Arvanitis Demetrios A, Vafiadaki Elizabeth, Fan Guo-Chang, Mitton Bryan A, Gregory Kimberly N, Del Monte Federica, Kontrogianni-Konstantopoulos Aikaterini, Sanoudou Despina, Kranias Evangelia G
Molecular Biology Division, Center for Basic Research, Foundation for Biomedical Research of the Academy of Athens, Athens, Greece.
Am J Physiol Heart Circ Physiol. 2007 Sep;293(3):H1581-9. doi: 10.1152/ajpheart.00278.2007. Epub 2007 May 25.
Depressed cardiac Ca cycling by the sarcoplasmic reticulum (SR) has been associated with attenuated contractility, which can progress to heart failure. The histidine-rich Ca-binding protein (HRC) is an SR component that binds to triadin and may affect Ca release through the ryanodine receptor. HRC overexpression in transgenic mouse hearts was associated with decreased rates of SR Ca uptake and delayed relaxation, which progressed to hypertrophy with aging. The present study shows that HRC may mediate part of its regulatory effects by binding directly to sarco(endo)plasmic reticulum Ca-ATPase type 2 (SERCA2) in cardiac muscle, which is confirmed by coimmunostaining observed under confocal microscopy. This interaction involves the histidine- and glutamic acid-rich domain of HRC (320-460 aa) and the part of the NH(2)-terminal cation transporter domain of SERCA2 (74-90 aa) that projects into the SR lumen. The SERCA2-binding domain is upstream from the triadin-binding region in human HRC (609-699 aa). Specific binding between HRC and SERCA was verified by coimmunoprecipitation and pull-down assays using human and mouse cardiac homogenates and by blot overlays using glutathione S-transferase and maltose-binding protein recombinant proteins. Importantly, increases in Ca concentration were associated with a significant reduction of HRC binding to SERCA2, whereas they had opposite effects on the HRC-triadin interaction in cardiac homogenates. Collectively, our data suggest that HRC may play a key role in the regulation of SR Ca cycling through its direct interactions with SERCA2 and triadin, mediating a fine cross talk between SR Ca uptake and release in the heart.
肌浆网（SR）中心脏钙循环功能降低与收缩力减弱有关，而收缩力减弱可能会发展为心力衰竭。富含组氨酸的钙结合蛋白（HRC）是一种SR成分，它与三联蛋白结合，并可能通过兰尼碱受体影响钙释放。转基因小鼠心脏中HRC的过度表达与SR钙摄取速率降低和舒张延迟有关，随着年龄增长会发展为肥大。本研究表明，HRC可能通过直接与心肌中的肌浆（内质）网钙ATP酶2型（SERCA2）结合来介导其部分调节作用，并通过共聚焦显微镜下观察到的共免疫染色得到证实。这种相互作用涉及HRC富含组氨酸和谷氨酸的结构域（320 - 460个氨基酸）以及SERCA2的NH(2)-末端阳离子转运结构域中伸入SR腔的部分（74 - 90个氨基酸）。SERCA2结合结构域在人HRC的三联蛋白结合区域（609 - 699个氨基酸）的上游。使用人和小鼠心脏匀浆通过共免疫沉淀和下拉试验以及使用谷胱甘肽S-转移酶和麦芽糖结合蛋白重组蛋白通过印迹覆盖法验证了HRC和SERCA之间的特异性结合。重要的是，钙浓度升高与HRC与SERCA2的结合显著减少有关，而它们对心脏匀浆中HRC - 三联蛋白相互作用具有相反的影响。总体而言，我们的数据表明，HRC可能通过其与SERCA2和三联蛋白的直接相互作用在SR钙循环调节中发挥关键作用，介导心脏中SR钙摄取和释放之间的精细相互作用。
