Cooperative interactions between calcium-binding sites on glycerinated muscle fibers the influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca²⁺ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca²⁺-binding sites in the intact filament lattice. In the presence of 5 mM MgCl₂ (no ATP), reduction of filament overlap was associated with a reduced binding of Ca²⁺ over the entire range of free Ca²⁺ concentrations (5 · 10^?8 – 2 · 10^?5 M). With maximum filament overlap (sarcomere length 2.1–2.2 μm) the maximum bound Ca²⁺ was equivalent to 4 mol Ca²⁺/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8–4.4 μm) the maximum bound Ca²⁺ was equivalent to 3 μmol Ca²⁺/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca²⁺ binding site appear, but cooperative properties are imposed upon the binding sites.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The binding of Mn2+ and ADP to myosin

The metal ion requirement of myosin-ADP binding was investigated by use of Mn²⁺. Mn²⁺ binds to two sets of noninteracting sites on myosin which are characterized by affinity constants of 10⁶ and 10³, M⁻¹ at 0.016 M KCl concentration. The maximum number of sites is 2 for the high affinity and 20–25 for the low affinity set. Binding of Mn²⁺ to the high affinity sites increases the affinity of ADP binding to myosin. F-actin inhibits ADP binding (Kiely, B., and Martonosi, A., Biochim. Biophys. Acta 172: 158–170 [1969]), but even at F-actin concentrations much higher than that required to saturate the actin binding sites of myosin or its proteolytic fragments, significant ADP binding remained. The actin insensitive portion of ADP binding was inhibited by 10⁻⁴ M inorganic pyrophosphate or ATP. The results are discussed on the basis of a model in which actin and ADP bind to myosin at distinct but interacting sites.     

Calcium transport by rabbit skeletal muscle microsomes (“fragmented sarcoplasmic reticulum”)

Ca²⁺ binding by skeletal muscle microsomes in 5 mM ATP exhibited saturation kinetics in the range of Ca²⁺ concentrations between 3 · 10^?8 and 10^?5 M. Approximately 140 nmoles binding sites per mg protein were found. These had a Ca²⁺ binding constant of approximately 4.5 · 10⁶ M^?1 with half saturation at 2.2 · 10^?7 M Ca²⁺. In the presence of oxalate, much larger amounts of Ca²⁺ (approx. 6 μmoles/mg protein) were taken up by the microsomes (Ca²⁺ uptake), but the rate of Ca²⁺ uptake increased linearly with [Ca²⁺] when ionized Ca²⁺ concentrations were below 3 · 10^?6 M. At Ca²⁺ concentrations above 3 · 10^?6, Ca²⁺ uptake was inhibited. Double reciprocal plots of the Ca²⁺ dependence of the initial rates of Ca²⁺ uptake in the concentration range between 3 · 10^?7 M and 10^?5 M, unlike those of Ca²⁺ binding, did not demonstrate saturation kinetics, but could be interpreted as representing a non-saturable system with inhibition at higher Ca²⁺ concentrations. In view of these differences, and because Ca²⁺-binding sites were almost fully saturated at 10^?6 M Ca²⁺, whereas Ca²⁺ uptake rate increased linearly with increasing [Ca²⁺] to approximately 3 · 10^?6 M, the Ca²⁺-binding sites are not shown kinetically to participate in oxalate-dependent Ca²⁺ uptake.     

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.     

Phosphatidylinositol 4,5-bisphosphate and calcium at ER-PM junctions — Complex interplay of simple messengers

Endoplasmic reticulum-plasma membrane contact sites (ER-PM MCS) are a specialised domain involved in the control of Ca²⁺ dynamics and various Ca²⁺-dependent cellular processes. Intracellular Ca²⁺ signals are broadly supported by Ca²⁺ release from intracellular Ca²⁺ channels such as inositol 1,4,5-trisphosphate receptors (IP₃Rs) and subsequent store-operated Ca²⁺ entry (SOCE) across the PM to replenish store content. IP₃Rs sit in close proximity to the PM where they can easily access newly synthesised IP₃, interact with binding partners such as actin, and localise adjacent to ER-PM MCS populated by the SOCE machinery, STIM1–2 and Orai1–3, to possibly form a locally regulated unit of Ca²⁺ influx. PtdIns(4,5)P₂ is a multiplex regulator of Ca²⁺ signalling at the ER-PM MCS interacting with multiple proteins at these junctions such as actin and STIM1, whilst also being consumed as a substrate for phospholipase C to produce IP₃ in response to extracellular stimuli. In this review, we consider the mechanisms regulating the synthesis and turnover of PtdIns(4,5)P₂ via the phosphoinositide cycle and its significance for sustained signalling at the ER-PM MCS. Furthermore, we highlight recent insights into the role of PtdIns(4,5)P₂ in the spatiotemporal organization of signalling at ER-PM junctions and raise outstanding questions on how this multi-faceted regulation occurs.     

Does Actin Polymerization Status Modulate CaStorage in Human Neutrophils? Release and Coalescence of CaStores by Cytochalasins

The aim of this paper was to establish whether actin polymerization modulated cytosolic Ca²⁺storage in human neutrophils. Over the concentration ranges which inhibit actin polymerization, cytochalasins A, B, and D liberated Ca²⁺from membrane-bound stores within neutrophils. Two Ca²⁺storage sites were identified in neutrophils by the accumulation of the Ca²⁺binding probe, chlortetracycline: one at the center of the cell and the other at the cell periphery. Confocal imaging demonstrated that cytochalasins released Ca²⁺from the neutrophil periphery, but not from the central Ca²⁺store. Ca²⁺store release was coupled to Ca²⁺influx, suggesting that the peripheral site may be a physiological store containing a Ca²⁺influx factor. 3,3′-Dihexyloxacarbocyanine iodide staining organelles, which correlate with Ca²⁺release sites, coalesced in neutrophils after treatment with cytochalasins. We propose that peripheral Ca²⁺storage sites are restricted from coalescence by cortical polymerized actin and that Ca²⁺store coalescence and Ca²⁺release are coupled events.     

Membrane potential-dependent calcium transport in right-side-out plasma membrane vesicles from Zea mays L. roots

Right-side-out plasma membrane vesicles isolated from Zea mays roots were used to study membrane potential (ΔΨ)-dependent Ca²⁺ transport. Membrane potentials were imposed on the vesicles using either K⁺ concentration gradients and valinomycin or SCN concentration gradients, and the size of the imposed ΔΨ was measured with [¹⁴C]tetraphenylphosphonium. Uptake of ⁴⁵Ca²⁺ into the vesicles was stimulated by inside-negative ΔΨ. The rate of transport increased to a maximum at a ΔΨ of about -80 mV and then declined at more negative ΔΨ. When extravesicular Ca²⁺ concentration was varied, uptake was maximal in the range 100–200 μM Ca²⁺. Neither dihydropyridine nor phenylalkylamine Ca²⁺ channel blockers had any effect on Ca²⁺ uptake but 30 μM ruthenium red was completely inhibitory with half maximal inhibition at 10–15 μM ruthenium red. Calcium transport was also inhibited by inorganic cations. Zn²⁺, Gd³⁺ and Mg²⁺ inhibited by a maximum of 30% while La³⁺, Nd³⁺ and Mn²⁺ inhibited by 70%. The inhibitory effects of La³⁺ and Gd³⁺ were additive. Lanthanum-insensitive Ca²⁺ five Ca²⁺ transport was totally inhibited by 80 μM Gd³⁺ and showed maximum activity at a ΔΨ of -60 mV, with less uptake at both higher and lower ΔΨ. Lanthanum and Gd³⁺ also inhibited Ca²⁺ uptake into protoplasts isolated from Zea roots and their individual and combined effects were similar in extent to those observed with plasma membrane vesicles. It is concluded that maize root plasma membrane contains two Ca²⁺-permeable channels that can be distinguished by their susceptibility to inhibition by La³⁺ and Gd³⁺. Both are inhibited by ruthenium red but not by other organic Ca²⁺ channel blockers.     

The binding of calcium and yttrium ions to a glycoprotein from bovine cortical bone

The binding of Ca²⁺ and Y³⁺ to an acidic glycoprotein from bovine cortical bone, bone sialoprotein, was determined from the titration curves at I 0·2 in the presence and absence of the cations. The binding of Y³⁺ was greater than that of Ca²⁺. The value for the association constant, k, for the interaction with Y³⁺ increased with pH, from log k 2·93 at pH3·4 to log k 3·50 at pH4·4, and the number of binding sites/mol. increased from 4·6 at pH3·4 to 9·1 at pH4·4. It is proposed that the binding site consists of three carboxyl groups, but it is likely that the binding is a strong electrostatic interaction rather than a co-ordination linkage. A chondroitin sulphate–protein complex also extracted from bovine cortical bone interacted with Y³⁺ and Ca²⁺ to a similar extent as did bone sialoprotein. It is suggested that these materials are present in bone at the resting and resorbing surfaces and that they contribute to the deposition of yttrium, americium and plutonium at these sites.     

Ca2+ attenuates nucleation activity of leiomodin

A transient increase in Ca²⁺ concentration in sarcomeres is essential for their proper function. Ca²⁺ drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin‐binding protein C were also found to be Ca²⁺ sensitive. However, the effects of Ca²⁺ on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene‐actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca²⁺. Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin‐binding sites binds Ca²⁺. We propose that Ca²⁺ binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca²⁺ as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function.Summary StatementCa²⁺ fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca²⁺ on the activity of leiomodin—an actin nucleator and thin filament length regulator—have remained unknown. In this study, we demonstrate that Ca²⁺ binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca²⁺ in the regulation of the sarcomeric protein interactions.     

Purification of calcium-sensitive regulatory protein of platelets which inhibits the gelation of actin

Ca²⁺-sensitive regulatory protein of human platelets which inhibits the gelation of actin was purified by DEAE-Sepharose and an affinity column using actin as a ligand. The protein was a single polypeptide chain with an average molecular weight of 90,000 and it bound to actin and inhibited its gelation at concentration from 10^?6–10^?7M of free calcium. Since the protein existed in the form of a complex with actin even though at concentration lower than 10^?7M of free calcium, binding and dissociation of actin and the protein appeared to be dependent on the concentration of free calcium, and complete dissociation was not seen.     

Calcium Binding to Plasma Membranes from Normal and SV40 Transformed Hamster Lymphocytes

Abstract

The calcium binding properties of isolated plasma membranes from normal and SV40 transformed hamster lymphocytes were compared over the Ca²⁺ concentration range of 10^?5M to 5 × 10^?3M and at physiological ionic strength. At all Ca²⁺ concentrations, normal membranes bound more Ca²⁺ than tumor membranes; at blood Ca²⁺ levels (1–2 mM) plasma membranes of normal cells bind twice as much as membranes from tumor cells. Normal plasma membranes demonstrated positive cooperative Ca²⁺ binding whereas tumor membranes displayed non-interacting Ca²⁺-binding sites. Ca²⁺ binding to both membranes was insensitive to Mg²⁺ (0.1 to 2.5 mM). A pH shift from 7 to 6 resulted in a 70% decrease of normal membrane-bound Ca²⁺ compared to a 40% decrease observed with tumor membranes. Extracellular surface Ca²⁺ binding to intact cells was also studied after a 72-hour equilibration of cells with ⁴⁵Ca²⁺ and with ethylene-glycol-bis-(β-amino-ethyl ether) N, N′-tetraacetate chelation as marker for surface Ca²⁺. Tumor cell surface Ca²⁺ binding was only 10% of that observed with quiescent lymphocytes. Normal lymphocytes stimulated to divide with phytohemagglutinin also showed a decreased level of surface Ca²⁺ (50%). However, plasma membranes isolated from non-dividing and phytohemagglutinin-stimulated lymphocytes exhibited equivalent Ca²⁺ binding.     

Asymmetric inhibition of spicule formation in sea urchin embryos with low concentrations of gadolinium ion

As gastrulation proceeds during sea urchin embryogenesis, primary mesenchyme cells (PMCs) fuse to form syncytial cables, within which calcium is deposited as CaCO₃, and a pair of spicules is formed. Earlier studies suggested that calcium, previously sequestered by primary mesenchyme cells, is secreted and incorporated into growing spicules. We examined the effects of gadolinium ion (Gd³⁺), a Ca²⁺ channel blocker, on spicule formation. Gd³⁺ did not lead to a retardation of embryogenesis prior to the initiation of gastrulation and did not inhibit the ingression of PMCs from the blastula wall or their migration along the inner blastocoel surface. However, when embryos were raised in seawater containing submicromolar to a few micromolar Gd³⁺, of which levels are considered to be insufficient to block Ca²⁺ channels, a pair of triradiate spicules was formed asymmetrically. At 1–3 μmol/L Gd³⁺, many embryos formed only one spicule on either the left or right side, or embryos formed a very small second spicule. Induction of the spicule abnormality required the presence of Gd³⁺ specifically during late blastula stage prior to spicule formation. An accumulation or adsorption of Gd³⁺ was not detected anywhere in the embryos by X‐ray microanalysis, which suggests that Ca²⁺ channels were not inhibited. These results suggest that Gd³⁺ exerts an inhibitory effect on spicule formation through a mechanism that does not involve inhibition of Ca²⁺ channels.     

Actin assembly by cadmium ions

Cadmium is a highly toxic metal entering cells by a variety of mechanisms. Its toxic action is far from being completely understood, although specific interaction with the cellular calcium metabolism has been indicated. Metal ions that influence intracellular Ca²⁺ concentrations or compete with Ca²⁺ for protein binding sites may exert an effect on actin filaments, whose assembly and disassembly are both regulated by a number of calcium-dependent factors. Cadmium is such a metal. Much evidence demonstrates that cadmium interferes with the dynamics of actin filaments in various types of cells. Here we show that, at high (0.8–1.0 mM) concentrations, CdCl₂ causes actin denaturation. At such Cd²⁺ concentrations, actin precipitates (really actin, as shown by SDS-PAGE, see Fig. 1B) in the form of irregular, disordered clots, clearly appreciable by electron microscopy. Denaturation seems to be reversible since, after Cd²⁺ removal by dialysis, the polymerizability of sedimented actin is restored almost completely. On the other hand, at concentrations ranging from 0.25 to 0.6 mM, CdCl₂ is more effective as an actin polymerizing agent than both MgCl₂ and CaCl₂. The Cd-related increase in the actin assembly rate is ascribable to an enhanced nucleation rather than to an increased monomer addition to filament growing ends. The latter, in contrast, appears quite slow. Critical concentration measurements revealed that the extent of polymerization of both Mg- and Cd-assembled actin are very close (C_c ranges from 0.25 to 0.5 μM), while Ca-polymerized actin shows a polymerization extent markedly lower (C_c=4.0 μM). By both the fluorescent Ca²⁺ chelator Quin-2 assay and limited proteolysis of actin by trypsin and α-chymotrypsin, the real substitution of G-actin-bound Ca²⁺ by Cd²⁺ has been appreciated. The increase in Quin-2 fluorescence after addition of excess CdCl₂ indicates that, in our experimental conditions, Ca²⁺ tightly-bound to actin is partially (60–70%) replaced by Cd²⁺, forming Cd-actin. Electrophoretic patterns after limited proteolysis reveal that the trypsin cleavage sites in the segment 61–69 of the actin polypeptide chain are less accessible in Cd-actin than in Ca-actin, although the cation-dependent effect is less pronounced in Cd-actin than in Mg-actin. Our results are consistent with some of the consequences on microfilament organization observed in Cd²⁺-treated cells; however, considering the positive effect of Cd²⁺ on actin polymerization in solution we have noticed that this was never observed in vivo. A different indirect effect of Cd²⁺ on some cellular event(s) influencing cytoplasmic actin polymerization appears to be reasonable. © 1997 Elsevier Science B.V. All rights reserved.     

Sphingosine inhibits muscarinic cholinergic receptor binding in rat parotid acinar cells

1. Sphingosine inhibited the binding of [³H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells.2. The inhibition of [³H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites.3. The effect of Sphingosine on the binding was not affected by the chelation of extracellular Ca²⁺.4. H-7, an inhibitor of protein kinase C, failed to decrease [³H]QNB binding.5. Stearylamine, an analogue of Sphingosine, was as effective as Sphingosine in inhibiting [³H]QNB binding.6. These results suggest that Sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca²⁺ and protein kinase C.     

Helix Straightening as an Activation Mechanism in the Gelsolin Superfamily of Actin Regulatory Proteins

Villin and gelsolin consist of six homologous domains of the gelsolin/cofilin fold (V1–V6 and G1–G6, respectively). Villin differs from gelsolin in possessing at its C terminus an unrelated seventh domain, the villin headpiece. Here, we present the crystal structure of villin domain V6 in an environment in which intact villin would be inactive, in the absence of bound Ca²⁺ or phosphorylation. The structure of V6 more closely resembles that of the activated form of G6, which contains one bound Ca²⁺, rather than that of the calcium ion-free form of G6 within intact inactive gelsolin. Strikingly apparent is that the long helix in V6 is straight, as found in the activated form of G6, as opposed to the kinked version in inactive gelsolin. Molecular dynamics calculations suggest that the preferable conformation for this helix in the isolated G6 domain is also straight in the absence of Ca²⁺ and other gelsolin domains. However, the G6 helix bends in intact calcium ion-free gelsolin to allow interaction with G2 and G4. We suggest that a similar situation exists in villin. Within the intact protein, a bent V6 helix, when triggered by Ca²⁺, straightens and helps push apart adjacent domains to expose actin-binding sites within the protein. The sixth domain in this superfamily of proteins serves as a keystone that locks together a compact ensemble of domains in an inactive state. Perturbing the keystone initiates reorganization of the structure to reveal previously buried actin-binding sites.Actin is crucial to such processes as cell movement, cell division, and apoptosis, which are regulated by numerous actin-binding proteins, including gelsolin, Arp2/3, and profilin (for review, see Ref. 1). Gelsolin, the most potent actin filament-severing protein known, can bind to, sever, cap, and nucleate actin filaments in a calcium-, pH-, ATP-, and phospholipid-dependent manner (for review, see Ref. 2). Villin, found in microvilli of absorptive epithelium, is a second member of the gelsolin family of actin-binding proteins. In addition to standard gelsolin-type activities, villin is able to bundle actin filaments and is subject to regulation by tyrosine phosphorylation as well as by Ca²⁺ and phosphatidylinositol 4,5-bisphosphate (for review, see Ref. 3). Many comparisons have been made between gelsolin and villin. The two share 50% amino acid sequence identity and show similar proteolytic cleavage patterns (4). Both contain six similarly folded domains, but villin possesses a seventh domain at its C terminus, the headpiece (HP)² domain, which folds into a compact structure that introduces a second F-actin-binding site into the protein. Recent studies indicate that villin uses the HP F-actin-binding sites to achieve bundling (5). In an environment devoid of free Ca²⁺, gelsolin and villin assume inactive conformations. After binding Ca²⁺, both undergo conformational rearrangements that expose their binding sites for F-actin. In villin, this includes revealing the HP actin-binding site through a “hinge mechanism” (6).Biochemical and structural studies have revealed eight Ca²⁺-binding sites of two types in gelsolin (for review, see Ref. 7). Each of the six domains contains a complete and evolutionarily conserved site, termed type 2, whereas G1 and G4 provide partial Ca²⁺ coordination at interfaces with actin through sites termed type 1. Sequential mutagenesis of these sites in villin has identified six functional Ca²⁺-binding sites (8): two major sites, one each of type 1 and type 2, in V1, plus four type 2 sites in V2–V6. The type 1 site in V1 regulates F-actin-capping and F-actin-severing activities, whereas the lower affinity type 2 site in V1 only affects severing (9). The other four sites are involved in stabilizing villin conformation, but they do not directly influence actin-severing activity. NMR studies of a fragment of villin that consists of V6 and the HP domain have implicated V6 residues Asn⁶⁴⁷, Asp⁶⁴⁸, and Glu⁶⁷⁰ in binding Ca²⁺ (10). These experiments also revealed the first 80 residues of V6 to undergo significant conformational change as a result of Ca²⁺ binding.Nanomolar to micromolar concentrations of free Ca²⁺ govern the actin-binding activities of gelsolin. In contrast, micromolar and millimolar concentrations of calcium ions are required for villin to exhibit capping and severing, respectively. However, after tyrosine phosphorylation, villin can sever actin filaments even at nanomolar Ca²⁺ concentrations (11). Furthermore, although the actin-severing ability of the N-terminal half of villin is calcium-dependent, that by the N-terminal half of gelsolin is not. In contrast, the binding of G-actin of the C-terminal half of both villin and gelsolin requires Ca²⁺. Creation of hybrid proteins demonstrated that the domains of villin and gelsolin are not interchangeable (12).Abundant x-ray crystallographic structural information exists for gelsolin, including the calcium ion-free (Ca²⁺-free), inactive structure of the intact protein (13), the activated N- and C-terminal halves, each in a bimolecular complex with actin (7, 14), and the activated C-terminal half on its own (15, 16). Structural data for intact villin are unavailable and are limited to fragment V1 (17), solved using NMR methods, and the HP domain, solved by NMR and x-ray crystallography (18, 19). NMR experiments also indicate that HP is connected to V6 by a 40-residue disordered linker. As a result, HP has been proposed to bind actin independently of the remainder of the protein (10).In this report, we present the structure of Ca²⁺-free, isolated villin V6, which exhibits a typical gelsolin domain fold. The long helix in V6 in this structure is straight, unlike the corresponding helix in G6 of intact Ca²⁺-free gelsolin, which is bent, and only straightens on calcium activation of the intact protein. Hence, V6 appears to be in an active conformation in the absence of Ca²⁺. Molecular dynamics simulations indicate that the preferred state of the long helix is also straight for isolated G6 in the absence of Ca²⁺. Furthermore, they suggest a bistable mechanism of helix conformational change regulated by the presence of the remaining domains, by calcium ions, and by other interactants. We therefore propose a mechanism for the gelsolin family proteins whereby Ca²⁺ triggers the straightening of the domain 6 helix in the native conformation of the inactive proteins to propagate more widespread conformational changes.     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

Simulations of calcium channel block by trivalent cations: Gd competes with permeant ions for the selectivity filter

Current through L-type calcium channels (Ca_V1.2 or dihydropyridine receptor) can be blocked by micromolar concentrations of trivalent cations like the lanthanide gadolinium (Gd³⁺). It has been proposed that trivalent block is due to ions competing for a binding site in both the open and closed configuration, but possibly with different trivalent affinities. Here, we corroborate this general view of trivalent block by computing conductance of a model L-type calcium channel. The model qualitatively reproduces the Gd³⁺ concentration dependence and the effect that substantially more Gd³⁺ is required to produce similar block in the presence of Sr²⁺ (compared to Ba²⁺) and even more in the presence of Ca²⁺. Trivalent block is explained in this model by cations binding in the selectivity filter with the charge/space competition mechanism. This is the same mechanism that in the model channel governs other selectivity properties. Specifically, selectivity is determined by the combination of ions that most effectively screen the negative glutamates of the protein while finding space in the midst of the closely packed carboxylate groups of the glutamate residues.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle