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Faithful reporting of temporal patterns of intracellular Ca 2+  dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca 2+  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 Ca 2+  affinities and accelerated kinetics by weakening the Ca 2+ -calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3  fast   is 40-fold faster than GCaMP3 with Ca 2+  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 Ca 2+  dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca 2+  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 Ca 2+  kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.

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