title: Characterization of Ca(2+)-Dependent Protein-Protein Interactions within the Ca(2+) Release Units of Cardiac Sarcoplasmic Reticulum.
abstract: In the heart, excitation-contraction (E-C) coupling is mediated by Ca(2+) release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the Ca(2+) release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich Ca(2+) binding protein (HRC) are known to bind the charged luminal region of triadin (TRN) and thus directly or indirectly regulate ryanodine receptor 2 (RyR2) activity. However, the mechanisms of CSQ and HRC mediated regulation of RyR2 activity through TRN have remained unclear. We first examined the minimal KEKE motif of TRN involved in the interactions with CSQ2, HRC and RyR2 using TRN deletion mutants and in vitro binding assays. The results showed that CSQ2, HRC and RyR2 share the same KEKE motif region on the distal part of TRN (aa 202-231). Second, in vitro binding assays were conducted to examine the Ca(2+) dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped Ca(2+) dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such Ca(2+) dependence pattern. Third, competitive binding was conducted to examine whether CSQ2, HRC, or RyR2 affects the TRN-HRC or TRN-CSQ2 binding at pCa4. Among them, only CSQ2 or RyR2 competitively inhibited TRN-HRC binding, suggesting that HRC can confer functional refractoriness to CRU, which could be beneficial for reloading of Ca(2+) into SR at intermediate Ca(2+) concentrations.
authors.affiliation: School of Life Sciences and Systems Biology Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
authors.forename: Shilpa
authors.initials: S
authors.lastname: Rani
authors.affiliation: School of Life Sciences and Systems Biology Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
authors.forename: Chang Sik
authors.initials: CS
authors.lastname: Park
authors.affiliation: School of Life Sciences and Systems Biology Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
authors.forename: Pradeep Kumar
authors.initials: PK
authors.lastname: Sreenivasaiah
authors.affiliation: School of Life Sciences and Systems Biology Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
authors.forename: Do Han
authors.initials: DH
authors.lastname: Kim
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002155:Calsequestrin / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D002352:Carrier Proteins; C489418:Hrc protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C498270:Trdn protein, mouse; C000592748:ryanodine receptor 2. mouse; D002118:Calcium
keywords: calsequestrin; histidine rich Ca2+ binding protein, and triadin; junctin; ryanodine receptor