Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain

Synaptotagmin I has two tandem Ca(2+)-binding C(2) domains, which are essential for fast synchronous synaptic transmission in the central nervous system. We have solved four crystal structures of the C(2)B domain, one of them in the cation-free form at 1.50 A resolution, two in the Ca(2+)-bound form at 1.04 A (two bound Ca(2+) ions) and 1.65 A (three bound Ca(2+) ions) resolution and one in the Sr(2+)-bound form at 1.18 A (one bound Sr(2+) ion) resolution. The side chains of four highly conserved aspartic acids (D303, D309, D363, and D365) and two main chain oxygens (M302:O and Y364:O), together with water molecules, are in direct contact with two bound Ca(2+) ions (sites 1 and 2). At higher Ca(2+) concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca(2+) coordination site (site 3). Divalent cation binding sites 1 and 2 in the C(2)B domain were previously identified from NMR NOE patterns and titration studies, supplemented by site-directed mutation analysis. One difference between the crystal and NMR studies involves D371, which is not involved in coordination with any of the identified Ca(2+) sites in the crystal structures, while it is coordinated to Ca(2+) in site 2 in the NMR structure. In the presence of Sr(2+), which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.     

Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.     

SNARE complex alters the interactions of the Ca2+ sensor synaptotagmin 1 with lipid bilayers

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca²⁺ sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca²⁺ binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca²⁺-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca²⁺ bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.     

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.     

The C2B domain of synaptotagmin is a Ca(2+)-sensing module essential for exocytosis

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca(2+) ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca(2+)-sensing module. Here, we report that Ca(2+) drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca(2)+ are mediated by a set of conserved acidic Ca(2)+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca(2)+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.     

The C2B domain of synaptotagmin I is a Ca2+-binding module

Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.     

Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C2 domains of synaptotagmin 1

Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domain beta-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.     

The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin-SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin-SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.     

The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln(197)-Arg(198), is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca(2+)-dependent interactions between synaptotagmin and SNARE protein complexes.     

Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes

The synaptic vesicle protein synaptotagmin I binds Ca2+ and is required for efficient neurotransmitter release. Here, we measure the response time of the C2 domains of synaptotagmin to determine whether synaptotagmin is fast enough to function as a Ca2+ sensor for rapid exocytosis. We report that synaptotagmin is "tuned" to sense Ca2+ concentrations that trigger neuronal exocytosis. The speed of response is unique to synaptotagmin I and readily satisfies the kinetic constraints of synaptic vesicle membrane fusion. We further demonstrate that Ca2+ triggers penetration of synaptotagmin into membranes and simultaneously drives assembly of synaptotagmin onto the base of the ternary SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment receptor) complex, near the transmembrane anchor of syntaxin. These data support a molecular model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex.     

Examining synaptotagmin 1 function in dense core vesicle exocytosis under direct control of Ca2+

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.     

Distinct roles of the C2A and the C2B domain of the vesicular Ca2+ sensor synaptotagmin 9 in endocrine beta-cells

Synaptotagmins form a family of calcium-sensor proteins implicated in exocytosis, and these vesicular transmembrane proteins are endowed with two cytosolic calcium-binding C2 domains, C2A and C2B. Whereas the isoforms syt1 and syt2 have been studied in detail, less is known about syt9, the calcium sensor involved in endocrine secretion such as insulin release from large dense core vesicles in pancreatic beta-cells. Using cell-based assays to closely mimic physiological conditions, we observed SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)-independent translocation of syt9C2AB to the plasma membrane at calcium levels corresponding to endocrine exocytosis, followed by internalization to endosomes. The use of point mutants and truncations revealed that initial translocation required only the C2A domain, whereas the C2B domain ensured partial pre-binding of syt9C2AB to the membrane and post-stimulatory localization to endosomes. In contrast with the known properties of neuronal and neuroendocrine syt1 or syt2, the C2B domain of syt9 did not undergo calcium-dependent membrane binding despite a high degree of structural homology as observed through molecular modelling. The present study demonstrates distinct intracellular properties of syt9 with different roles for each C2 domain in endocrine cells.     

Structure of human synaptotagmin 1 C2AB in the absence of Ca2+ reveals a novel domain association

Release of neurotransmitter from synaptic vesicles requires the Ca2+/phospholipid-binding protein synaptotagmin 1. There is considerable evidence that cooperation between the tandem C2 domains of synaptotagmin is a requirement of regulated exocytosis; however, high-resolution structural evidence for this interaction has been lacking. The 2.7 A crystal structure of the cytosolic domains of human synaptotagmin 1 in the absence of Ca2+ reveals a novel closed conformation of the protein. The shared interface between C2A and C2B is stabilized by a network of interactions between residues on the C-terminal alpha-helix of the C2B domain and residues on loops 1-3 of the Ca2+-binding region of C2A. These interactions alter the overall shape of the Ca2+-binding pocket of C2A, but not that of C2B. Thus, synaptotagmin 1 C2A-C2B may utilize a novel regulatory mechanism whereby one C2 domain could regulate the other until an appropriate triggering event decouples them.     

Structural transitions in the synaptic SNARE complex during Ca2+-triggered exocytosis

The synaptic SNARE complex is a highly stable four-helix bundle that links the vesicle and plasma membranes and plays an essential role in the Ca(2+)-triggered release of neurotransmitters and hormones. An understanding has yet to be achieved of how this complex assembles and undergoes structural transitions during exocytosis. To investigate this question, we have mutated residues within the hydrophobic core of the SNARE complex along the entire length of all four chains and examined the consequences using amperometry to measure fusion pore opening and dilation. Mutations throughout the SNARE complex reduced two distinct rate processes before fusion pore opening to different degrees. These results suggest that two distinct, fully assembled conformations of the SNARE complex drive transitions leading to open fusion pores. In contrast, a smaller number of mutations that were scattered through the SNARE complex but were somewhat concentrated in the membrane-distal half stabilized open fusion pores. These results suggest that a structural transition within a partially disassembled complex drives the dilation of open fusion pores. The dependence of these three rate processes on position within the SNARE complex does not support vectorial SNARE complex zipping during exocytosis.     

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4

The neuronal protein synaptotagmin 1 functions as a Ca(2+) sensor in exocytosis via two Ca(2+)-binding C(2) domains. The very similar synaptotagmin 4, which includes all the predicted Ca(2+)-binding residues in the C(2)B domain but not in the C(2)A domain, is also thought to function as a neuronal Ca(2+) sensor. Here we show that, unexpectedly, both C(2) domains of fly synaptotagmin 4 exhibit Ca(2+)-dependent phospholipid binding, whereas neither C(2) domain of rat synaptotagmin 4 binds Ca(2+) or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca(2+) ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C(2)B domain unable to form full Ca(2+)-binding sites. These results indicate that synaptotagmin 4 is a Ca(2+) sensor in the fly but not in the rat, that the Ca(2+)-binding properties of C(2) domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.     

Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex

The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface.     

Myosin-Va regulates exocytosis through the submicromolar Ca2+-dependent binding of syntaxin-1A

Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 microM Ca2+. Interference with formation of the syntaxin-1A-myosin-Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca2+ depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca2+.     

The pilus-induced Ca2+ flux triggers lysosome exocytosis and increases the amount of Lamp1 accessible to Neisseria IgA1 protease

The IgA1 protease secreted by the pathogenic Neisseriae cleaves Lamp1, a major integral membrane glycoprotein of lysosomes, and significantly reduces its steady-state levels in an infected cell. IgA1 protease hydrolysis of Lamp1 is inefficient at the low pH of lysosomes, strongly suggesting that the enzyme is unlikely to reduce Lamp1 levels within lysosomes to any appreciable extent. We therefore explored the possibility that the protease may reach Lamp1 through an alternative route. We demonstrate that Neisseria pili induce a transient increase in the levels of cytosolic free Ca²⁺ in A431 human epithelial cells, as demonstrated previously for ME180 cells. This Ca²⁺ flux triggers lysosome exocytosis, quickly altering the cellular distribution of Lamp1 and increasing surface Lamp1 levels. Finally, we demonstrate that surface Lamp1 is cleaved by IgA1 protease secreted by adherent bacteria. We conclude that the pilus-induced Ca²⁺ flux increases the amount of Lamp1 that is cleavable by the IgA1 protease.     

Mutations in the effector binding loops in the C2A and C2B domains of synaptotagmin I disrupt exocytosis in a nonadditive manner

The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2 domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca2+ requirements for syt-liposome interactions from approximately 20 to approximately 40 microm Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in an approximately 50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full-length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2 domains of syt cooperate with one another to trigger release via loop-mediated electrostatic interactions with effector molecules.     

The effect of Mg2+ on the Ca2+ binding to troponin C in rabbit fast skeletal myofibrils.

The effect of Mg2+ on the Ca2+ binding to rabbit fast skeletal troponin C and the CA2+ dependence of myofibrillar ATPase activity was studied in the physiological state where troponin C was incorporated into myofibrils. The Ca2+ binding to troponin C in myofibrils was measured directly by 45Ca using the CDTA-treated myofibrils as previously reported (Morimoto, S. and Ohtsuki, I. (1989) J. Biochem. 105, 435-439). It was found that the Ca2+ binding to the low and high affinity sites of troponin C in myofibrils was affected by Mg2+ competitively and the Ca2(+)- and Mg2(+)-binding constants were 6.20 x 10(6) and 1.94 x 10(2) M-1, respectively, for the low affinity sites, and 1.58 x 10(8) and 1.33 x 10(3) M-1, respectively, for the high affinity sites. The Ca2+ dependence of myofibrillar ATPase was also affected by Mg2+, with the apparent Ca2(+)- and Mg2(+)-binding constants of 1.46 x 10(6) and 276 x 10(2) M-1, respectively, suggesting that the myofibrillar ATPase was modulated through a competitive action of Mg2+ on Ca2+ binding to the low affinity sites, though the Ca2+ binding to the low affinity sites was not simply related to the myofibrillar ATPase.