Interaction of HRC (Histidine-rich Ca2+-Binding Protein) and Triadin in the Lumen of Sarcoplasmic Reticulum

Abstract

HRC (histidine-rich Ca2+binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca2+ with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca2+-sensitive. Our data suggest that HRC may play a role in the regulation of Ca2+release from the sarcoplasmic reticulum by interaction with triadin. HRC (histidine-rich Ca2+binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca2+ with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca2+-sensitive. Our data suggest that HRC may play a role in the regulation of Ca2+release from the sarcoplasmic reticulum by interaction with triadin. histidine-rich Ca2+-binding protein sarcoplasmic reticulum polyacrylamide gel electrophoresis glutathioneS-transferase polymerase chain reaction sarcoplasmic reticulum Ca2+-ATPase HRC1 (histidine rich Ca2+-binding protein) was first identified from rabbit skeletal muscle because of its ability to bind low density lipoprotein with high affinity. However, HRC did not appear to function as a lipoprotein receptor, as it was localized to the sarcoplasmic reticulum (SR) where it has no access to plasma lipoproteins (1Hofmann S.L. Goldstein J.L. Orth K. Moomaw C.R. Slaughter C.A. Brown M.S. J. Biol. Chem. 1989; 264: 18083-18090Abstract Full Text PDF PubMed Google Scholar). We have previously reported compelling evidence supporting that HRC resides in the lumen of the SR (2Suk J.Y. Kim Y.S. Park W.J. Biochem. Biophys. Res. Commun. 1999; 263: 667-671Crossref PubMed Scopus (29) Google Scholar), consistent with the results of the earlier immunofluorescence and immunoelectron microscopic studies (3Hofmann S.L. Brown M.S. Lee E. Pathak R.K. Anderson R.G. Goldstein J.L. J. Biol. Chem. 1989; 264: 8260-8270Abstract Full Text PDF PubMed Google Scholar). This suggests that HRC may play a role in Ca2+ homeostasis in the SR, although its physiological function is largely unknown. The deduced amino acid sequence of a full-length HRC cDNA reveals several unique features (1Hofmann S.L. Goldstein J.L. Orth K. Moomaw C.R. Slaughter C.A. Brown M.S. J. Biol. Chem. 1989; 264: 18083-18090Abstract Full Text PDF PubMed Google Scholar): 1) an amino-terminal signal sequence; 2) in the middle, nine nearly identical repeats of 29 residues containing a histidine-rich sequence HRHRGH and a stretch of 10 to 11 acidic amino acids; 3) a 13-residue stretch of polyglutamic acid near the carboxyl terminus; and 4) a cluster of 14 closely spaced cysteine residues in the carboxyl terminus. High contents of acidic amino acids may account for the high capacity Ca2+ binding of HRC, which is reminiscent of calsequestrin, a well characterized Ca2+-binding protein of the SR. Triadin was also first identified from rabbit skeletal muscle and found to be a single span membrane protein localized to skeletal muscle triads (4Caswell A.H. Brandt N.R. Brunschwig J.P. Purkerson S. Biochemistry. 1991; 30: 7507-7513Crossref PubMed Scopus (134) Google Scholar, 5Knudson C.M. Stang K.K. Moomaw C.R. Slaughter C.A. Campbell K.P. J. Biol. Chem. 1993; 268: 12646-23654Abstract Full Text PDF PubMed Google Scholar). Triadin was shown to be associated into a stable quaternary complex with the ryanodine receptor, calsequestrin, and junctin at the junctional membrane (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Previous studies suggest that calsequestrin, the high capacity Ca2+-binding protein, actively participates in muscle contraction by regulating the amounts of Ca2+ released by the ryanodine receptor, the Ca2+ release channel (7Ikemoto N. Ronjat M. Meszaros L.G. Koshita M. Biochemistry. 1989; 28: 6764-6771Crossref PubMed Scopus (187) Google Scholar, 8Ikemoto N. Antoniu B. Kang J.J. Meszaros L.G. Ronjat M. Biochemistry. 1991; 30: 5230-5237Crossref PubMed Scopus (82) Google Scholar, 9Kawasaki T. Kasai M. Biochem. Biophys. Res. Commun. 1994; 199: 1120-1127Crossref PubMed Scopus (93) Google Scholar, 10Donoso P. Beltran M. Hidalgo C. Biochemistry. 1996; 35: 13419-13425Crossref PubMed Scopus (70) Google Scholar). This regulatory effect was suggested to be regulated by calsequestrin-anchoring proteins, triadin (11Guo W. Campbell K.P. J. Biol. Chem. 1995; 270: 9027-9030Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 12Guo W. Jorgensen A.O. Campbell K.P. Soc. Gen. Physiol. Ser. 1996; 51: 19-28PubMed Google Scholar) and junctin (13Mitchell R.D. Simmerman H.K. Jones L.R. J. Biol. Chem. 1988; 263: 1376-1381Abstract Full Text PDF PubMed Google Scholar, 14Jones L.R. Zhang L. Sanborn K. Jorgensen A.O. Kelley J. J. Biol. Chem. 1995; 270: 30787-30796Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Several triadin isoforms appear to arise from alternative splicing of the single triadin gene (5Knudson C.M. Stang K.K. Moomaw C.R. Slaughter C.A. Campbell K.P. J. Biol. Chem. 1993; 268: 12646-23654Abstract Full Text PDF PubMed Google Scholar, 15Guo W. Jorgensen A.O. Jones L.R. Campbell K.P. J. Biol. Chem. 1996; 271: 458-465Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 16Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1999; 274: 28660-28668Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). All triadin isoforms share identical sequences over the first 250–260 residues. This common region encompasses the short amino-terminal cytoplasmic region, the membrane-spanning region, and the highly charged lumenal domain. Particularly, the lumenal domain contains several clusters of charged amino acids, which are referred to as “KEKE” motifs (17Realini C. Rogers S.W. Rechsteiner M. FEBS Lett. 1994; 348: 109-113Crossref PubMed Scopus (158) Google Scholar) and have been proposed to facilitate protein-protein interactions by acting as polar zippers (18Realini C. Rechsteiner M. J. Biol. Chem. 1995; 270: 29664-29667Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 19Perutz M.F. Johnson T. Suzuki M. Finch J.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5355-5358Crossref PubMed Scopus (941) Google Scholar, 20West Jr., R.W. Kruger B. Thomas S. Ma J. Milgrom E. Gene. 2000; 243: 195-205Crossref PubMed Scopus (18) Google Scholar). A recent study has demonstrated that a single KEKE motif of the lumenal domain of triadin is indeed essential for the binding of triadin to calsequestrin (21Kobayashi Y.M. Alseikhan B.A. Jones L.R. J. Biol. Chem. 2000; 275: 17639-17646Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Since HRC shares many biochemical and structural features with calsequestrin, we reasoned that HRC might be involved in Ca2+ release from the SR by playing a similar role as calsequestrin. In addition, several lines of circumstantial evidence suggested that HRC might be associated with triadin (22Kim K.C. Caswell A.H. Talvenheimo J.A. Brandt N.R. Biochemistry. 1990; 29: 9281-9289Crossref PubMed Scopus (106) Google Scholar, 23Damiani E. Picello E. Saggin L. Margreth A. Biochem. Biophys. Res. Commun. 1995; 209: 457-465Crossref PubMed Scopus (44) Google Scholar, 24Sacchetto R. Turcato F. Damiani E. Margreth A. J. Muscle Res. Cell Motil. 1999; 20: 403-415Crossref PubMed Scopus (27) Google Scholar). In the present study, we have utilized immunoprecipitation and fusion protein binding assays to investigate the alleged interaction between HRC and triadin. Our data demonstrate that HRC indeed binds to triadin, and this binding occurs between the histidine-rich acidic region of HRC and the lumenal region of triadin containing the KEKE motif, which is also critical for binding to calsequestrin. Heavy SR vesicles were isolated from skeletal muscle of male New Zealand White rabbit according to the heavy SR preparation method described previously (25Kim D.H. Sreter F.A. Ohnishi S.T. Ryan J.F. Roberts J. Allen P.D. Meszaros L.G. Antoniu B. Ikemoto N. Biochim. Biophys. Acta. 1984; 775: 320-327Crossref PubMed Scopus (80) Google Scholar). Protein assays were performed using bovine serum albumin as a standard (26Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). Rabbit skeletal heavy SR vesicles were solubilized at a protein concentration of 5 mg/ml in a buffer containing 2% Triton X-100, 1 m NaCl, 1 mmdithiothreitol, 20 mm Tris-Cl (pH 7.4), and protease inhibitor mixture (Roche Molecular Biochemicals) for 1 h at 4 °C. Solubilized proteins were obtained by centrifugation (a Beckman TLA-100.3 rotor) at 60,000 rpm for 45 min at 4 °C. Triton X-100 solubilized rabbit skeletal heavy SR proteins were diluted 10-fold in 20 mmTris-Cl (pH 7.4), 1 mm dithiothreitol, and protease inhibitor mixture (Roche Molecular Biochemicals). One ml of the diluted extracts was incubated with purified sheep polyclonal antibody raised against HRC (a gift from Dr. Sandra Hoffman, Southwestern Medical Center) or sheep polyclonal antibody raised against triadin (a gift from Dr. Kevin Campbell, University of Iowa College of Medicine) for 2 h at 4 °C, followed by further incubation with 50 µl of protein G-agarose beads (Roche Molecular Biochemicals) for 4 h at 4 °C. Immunoprecipitates were collected and washed several times with a buffer containing 20 mm Tris-Cl (pH 7.4), 0.15m NaCl, and 0.2% Triton X-100. Proteins were eluted from the affinity beads by boiling in SDS-PAGE sample buffer (60 mm Tris-Cl (pH 6.8), 2% SDS, 10% glycerol, 100 mm dithiothreitol, and 0.01% bromphenol blue) and subjected to SDS-PAGE. Purified sheep preimmune IgGs were used as a control. Fragments of HRC and triadin were amplified by PCR, digested with EcoRI andXhoI and subcloned into the EcoRI-XhoI sites of pGEX 4T-1 (Pharmacia). Primer sets used for PCR are as follows: HRC-N (27–199 residues), 5′-ATCCG AATTC ACCCA GCAGC TGAGA GG-3′, 5′-ATTCG TCGAC CTGTG CTCTG GGGAG ACC-3′; HRC-M (193–357 residues), 5′-TTCGG AATTC GAGGA GGTCT CCCCA GAGC-3′, 5′-AACGG TCGAC GACGT GATGG TGTTC ACC-3′; HRC-C (471–847 residues), 5′-ATGCG AATTC CACCA TCACG TCCCT CACC-3′, 5′-TTAGC TCGAG GGCGT CTCCA GCATG TCC-3′; Tri-a (1–66 residues), 5′ ATGAAT TCATGACTGAGATCACTGC-3′, 5′ATGTC GACTA ACAAC GGCAA CTGC-3′; Tri-b (79–160 residues), 5′-ATCCG AATTC TCTAT TGCCA AGATG GGC-3′, 5′-TTAGC TCGAG TGGTA TTTTC CTCTC AGG-3′; Tri-c(151–260 residues), 5′-AGTCG AATTC CAGGA AAAAC CTGAG AGG-3′, 5′-TTAGC TCGAG GTTTT GAAAC AGCAG CAG-3′; Tri-c1 (151–204 residues), 5′-AGTCG AATTC CAGGA AAAAC CTGAG AGG-3′, 5′-TCTTC TCGAG TTTTG TCACT GTCTT TGTTT CTGG-3′; Tri-c2 (204–260 residues), 5′-AGTCG AATTC GAGGA GAAGA AAGCT CG-3′, 5′-TTAGC TCGAG GTTTT GAAAC AGCAG CAG-3′. To produce more GST fusion proteins containing various numbers of histidine-rich acidic repeats of HRC, one forward and two backward primers were synthesized in addition to the HRC-M primer set. The primers were used in combinations. The additional forward primer is: 5′-TTCGG AATTC CACAG GCACC GGGGC CAC-3′. The additional backward primers are: 5′-AACGG TCGAC GGCCC TGGTG ACGGT CGCT-3′ and 5′-AACGG TCGAC TTCAC CTTCG TCATC ATC-3′. GST fusion constructs were induced in Escherichia coli JM109 with 0.5 mmisopropyl β-d-thiogalactopyranoside for 3 h at 30 °C. Cells were harvested, resuspended in phosphate buffered saline containing 1 mm phenylmethylsulfonyl fluoride, lysed by sonication on ice, and then incubated in 1% Triton X-100 for 1 h. The soluble fraction was obtained by centrifugation at 13,000 rpm for 10 min at 4 °C. The fusion proteins were immobilized by incubating 1 ml of the soluble E. coli fraction with 50 µl of bead volume of glutathione-Sepharose 4B (Pharmacia) for 3 h and washed times with 1 ml The immobilized fusion proteins were incubated with 1 ml of heavy SR proteins solubilized with Triton X-100 for 5 h at 4 °C. were then washed with 20 mm Tris-Cl (pH 7.4), and 0.2% Triton X-100. proteins were eluted by boiling in SDS-PAGE sample buffer and subjected to SDS-PAGE. Protein were by or 10% SDS-PAGE and then to by a by the The were with buffer mm Tris-Cl (pH mm and with bovine serum and then incubated with the antibody in bovine serum for 1 h at with The were washed with of for min and incubated with the antibody in bovine serum for 1 h at with The were then washed times with for 20 min were using the against rabbit skeletal triadin and were from The interaction of HRC and triadin was first by co-immunoprecipitation experiments with Solubilized heavy SR proteins were with or The proteins were then by 1) with or of HRC 1 4) to the of triadin preimmune serum was to of proteins 1 immunoprecipitation of triadin 1 4) to the of HRC 1 preimmune serum of proteins 1 and These results show that HRC indeed binds to triadin. SR protein, was by HRC triadin and that the interaction between HRC and triadin is of HRC were and purified as GST fusion proteins to the triadin binding domain of HRC HRC-N not HRC-M contains of the nine nearly identical histidine-rich acidic repeats of a histidine-rich sequence a stretch of 10 to 11 acidic amino acids and a sequence containing two and one in a charged HRC-C contains a 13-residue stretch of polyglutamic acids and a cluster of 14 closely spaced cysteine residues. The GST fusion proteins and a GST were incubated with solubilized heavy SR and using The were by SDS-PAGE and with 2 and the were to and with antibody The that the HRC-M region binds to triadin. the histidine-rich acidic repeats may represent a novel domain that is involved in protein-protein The repeats are highly in amino acid sequences and the is an one To the triadin binding domain in the repeats with we have more GST fusion proteins containing various sets of repeats by PCR 3 of repeats in fusion proteins might arise to the highly of the Protein binding assays were performed as described 3 GST fusion proteins a did not bind to triadin, that no is essential for the triadin This is consistent with the that the repeats are nearly identical with a amino acid or GST fusion proteins, more and a repeats and were shown to bind to triadin in 2 is identical to the fusion protein on we suggest that more and a repeats are for the triadin binding of structural studies be to the of the protein binding of the of the histidine-rich acidic repeats of HRC for triadin sequences of the histidine-rich acidic repeats of HRC are of the fusion proteins are the fusion proteins were incubated with solubilized heavy SR. The proteins were by SDS-PAGE and with or with antibody GST and GST fusion proteins are by a Ca2+-binding protein of SR, binds to triadin and junctin in a We the interaction between HRC and triadin is also by Ca2+ HRC was from solubilized heavy SR in the of or various of The proteins were by to and with and The interaction between HRC and triadin was in the of Ca2+ and by of This is similar to the interaction between calsequestrin and triadin. results were obtained the HRC-M fusion protein was utilized to the triadin protein 4 that HRC-M is the domain responsible for triadin binding of HRC in a Ca2+ All triadin isoforms in a have identical amino acid sequence over the first 250–260 residues. This common region contains a short amino-terminal cytoplasmic a membrane-spanning region, and highly charged The charged domain has been shown to be responsible for binding to calsequestrin and the ryanodine We that this common region might also the HRC binding domain. GST fusion proteins were first Tri-a contains the amino-terminal cytoplasmic region and of the membrane-spanning Tri-b contains the of the common and contains the of the common domain GST fusion proteins and GST were purified and incubated with solubilized heavy SR. The protein were then on on and then with 5 or with antibody or antibody the fusion protein was to We then two more GST fusion proteins, Tri-c1 and which are the and of the region, The results of a similar binding assay that the Tri-c2 region contains the HRC binding 5 the Tri-c2 region contains the KEKE motif that was previously shown to bind directly to calsequestrin (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). However, it is not that this KEKE motif also binds to HRC as well as to calsequestrin. fusion protein also triadin 5 that triadin a the and the for appear to be the The Tri-c2 fusion protein was also used to the interaction between HRC and triadin is as shown in the interaction was in the of Ca2+ and by The was was shown that the ryanodine receptor, calsequestrin, triadin, and junctin a stable quaternary at the of the junctional SR membrane (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). In this protein triadin and junctin are suggested to as a that the Ca2+-binding protein into the of the Ca2+ channel of this structural it is that a high concentration of Ca2+ be at the of the junctional SR and Ca2+ be released the ryanodine of Ca2+release in muscle In the present study, we have shown that HRC, SR Ca2+-binding protein, directly binds to triadin, that HRC be an additional of this protein HRC is a high capacity and low affinity Ca2+-binding protein E. Damiani E. Margreth A. Biochem. Biophys. Res. Commun. PubMed Scopus (44) Google Scholar) calsequestrin. We not the role of HRC, as the more Ca2+-binding protein calsequestrin to its role as a Ca2+ was shown that calsequestrin a role in the regulation of the ryanodine M. K. A. S. M. A. Biochemistry. PubMed Scopus Google Scholar, S. M. G. A. M. Ronjat M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) in addition to the Ca2+ HRC may also have a regulatory to be this is or HRC as a Ca2+ The first evidence of an interaction between HRC and triadin was in an earlier study (22Kim K.C. Caswell A.H. Talvenheimo J.A. Brandt N.R. Biochemistry. 1990; 29: 9281-9289Crossref PubMed Scopus (106) Google Scholar). In this study, the by using affinity that a protein, as triadin, to the of the and to a novel This protein was with which Ca2+-binding proteins K.P. D.H. Jorgensen A.O. J. Biol. Chem. Full Text PDF PubMed Google Scholar). HRC similar on and it is the SR protein in this we reasoned that this protein is R. Turcato F. Damiani E. Margreth A. J. Muscle Res. Cell Motil. 1999; 20: 403-415Crossref PubMed Scopus (27) Google Scholar) performed binding assays with HRC and the that HRC binds to the short amino-terminal cytoplasmic region of triadin. This is with data and also with the earlier and microscopic studies (3Hofmann S.L. Brown M.S. Lee E. Pathak R.K. Anderson R.G. Goldstein J.L. J. Biol. Chem. 1989; 264: 8260-8270Abstract Full Text PDF PubMed Google Scholar) and and experiments (2Suk J.Y. Kim Y.S. Park W.J. Biochem. Biophys. Res. Commun. 1999; 263: 667-671Crossref PubMed Scopus (29) Google Scholar), which suggested that HRC resides in the lumen of the SR. is that of the protein the binding assay may the of protein-protein interactions and the results of binding assays be with Our data show that HRC binds to the lumenal domain of triadin, which is highly in charged amino We further the interaction domain to the Tri-c2 region of triadin Notably, this region contains the KEKE motif that was shown to directly bind to calsequestrin (21Kobayashi Y.M. Alseikhan B.A. Jones L.R. J. Biol. Chem. 2000; 275: 17639-17646Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). KEKE motifs are as of sequences residues in of and with more and residues and or charged residues in a (17Realini C. Rogers S.W. Rechsteiner M. FEBS Lett. 1994; 348: 109-113Crossref PubMed Scopus (158) Google Scholar). This motif was suggested to between proteins (18Realini C. Rechsteiner M. J. Biol. Chem. 1995; 270: 29664-29667Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 19Perutz M.F. Johnson T. Suzuki M. Finch J.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5355-5358Crossref PubMed Scopus (941) Google Scholar, 20West Jr., R.W. Kruger B. Thomas S. Ma J. Milgrom E. Gene. 2000; 243: 195-205Crossref PubMed Scopus (18) Google Scholar). The and Tri-c2 of triadin and two KEKE the KEKE motif of Tri-c2 is responsible for interaction with calsequestrin by an in Our data are not to this KEKE motif the or KEKE motifs are involved in the HRC contains nine nearly identical repeats of 29 of a histidine-rich sequence a stretch of 10 to 11 acidic amino acids, and a sequence containing two and one in a charged (1Hofmann S.L. Goldstein J.L. Orth K. Moomaw C.R. Slaughter C.A. Brown M.S. J. Biol. Chem. 1989; 264: 18083-18090Abstract Full Text PDF PubMed Google Scholar). Our data show that this domain directly binds to triadin and to represent a novel motif for protein-protein However, the the interaction between HRC and triadin is was suggested that KEKE motifs protein-protein interactions by acting as polar that the histidine-rich acidic repeats are to a the interaction may be by the interaction and the interaction (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, R.D. Simmerman H.K. Jones L.R. J. Biol. Chem. 1988; 263: 1376-1381Abstract Full Text PDF PubMed Google Scholar, 14Jones L.R. Zhang L. Sanborn K. Jorgensen A.O. Kelley J. J. Biol. Chem. 1995; 270: 30787-30796Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) are that a common of the triadin binding region of calsequestrin was localized to the stretch of residues Ma J. Kim D.H. FEBS Lett. 2000; PubMed Scopus Google Scholar). In a similar the stretch of acidic amino acids in the histidine-rich acidic repeats of HRC may be essential for triadin binding of However, it be that the stretch of residues in HRC not bind to triadin, that the of and interactions more We that occurs the region of triadin and this interaction is not by high Ca2+ triadin was shown to with junctin in a (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). We that triadin and junctin a by with and and that the of this is not by in Ca2+ that or interactions are by polar zippers that are in this In we have shown that HRC binds to triadin in a in SR This and biochemical of HRC suggest that HRC may play a similar role to calsequestrin be to this of that HRC, which is in