Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes
Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes
复制标题
DOI：
10.1016/j.yjmcc.2013.07.007
复制
发表时间：
2013-10-01
影响因子：
5
通讯作者：
Oceandy, Delvac
中科院分区：
文献类型：
--
作者：
Mohamed, Tamer M. A.;Abou-Leisa, Riham;Oceandy, Delvac
质膜钙/钙调蛋白依赖性ATP酶（PMCA 4）的亚型4最近已成为心脏中几个关键病理生理过程（如收缩性和肥大）的重要调节剂。然而，由于缺乏分子工具，直接监测PMCA 4活性和评估心肌细胞中其附近的钙动力学是困难的。在这项研究中，我们开发了新的钙荧光指示剂，通过融合GCaMP 2钙传感器到PMCA 4的N-末端，产生PMCA 4-GCaMP 2融合分子。我们还鉴定了一种新的PMCA 4特异性抑制剂，这可能有助于研究该分子在心肌细胞和其他细胞类型中的作用。该融合分子被正确地靶向质膜并与小窝蛋白-3共定位。它可以监测电刺激心肌细胞的信号振荡。在电刺激的新生和成年大鼠心肌细胞中，PMCA 4-GCaMP 2产生的信号幅度更高，信号衰减速率更快，与突变的无活性PMCA 4（mut）GCaMP 2融合蛋白相比。小分子文库筛选使我们能够鉴定PMCA 4的新型选择性抑制剂，我们发现其降低PMCA 4-GCaMP 2的信号幅度并将信号衰减时间（Tau）延长至与PMCA 4（mut）GCaMP 2产生的信号相当的水平。此外，PMCA 4-GCaMP 2（而非突变形式）对β-肾上腺素能刺激产生增强的信号。总之，PMCA 4-GCaMP 2和PMCA 4（mut）GCaMP 2分别在活性或非活性条件下显示了泵附近的钙动力学。总之，PMCA 4-GCaMP 2与新的特异性抑制剂一起提供了监测心肌细胞中钙转运蛋白附近的钙动力学的新手段，并且可能成为进一步研究PMCA 4的生物学功能的有用工具。此外，类似的方法可能有助于研究心脏兴奋-收缩偶联过程中其他钙转运蛋白的活性。(C)2013爱思唯尔有限公司保留所有权利。
Isoform 4 of the plasma membrane calcium/calmodulin dependent ATPase (PMCA4) has recently emerged as an important regulator of several key pathophysiological processes in the heart, such as contractility and hypertrophy. However, direct monitoring of PMCA4 activity and assessment of calcium dynamics in its vicinity in cardiomyocytes are difficult due to the lack of molecular tools. In this study, we developed novel calcium fluorescent indicators by fusing the GCaMP2 calcium sensor to the N-terminus of PMCA4 to generate the PMCA4-GCaMP2 fusion molecule. We also identified a novel specific inhibitor of PMCA4, which might be useful for studying the role of this molecule in cardiomyocytes and other cell types.Using an adenoviral system we successfully expressed PMCA4-GCaMP2 in both neonatal and adult rat cardiomyocytes. This fusion molecule was correctly targeted to the plasma membrane and co-localised with caveolin-3. It could monitor signal oscillations in electrically stimulated cardiomyocytes. The PMCA4-GCaMP2 generated a higher signal amplitude and faster signal decay rate compared to a mutant inactive PMCA4(mut)GCaMP2 fusion protein, in electrically stimulated neonatal and adult rat cardiomyocytes. A small molecule library screen enabled us to identify a novel selective inhibitor for PMCA4, which we found to reduce signal amplitude of PMCA4-GCaMP2 and prolong the time of signal decay (Tau) to a level comparable with the signal generated by PMCA4(mut)GCaMP2. In addition, PMCA4-GCaMP2 but not the mutant form produced an enhanced signal in response to beta-adrenergic stimulation. Together, the PMCA4-GCaMP2 and PMCA4(mut)GCaMP2 demonstrate calcium dynamics in the vicinity of the pump under active or inactive conditions, respectively.In summary, the PMCA4-GCaMP2 together with the novel specific inhibitor provides new means with which to monitor calcium dynamics in the vicinity of a calcium transporter in cardiomyocytes and may become a useful tool to further study the biological functions of PMCA4. In addition, similar approaches could be useful for studying the activity of other calcium transporters during excitation-contraction coupling in the heart. (C) 2013 Elsevier Ltd. All rights reserved.