Characterizing the response of calcium signal transducers to generated calcium transients
Characterizing the response of calcium signal transducers to generated calcium transients
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DOI：
10.1021/bi982495z
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发表时间：
1999-03-30
期刊：
影响因子：
2.9
通讯作者：
Johnson, JD
中科院分区：
文献类型：
--
作者：
Davis, JP;Tikunova, SB;Johnson, JD
细胞Ca 2+瞬变和Ca 2+结合蛋白调节多种生理现象，如肌肉收缩、神经分泌和细胞分裂。当Ca 2+与缓慢的Ca 2+螯合剂EGTA或Mg 2 +/EDTA快速混合时，可以产生不同持续时间（0.1-50 ms半宽（hws））和幅度的人工Ca 2+瞬变（ACT）。我们已经暴露了几个Ca 2+指标，Ca 2+结合蛋白，和Ca 2+依赖性酶的ACT的各种持续时间，并观察到他们的瞬时结合的Ca 2+，复合物的形成，和/或激活。0.1 ms hw ACT瞬时占据肌钙蛋白C N-末端调节位点的70%，与其快速Ca 2+结合速率（8.7 +/- 2.0 x 10（7）M-1 s（-1））一致。1.1 ms hw ACT产生的钙调蛋白（CaM）的N-末端与RS-20肽的瞬时结合率接近90%，但CaM的C-末端与RS-20的结合率很低。0.6 ms hw ACT足以在CaM的N-末端瞬时结合类似于60%的肌球蛋白轻链激酶（MLCK），而1.8 ms hw ACT产生类似于22%的肌浆网（SR）Ca 2 +/ATP酶的瞬时激活。ACT在CaM与MLCK或SR Ca 2 +/ATP酶激活的最大结合发生之前已经回落至基线，类似于10-30 ms，并且在Ca瞬变消退之后结合和酶激活持续很长时间。ACTs的使用使我们能够可视化的Ca 2 +-结合蛋白的Ca 2 +-交换率如何决定其Ca 2 +-诱导的构象变化，Ca 2 +-诱导的蛋白质/肽和蛋白质/蛋白质的相互作用，以及酶的激活和失活，响应于不同幅度和持续时间的Ca 2+瞬变。通过表征这些蛋白质对ACT的反应，我们可以更确定地预测它们将如何对天然Ca 2+瞬变反应以调节细胞现象。
Cellular Ca2+ transients and Ca2+-binding proteins regulate physiological phenomena as diverse as muscle contraction, neurosecretion, and cell division. When Ca2+ is rapidly mixed with slow Ca2+ chelators, EGTA, or Mg2+/EDTA, artificial Ca2+ transients (ACTs) of varying duration (0.1-50 ms half-widths (hws)) and amplitude can be generated. We have exposed several Ca2+ indicators, Ca2+-binding proteins, and a Ca2+-dependent enzyme to ACTs of various durations and observed their transient binding of Ca2+, complex formation, and/or activation. A 0.1 ms hw ACT transiently occupied similar to 70% of the N-terminal regulatory sites of troponin C consistent with their rapid Ca2+ on-rate (8.7 +/- 2.0 x 10(7) M-1 s(-1)). A 1.1 ms hw ACT produced similar to 90% transient binding of the N-terminal of calmodulin (CaM) to the RS-20 peptide, but little binding of CaM's C-terninal to RS-20. A 0.6 ms hw ACT was sufficient fur the N-terminal of CaM to transiently bind similar to 60% of myosin light chain kinase (MLCK), while a 1.8 ms hw ACT produced similar to 22% transient activation of the sarcoplasmic reticulum (SR) Ca2+/ATPase, Ln both cases, the ACT had fallen back to baseline similar to 10-30 ms before maximal binding of CaM to MLCK or SR Ca2+/ATPase activation occurred and binding and enzyme activation persisted long after the Ca transient had subsided. The use of ACTs has allowed us to visualize how the Ca2+-exchange rates of Ca2+-binding proteins dictate their Ca2+-induced conformational changes, Ca2+-induced protein/peptide and protein/protein interactions, and enzyme activation and inactivation, in response to Ca2+ transients of various amplitude and duration. By characterizing the response of these proteins to ACTs, we can predict with greater certainty how they would respond to natural Ca2+ transients to regulate cellular phenomena.