Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein
Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein
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DOI：
10.1074/jbc.m312751200
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发表时间：
2004-04-02
影响因子：
4.8
通讯作者：
Griesbeck, O
中科院分区：
文献类型：
--
作者：
Heim, N;Griesbeck, O
遗传钙探针在神经科学、细胞生物学和药物筛选领域具有巨大的潜力。以前，已经描述了细胞钙动力学的比率和非比率度量指标，其由作为荧光团的绿色荧光蛋白（GFP）和作为钙结合部分的钙调蛋白的突变体以几种构型组成。然而，这些基于钙调蛋白的探针类型具有一系列缺陷，例如当在转基因生物体中表达时动态范围减小以及在某些靶向中缺乏钙敏感性。我们基于骨骼肌和心肌的肌钙蛋白C变体开发了新型钙探针。这些指示剂具有高达140%的比率变化，K（d）s范围从470 nM至29 μ M，并且改善了亚细胞靶向性质。我们将指示剂靶向HEK293细胞和原代海马神经元的质膜。在长期持续的去极化，海马神经元的膜下钙水平被发现是在与散装胞质钙水平的平衡，这表明没有常设梯度持续从膜向胞质。我们期望这种使用专门的钙敏感蛋白的新指标将与细胞生化机制的相互作用最小化。
Genetic calcium probes offer tremendous potential in the fields of neuroscience, cell biology, and pharmaceutical screening. Previously, ratiometric and non-ratio-metric indicators of cellular calcium dynamics have been described that consist of mutants of the green fluorescent protein (GFP) as fluorophores and calmodulin as calcium-binding moiety in several configurations. However, these calmodulin-based types of probes have a series of deficiencies, such as reduced dynamic ranges, when expressed within transgenic organisms and lack of calcium sensitivity in certain targetings. We developed novel types of calcium probes based on troponin C variants from skeletal and cardiac muscle. These indicators have ratio changes up to 140%, K(d)s ranging from 470 nM to 29 muM, and improved subcellular targeting properties. We targeted the indicators to the plasma membrane of HEK293 cells and primary hippocampal neurons. Upon long lasting depolarization, submembrane calcium levels in hippocampal neurons were found to be in equilibrium with bulk cytosolic calcium levels, suggesting no standing gradient persists from the membrane toward the cytosol. We expect that such novel indicators using specialized calcium sensing proteins will be minimally interacting with the cellular biochemical machinery.