Abstract
Sarcoplasmic reticulum (SR) luminal proteins play an important role in calcium buffering and in regulation of Ca2+ release. Recently, we showed that knockdown of calumenin enhanced Ca2+ cycling in the SR (Sahoo et al., (2009) J. Biol.Chem. 284, 31109-31121). To identify the molecular mechanism behind the enhanced Ca2+ release in calumenin knockdown cells, we investigated the role of calumenin on RyR2-mediated Ca2+ release. Using immunprecipitation assay we found that calumenin interacts with RyR2 in mouse heart and calumenin and RyR2 are co-localized in cardiomyocytes. GST-pull down assay of calumenin deletion fragments and GST-RyR2 intra-luminal loops suggested that the 132-222 amino acid region of calumenin and RyR2-Loop I (4520-4577 amino acids) are the major binding partners. Knockdown of calumenin enhanced the binding of ryanodine in calumenin knockdown HL-1 cells, as depicted by [3H]ryanodine binding assay. Single channel recordings of RyR2 in rat cardiac SR showed that addition of purified GST-calumenin or GST-calumenin-B (132-222 amino acid) to the trans side of the lipid bilayer decreased open probability of RyR2 channels. Taken together, the present results show that calumenin inhibits RyR2 activity in cardiomyocytes by a direct interaction with RyR2. Supported by Korea NRF Grant (2010-0002159), GIST Systems Biology Infrastructure Establishment Grant (2010) and KISTI-KREONET (2010).
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