Phospholipid-dependent Ca2+ binding by the 36-kDa tyrosine kinase substrate (calpactin) and its 33-kDa core

A 36-kDa protein, which is a component of the membrane skeleton, has been shown to co-localize with spectrin in addition to serving as a major substrate for tyrosine-protein kinases. This protein, which will be referred to as calpactin (for calcium-dependent phospholipid and actin binding protein), was isolated from bovine intestine as the complex with a 10-kilodalton light chain and the Ca2+ binding was analyzed by equilibrium dialysis with 45Ca2+ in the presence or absence of phospholipid. Although Ca2+ binding by calpactin alone was negligible at micromolar free Ca2+, it was greatly enhanced by liposomes containing phosphatidylserine or phosphatidylinositol. A proteolytic derivative of calpactin, termed the "core," which has lost the site of association with the light chain in addition to the site of tyrosine phosphorylation by pp60src, was also found to contain this high affinity phospholipid enhanced Ca2+-binding activity. Scatchard plots reveal that each calpactin monomer or core polypeptide bound 2 Ca2+ ions with a Kd of 4.5 X 10(-6) M at 200 micrograms of phosphatidylserine/ml. Liposome binding experiments confirmed that calpactin as a complex with light chain as well as calpactin monomer or the 33-kDa core interact with phosphatidylserine liposomes in a Ca2+-dependent manner.     

Calpactin-like proteins in human spermatozoa

Polyclonal antibodies directed against human calpactin I (p36) and calpactin II (p35) have been employed to investigate the distribution of calpactin-like proteins in human spermatozoa. Calpactins are a family of Ca2+-regulated cytoskeletal proteins that are major substrates of oncogene and growth factor receptor protein tyrosine kinases. The existence of a Triton-soluble 37-kDa protein antigenically related to calpactin II from somatic cells was revealed by Western blot analysis of human sperm extracts. The 37-kDa protein was not released from spermatozoa after experimental induction of the acrosome reaction by A23187 and Ca2+. Treatment of sperm homogenates with an EGTA-containing buffer partially solubilized the 37-kDa protein from the corpuscolate matter. Indirect immunofluorescence microscopy showed that anticalpactin II binds specifically to the sperm tail and to a band-like structure encircling the sperm head at the equatorial segment. In contrast, antibodies to calpactin I were found to bind to the tail midpiece, but failed to bind to Western blots of sperm proteins. This is the first immunological and biochemical report on the presence of calpactin proteins in a germ cell, the human spermatozoon.     

Isolation of Ca2+ sensitive proteins linked to the membrane fraction of the brain cortex and the spinal cord in pigs

Four Ca2+-sensitive proteins of respective subunit molecular weights 67 kDa, 37 kDa, 36 kDa and 32 kDa were purified from pig brain and spinal cord. Associated to the particulate fraction at millimolar concentrations of free Ca2+, they were solubilized using an EGTA-containing buffer and purified by a selective Ca2+-dependent precipitation. The 36 kDa protein is present in the tissues in a tetrameric form of (2 X 36 kDa + 2 X 13 kDa) and in a monomeric form. These proteins with the 37 kDa protein share the functional properties of the two well-known Ca2+-binding proteins, named calpactin I and calpactin II; they were able to interact with F-actin, brain spectrin (fodrin) and phosphatidylserine-liposomes in a Ca2+-dependent manner. The 67 kDa protein depolymerizes the actin filament in presence of Ca2+, it also binds to tubulin and to the neurofilament subunit NF-70, but not to brain spectrin. The 32 kDa protein does not share any association with F-actin and brain spectrin.     

Epidermal-growth-factor-stimulated phosphorylation of calpactin II in membrane vesicles shed from cultured A-431 cells.

Membrane vesicles shed from intact A-431 epidermoid carcinoma cells and harvested in the presence of Ca2+ contained epidermal-growth-factor (EGF) receptor/kinase substrates of apparent molecular masses 185, 85, 70, 55, 38 and 27 kDa. The 38 kDa substrate (p38) was recognized by an antibody that had been raised against the human placental EGF receptor/kinase substrate calpactin II (lipocortin I). The A-431 and placental substrates, isolated by immunoprecipitation after phosphorylation in situ, yielded identical phosphopeptide maps upon limited proteolytic digestion with each of five different enzymes. The A-431-cell vesicular p38 is therefore calpactin II. EGF treatment of the intact A-431 cells before inducing vesiculation was not necessary for the substrate to be present within the vesicles. Our data thus indicate that receptor internalization is not a prerequisite for receptor-mediated phosphorylation of calpactin II. The ability of the protein to function as a substrate for the receptor/kinase depended upon the continued presence of Ca2+ during the vesicle-isolation procedure. EGF-stimulated phosphorylation of calpactin II was much less pronounced in vesicles prepared from A-431 cells in the absence of Ca2+, although comparable amounts of the protein were detectable by immunoblotting. Calpactin II therefore appears to be sequestered in a Ca2+-modulated manner within shed vesicles, along with at least four other major targets for the EGF receptor/kinase. The vesicle preparation may be a useful model system in which to study the phosphorylation and function of potentially important membrane-associated substrates for the receptor.     

Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src.

Calpactins I and II are proteins that bind Ca2+, phospholipids, actin and spectrin; they are also major substrates of oncogene and growth-factor-receptor tyrosine kinases. Since calpactins have been proposed to provide a link between membrane lipids and the cytoskeleton, we examined in detail the interactions between purified calpactin I and phospholipid liposomes. We focused on the Ca2+-dependence, the effects of phosphorylation of calpactin I by p60v-src (the protein kinase coded for by the Rous-sarcoma-virus oncogene), and the effects of the binding of calpactin I light chain to calpactin I heavy chain. Binding of the light chain to the heavy chain increased the affinity of calpactin I for phosphatidylserine (PS) liposomes. The opposite effect was observed for phosphorylation by p60v-src; phosphorylation decreased the affinity of calpactin I for PS liposomes. These two opposite effects appeared to be independent, since phosphorylation did not prevent light-chain binding to the heavy chain. Calpactin I was found, by the use of three different techniques, to bind to phospholipid liposomes at less than 10(-8) M free Ca2+. This result is in contrast with those of previous studies, which indicated that greater than 10(-6) M free Ca2+ was required. Our findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src.     

Identification of phospholipid-dependent calcium-binding proteins as constituents of matrix vesicles

Uptake of mineral ions by isolated matrix vesicles (MV) incubated in synthetic cartilage lymph follows a consistent pattern. After an initial lag period, MV rapidly accumulate large amounts of Ca2+ and Pi before the appearance of crystalline mineral. The ability of MV to accumulate Ca2+ is readily destroyed by proteases, indicating that proteins are important in Ca2+ accumulation. Since MV contain significant amounts of phosphatidylserine (PS), an acidic phospholipid with affinity for Ca2+, it seemed probable that this lipid might also contribute to Ca2+ binding. The development of methods for reproducible isolation of pure active MV enabled us to search for factors responsible for the rapid accumulation of Ca2+. Reported here are studies which reveal that a set of intensely staining MV proteins, extractable with EGTA, selectively bind to Ca2+, but only in the presence of acidic phospholipids. These 30-36-kDa proteins form readily sedimentable insoluble ternary complexes of protein, Ca2+, and lipid in the presence of low levels of Ca2+. With liposomes composed of PS, alone or in combination with phosphatidylethanolamine, submicromolar levels of Ca2+ or certain other divalent cations, but not Mg2+, are sufficient to form the complexes. The physical and chemical properties of these MV proteins appear to be like those of the calpactin family of membrane-associated proteins. In fact, these MV proteins were found to cross-react with antibodies to calpactin II. Thus, calpactins appear to be important protein constituents of avian growth plate MV. This finding helps explain the enrichment in PS previously noted in MV and may also point to the mechanism by which MV rapidly accumulate Ca2+.     

Purification, characterization, and partial sequence analysis of 32-kDa calcimedin from chicken gizzard

Calcimedin is a group of proteins which has a binding ability to several hydrophobic matrices or cellular membrane fractions in the presence of Ca2+. Although the molecular properties were partially clarified, the physiological functions of calcimedins have not been clearly defined. In this study, we describe the isolation and characterization of 32-kDa calcimedin from chicken gizzard. Both structural and functional studies establish that 32-kDa calcimedin is a member of the calpactin/lipocortin family. The 32-kDa calcimedin displays phospholipase A2 inhibitory activity, Ca2(+)-dependent F-actin binding activity, and phospholipid binding activity similar to those of calpactins/lipocortins. Antiendonexin II antibody recognized 32-kDa calcimedin. However, antibodies against calpactin I (lipocortin II), calpactin II (lipocortin I), 35-kDa calcimedin, and 67-kDa calcimedin did not cross-react with 32-kDa calcimedin. One-dimensional peptide maps of the 32-kDa calcimedin and the 35-kDa calcimedin are different, confirming that they are distinct proteins. By comparing the sequence of 32-kDa calcimedin with the predicted sequence of endonexin II, we concluded that the primary structure of the 32-kDa protein is highly conserved. In particular, the sequences AMKGMGTDDEXEIXL, GMGTDEEEIL, VLTEILASR, and ILTSR conform to the endonexin consensus sequence, which is characteristic of the calpactin/lipocortin family.     

Two Ca2(+)-binding proteins in the mitotic apparatus of sea urchin eggs

Two Ca2(+)-binding proteins of sea urchin eggs were purified and partially characterized. They showed Ca2(+)-dependent binding to actin filaments and Ca2(+)-dependent changes of fluorescence intensity which was used to estimate the affinity constant of these proteins to Ca2+ ions. Ca2+ ions did not increase phospholipid binding ability of these proteins. Therefore these proteins are distinguished from the calpactin family. Staining of sections of metaphase eggs embedded in paraffin showed their localization in the mitotic apparatus. Furthermore, staining of whole mount eggs with anti-tubulin and antibodies against these proteins, followed by observations with confocal laser-scanning microscopy showed their co-localization with microtubules more clearly. In vitro co-sedimentation assay of microtubules with these proteins, however, showed no interaction between them. This suggested that some structures surrounding the mitotic apparatus microtubules are responsible for their localization.     

Evidence for Ca(2+)-mediated F-actin/phospholipid binding of human sperm calpactin II.

We have previously reported the presence in human spermatozoa of calpactin II, a calcium-binding component of the membrane skeleton (Berruti, Exper. Cell Res. 179, 374, 1988). Reported here are studies which show the ability of sperm calpactin II to interact with filamentous actin and acidic phospholipids in a Ca(2+)-dependent fashion. At high Ca2+ concentrations (greater than 1 mM) sperm calpactin II binds to actin filament and it is resolubilized by EGTA. Liposome binding experiments reveal that sperm calpactin II does associate with phospholipidic vesicles at micromolar free Ca2+ levels. These interaction properties together with the cellular distribution of the protein revealed by immunofluorescence analysis in Ca2+ and ionophore A23187-treated human spermatozoa allow to hypothesize a physiological role for calpactin II in sperm Ca(2+)-mediated events.     

Epidermal growth factor dependent phosphorylation of a 35-kilodalton protein in placental membranes

In human placental membranes isolated in the presence of ethylenediaminetetraacetic acid (EDTA), epidermal growth factor (EGF) stimulated the [gamma-32P]ATP-dependent phosphorylation of tyrosine residues on the 170-kilodalton (kDa) EGF receptor and on a 35-kDa protein. The initial rate of phosphorylation of these proteins in the presence of EGF was 5.2 and 3.5 nmol of phosphate min-1 (mg of receptor protein)-1, and this was approximately 10- and 6-fold higher than the basal rate, respectively. Half-maximal phosphorylation of both proteins occurred at about 2.5 nM EGF. In the presence of p-nitrophenyl phosphate, EGF stimulated the phosphorylation of the 35-kDa protein but not the EGF receptor, suggesting that hormone-stimulated autophosphorylation of the receptor/kinase was not required for kinase activation. The 35-kDa protein exists in two forms: (1) 35Keluate, which was associated with the membrane in the presence of Ca2+ but was eluted with EDTA, and (2) 35Kmemb, which was not eluted from membranes with EDTA. Both forms were immunologically related to a 35-kDa protein previously isolated from A431 cells. Antiserum against the 35-kDa protein also reacted with a protein with an apparent size of 66 kDa that was phosphorylated in an EGF-dependent manner. In phosphorylation reactions performed in the presence of Mg2+, Ca2+ was required for phosphorylation of the 35Keluate form, but Ca2+ was not required for phosphorylation of the 35Kmemb form. Phosphorylation appears to change the membrane-binding properties of the 35Kmemb form because 32P-labeled 35Kmemb could be eluted from the membrane by EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling

Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non-neoplastic cells. Immunoreactive calpactin II, a well-known substrate for EGF-receptor kinase, was found predominantly in the cytosol, although a second immunoreactive pool was found in a Triton X-100-extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2-5 min) formation of ruffles at the cell surface and at the cell-cell contacts. Both calpactin II and filamentous actin were found co-localized at the membrane ruffles. Immunoprecipitations of membrane-bound calpactin II from 32P-labeled cells indicate a transient EGF-dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2-5 min) function for calpactin II at the plasma membrane during the EGF-induced mitogenesis of T51B cells.     

Association of the S-100-related calpactin I light chain with the NH2-terminal tail of the 36-kDa heavy chain

Calpactin I, a Ca2+- and phospholipid-binding cytoskeletal protein, which serves as a major substrate of protein-tyrosine kinases, was isolated from bovine intestine and lung as a species containing two 36-kDa heavy chains and two 10-kDa light chains. The heavy chain is comprised of two distinct domains which can be identified by limited proteolysis: a COOH-terminal 33-kDa core, which contains the Ca2+- and phospholipid-binding sites, and an NH2-terminal tail, which contains the major site of phosphorylation by pp60v-src. To determine the site of association of the light chain on the heavy chain, we analyzed the association states of the light chain, core, and tail by sucrose gradient centrifugation after limited chymotryptic digestion. The core was not detected in higher Mr complexes with the light chain, and the tail cosedimented with a light chain dimer. The tail, isolated from chymotryptic digests and radiolabeled with 125I, was found to form a specific complex with the light chain, but not the core. The authentic tail and a synthetic peptide corresponding to residues 1-29 of the calpactin I heavy chain were both able to specifically inhibit the reassociation between heavy and light chain, whereas a synthetic peptide corresponding to residues 15-33 was inactive. These results suggest that the tail may serve as a site of regulation by light chain or phosphorylation.     

A calcium-dependent 35-kilodalton substrate for epidermal growth factor receptor/kinase isolated from normal tissue

We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, can serve as a substrate for the epidermal growth factor (EGF) receptor/tyrosine kinase (Fava, R.A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). We now report the detection of an antigenically related 35-kDa protein in a number, but not all, of rat, pig, and human tissues. These antigenically related proteins also can serve as substrates for the EGF receptor/kinase in the presence of Ca2+. All of these proteins share the property of reversible, Ca2+-dependent binding to the particulate fraction (presumably membranes) of cell homogenates. We have isolated the 35-kDa substrate from porcine lung and have demonstrated that it is a Ca2+-binding protein. The amino-terminal sequence and the site of tyrosine phosphorylation therein have been determined. The positions of the acidic amino acid residues amino-terminal to the tyrosine phosphorylation site bear a distinct resemblance to the sequence in the homologous region of a number of other substrates for tyrosine kinases. Based on available data, the 35-kDa protein clearly differs from the protein I complex derived from intestinal mucosa and thought to be related to the proteins isolated herein (Gerke, V., and Weber, K. (1985) J. Biol. Chem. 260, 1688-1695). Finally, we report a striking sequence homology between the porcine 35-kDa described herein and human lipocortin, a phospholipase A2 inhibitor.     

Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein

Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.     

Characterization of the Ca2+-binding sites of annexin II tetramer

Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains three type II and two type III Ca(2+)-binding sites which are thought to regulate the interaction of AIIt with anionic phospholipid, F-actin, and heparin. In the present study we utilized site-directed mutagenesis to create AIIt mutants with inactive type III (TM AIIt), type II (CM AIIt), and both type II and III Ca(2+)-binding sites (TCM AIIt). Surprisingly, we found that in the presence of Ca(2+), the TM, CM, and TCM AIIt bound phospholipid and F-actin with similar affinity to the wild type AIIt (WT AIIt). Furthermore, the TCM mutant, and to a lesser extent the TM and CM AIIt displayed dose-dependent Ca(2+)-independent phospholipid aggregation and binding. While the TM and CM AIIt demonstrated Ca(2+)-dependent binding to F-actin, the binding of the TCM AIIt was Ca(2+)-independent. These results suggest that the type II or type III Ca(2+)-binding sites do not directly participate in anionic phospholipid or F-actin binding. We therefore propose that in the absence of Ca(2+), the type II and type III Ca(2+)-binding sites of AIIt stabilize a conformation of AIIt that is unfavorable for binding phospholipid and F-actin. Ca(2+) binding to these sites, or the inactivation of these Ca(2+)-binding sites by site-directed mutagenesis, results in a conformational change that promotes binding to anionic phospholipid and F-actin. Since the TM, CM, and TCM AIIt require Ca(2+) for binding to heparin, we also propose that novel Ca(2+)-binding sites regulate this binding event.     

cDNA structure and expression of calpactin, a peptide involved in Ca2(+)-dependent cell aggregation in sponges.

Aggregation of cells of the marine sponge Geodia cydonium is mediated by an aggregation factor (AF) particle of Mr 1.3 X 10(8). It is now reported that the AF particle is associated with calpactin, which was ascribed a role in the cell-adhesion process. In order to identify the sequence similarity to other members of the lipocortin family, the cDNA of sponge calpactin was cloned and found to display an 80% sequence similarity to vertebrate calpactin II but only a 47% similarity to calpactin I. The calpactin gene, which contains the consensus sequence coding for the amino acids G-T-D-E, was expressed in Escherichia coli and subsequently purified to a 37000-Mr polypeptide. Both the p32 and the p37 are provided with approximately two Ca2+ ions/molecule and the property to bind to phospholipids. The dissociation constant (calpactin-Ca2+) was in the absence of phospholipids in the range 500-700 microM-Ca2+ but in their presence about 20-30 microM-Ca2+. On the basis of (i) inhibition studies with antibodies to calelectrin and (ii) competition experiments with soluble phospholipids (both chemically defined as well as total homologous membrane lipids) we conclude that the AF-associated calpactin and plasma-membrane-bound phospholipid(s) are involved in cell-cell aggregation in sponges.     

Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies.

Vitamin K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. To determine which regions in protein S are important for its cofactor activity, we have raised and characterized a large panel of monoclonal antibodies against human protein S. Several of the antibodies were directed against Ca2(+)-dependent epitopes, and they were found to be located either in the domain containing gamma-carboxyglutamic acid (Gla), the thrombin-sensitive region, or in the first epidermal growth factor (EGF)-like domain. The first two types of epitopes were exposed at approximately 1 mM Ca2+, whereas the epitope(s) in the EGF-like domains required less than 1 microM Ca2+, suggesting the presence of one or more high affinity Ca2(+)-binding site(s). The antibodies, as well as their Fab' fragments, against all three types of Ca2(+)-dependent epitopes efficiently inhibited the activated protein C cofactor function of protein S, but through different mechanisms. The antibodies against the Gla domain exerted their effects through inhibition of protein S binding to negatively charged phospholipid. Fab'-fragments of antibodies against the thrombin-sensitive region and the first EGF-like domain were the most potent inhibitors of the activated protein C cofactor function but did not inhibit phospholipid binding of protein S. In conclusion, we have identified the domains in protein S that are important for the activated protein C cofactor activity. The Gla domain is instrumental in the binding of protein S to phospholipid, whereas the thrombin-sensitive region and the first EGF-like domain may be directly involved in protein-protein interactions on the phospholipid surface.     

Differential fractionation of matrix vesicle proteins. Further characterization of the acidic phospholipid-dependent Ca2(+)-binding proteins

Matrix vesicles (MV) initiate de novo mineralization in a variety of vertebrate-calcifying tissues. In recent studies, a quantitatively major group of MV proteins, the acidic phospholipid-dependent Ca2(+)-binding proteins (APD-CaBP) were found to be immunologically related to the annexin family of proteins that possess phospholipase A2 inhibitory activity. This finding helped explain the enrichment of phosphatidylserine as well as the presence of large amounts of complexed Ca2+ noted previously in these structures. To characterize further these annexin-like proteins, preparations of both collagenase-released MV and MV-enriched microsomes were subjected to a differential fractionation process that led to the isolation and purification to homogeneity of two of the MV APD-CaBP, a 33-kDa protein and a 36-kDa calpactin II-like protein. Polyclonal antibodies raised to each pure protein were found not to cross-react with the other, thus indicating two distinctive proteins. Measurement of the phosphatidylserine-dependent Ca2(+)-binding properties of the proteins revealed apparent Kd values of 2.5 x 10(-7) and 5.0 x 10(-7) M for the 36- and 33-kDa proteins, respectively. Such high affinities indicate that both proteins would be normally bound to the membrane of MV. Immunological studies revealed the presence of both APD-CaBP in cultured growth plate chondrocytes but not in vesicles released into the culture medium. The finding of the 33-kDa but not the 36-kDa protein in vesicles released from the calcifying matrix of the chondrocyte cultures by collagenase digestion may indicate a role for this protein in MV mineralization.     

A 32-kDa protein associated with phospholipase A2-inhibitory activity from human placenta

Two monomeric 32-kDa proteins, termed 32K-I (pI 5.8) and 32K-II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+-chelator. Only 32K-I was associated with PLA2-inhibitory activity. CNBr peptide mapping indicated that 32K-I was distinct from 32K-II and two 36-kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2-inhibitory activity. 32K-I bound to PS in a Ca2+-dependent manner. 32K-I was detected in many tissues except brain, cardiac and skeletal muscle.     

Characterization of a Ca2+ binding and regulatory site in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.

A region in the skeletal muscle ryanodine receptor between amino acids 4014 and 4765 was expressed as a trpE fusion protein. Overlay studies revealed that this region bound Ca2+ and ruthenium red, an indicator of Ca(2+)-binding sites. Ca2+ binding was mapped to subregion 13b between amino acids 4246 and 4377, encompassing a predicted high affinity Ca(2+)-binding site, and to subregion 13c between amino acids 4364 and 4529, encompassing two predicted high affinity Ca(2+)-binding sites. Ca2+ binding was then mapped to three shorter sequences, 22(13b1), 36(13c1), and 35(13c2), amino acids long, each encompassing one of the three predicted Ca(2+)-binding sites. Site-directed polyclonal antibodies were raised against these three short sequences and purified on antigen affinity columns. The antibody against sequence 13c2, lying between residues 4478 and 4512, specifically recognized both denatured and native forms of the ryanodine receptor, suggesting that at least part of the 35 amino acid sequence containing the Ca(2+)-binding site is surface-exposed. The affinity purified antibody increased the Ca2+ sensitivity of ryanodine receptor channels incorporated into planar lipid bilayers, resulting in increased open probability and opening time without altering channel conductance. The antibody-activated channel was still modulated by Ca2+, Mg2+, ATP, ryanodine, and ruthenium red. These observations suggest that sequence 13c2 may be involved in Ca(2+)-induced Ca2+ release.