Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics

作者：Helassa Nordine; Zhang Xiao hua; Conte Ianina; Scaringi John; Esposito Elric; Bradley Jonathan; Carter Thomas; Ogden David; Morad Martin; Toeroek Katalin

来源：Scientific Reports, 2015, 5(1): 15978.

DOI：10.1038/srep15978

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 degrees 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.

• 出版日期2015-11-3
• 单位河北医科大学