文章基本信息

标题：Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics
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作者：Nordine Helassa ; Xiao-hua Zhang ; Ianina Conte 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2015
卷号：5
期号：1
DOI：10.1038/srep15978
语种：English
出版社：Springer Nature
摘要：Faithful reporting of temporal patterns of intracellular Ca²⁺ dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca²⁺ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca²⁺ affinities and accelerated kinetics by weakening the Ca²⁺-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3_fast is 40-fold faster than GCaMP3 with Ca²⁺ decay and rise times, t _1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3_fast revealed discreet transients with significantly faster Ca²⁺ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca²⁺ at 940 nm, GCaMP3_fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca²⁺ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.