nuclei. Salyrgan, an organic mercurial, either prevents or abolishes the ATP-induced contraction. Other nucleotide triphosphates such as guanosine triphosphate (GTP), inosine triphosphate (ITP) or uridine triphosphate (UTP) likewise induce a volume decrease of the glycerinated macronuclei, but to a distinctly lesser extent than ATP.The results indicate that the volume decrease caused by the ATP-contraction solution is not a passive osmotic process. The resemblance to actomyosin contractions suggests that the volume decrease reported here might also be the result of the reaction of nuclear actomyosin and ATP.

     

A mathematical model of airway and pulmonary arteriole smooth muscle

Airway hyperresponsiveness is a major characteristic of asthma and is believed to result from the excessive contraction of airway smooth muscle cells (SMCs). However, the identification of the mechanisms responsible for airway hyperresponsiveness is hindered by our limited understanding of how calcium (Ca²⁺), myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCP) interact to regulate airway SMC contraction. In this work, we present a modified Hai-Murphy cross-bridge model of SMC contraction that incorporates Ca²⁺ regulation of MLCK and MLCP. A comparative fit of the model simulations to experimental data predicts 1), that airway and arteriole SMC contraction is initiated by fast activation by Ca²⁺ of MLCK; 2), that airway SMC, but not arteriole SMC, is inhibited by a slower activation by Ca²⁺ of MLCP; and 3), that the presence of a contractile agonist inhibits MLCP to enhance the Ca²⁺ sensitivity of airway and arteriole SMCs. The implication of these findings is that murine airway SMCs exploit a Ca²⁺-dependent mechanism to favor a default state of relaxation. The rate of SMC relaxation is determined principally by the rate of release of the latch-bridge state, which is predicted to be faster in airway than in arteriole. In addition, the model also predicts that oscillations in calcium concentration, commonly observed during agonist-induced smooth muscle contraction, cause a significantly greater contraction than an elevated steady calcium concentration.     

Cyclic AMP-independent relaxation of depolarized rat ileal smooth muscle by isoproterenol.

Isoproterenol brings about a rapid transient relaxation of depolarized ileal smooth muscle as well as a longer lasting decline in tension. Only the latter effect is mimicked by dibutyryl cyclic AMP or by phosphodiesterase inhibitors. Beta adrenergic blocking agents cause a rapid transient contraction in preparations relaxed by isoproterenol but not in preparations relaxed by the other agents, and this effect persists when influx of Ca²⁺ from the extracellular space is prevented. The transient component of the isoproterenol-induced relaxation is therefore attributed to sequestration of Ca²⁺ at intracellular sites, and the contraction which follows the subsequent addition of beta blocking agents is due to release of Ca²⁺ from these sites.     

A three-dimensional simulation model of cardiomyocyte integrating excitation-contraction coupling and metabolism

Recent studies have revealed that Ca²⁺ not only regulates the contraction of cardiomyocytes, but can also function as a signaling agent to stimulate ATP production by the mitochondria. However, the spatiotemporal resolution of current experimental techniques limits our investigative capacity to understand this phenomenon. Here, we created a detailed three-dimensional (3D) cardiomyocyte model to study the subcellular regulatory mechanisms of myocardial energetics. The 3D cardiomyocyte model was based on the finite-element method, with detailed subcellular structures reproduced, and it included all elementary processes involved in cardiomyocyte electrophysiology, contraction, and ATP metabolism localized to specific loci. The simulation results were found to be reproducible and consistent with experimental data regarding the spatiotemporal pattern of cytosolic, intrasarcoplasmic-reticulum, and mitochondrial changes in Ca²⁺; as well as changes in metabolite levels. Detailed analysis suggested that although the observed large cytosolic Ca²⁺ gradient facilitated uptake by the mitochondrial Ca²⁺ uniporter to produce cyclic changes in mitochondrial Ca²⁺ near the Z-line region, the average mitochondrial Ca²⁺ changes slowly. We also confirmed the importance of the creatine phosphate shuttle in cardiac energy regulation. In summary, our 3D model provides a powerful tool for the study of cardiac function by overcoming some of the spatiotemporal limitations of current experimental approaches.     

Millisecond time-resolved changes occurring in Ca2+-regulated myosin filaments upon relaxation

Contraction of many muscles is activated in part by the binding of Ca²⁺ to, or phosphorylation of, the myosin heads on the surface of the thick filaments. In relaxed muscle, the myosin heads are helically ordered and undergo minimal interaction with actin. On Ca²⁺ binding or phosphorylation, the head array becomes disordered, reflecting breakage of the head-head and other interactions that underlie the ordered structure. Loosening of the heads from the filament surface enables them to interact with actin filaments, bringing about contraction. On relaxation, the heads return to their ordered positions on the filament backbone. In scallop striated adductor muscle, the disordering that takes place on Ca²⁺ binding occurs on the millisecond time scale, suggesting that it is a key element of muscle activation. Here we have studied the reverse process. Using time-resolved negative staining electron microscopy, we show that the rate of reordering on removal of Ca²⁺ also occurs on the same physiological time scale. Direct observation of images together with analysis of their Fourier transforms shows that activated heads regain their axial ordering within 20 ms and become ordered in their final helical positions within 50 ms. This rapid reordering suggests that reformation of the ordered structure, and the head-head and other interactions that underlie it, is a critical element of the relaxation process.     

Thin filaments of bivalve smooth muscle may contain a calponin-like protein

A novel 40-kDa calponin-like protein (CaP) was detected in thin filaments from catch muscles of the mussel Crenomytilus grayanus. The content of CaP in thin filaments depends on isolation conditions and varies from complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the CaP content in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22°C and optimal values of pH and ionic strength of the extraction solution, all CaP co-sediments with thin filaments. At 2°C it does not interact with thin filaments and remains in the supernatant. Like vertebrate smooth muscle calponin (33 kDa), the mussel CaP is thermostable, inhibits the Mg²⁺-ATPase activity of actomyosin, and can be phosphorylated, which is performed by endogenous (co-isolated) kinases in a Ca²⁺-independent manner. Thus, the C. grayanus CaP is a new member of the family of calponins, the function of which in muscle and nonmuscle cells is still obscure. We suggest that CaP is involved in Ca²⁺-independent regulation of smooth muscle contraction.