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KMID : 0578320160390020149
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Molecules and Cells
2016 Volume.39 No. 2 p.149 ~ p.155
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Characterization of Ca2+-Dependent Protein-Protein Interactions within the Ca2+ Release Units of Cardiac Sarcoplasmic Reticulum
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Rani Shilpa
Park Chang-Sik
Sreenivasaiah Pradeep Kumar
Kim Do-Han
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Abstract
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In the heart, excitation-contraction (E-C) coupling is mediated by Ca2+ release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the Ca2+ release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich Ca2+ 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 Ca2+ dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped Ca2+ dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such Ca2+ 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 Ca2+ into SR at intermediate Ca2+ concentrations.
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KEYWORD
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calsequestrin, histidine rich Ca2+ binding protein, and triadin, junctin, ryanodine receptor
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