Background and aims: Imaging of neuronal network activity in free-moving animals is the aim of much research to understand brain function and the underlying mechanisms. To visualise neuronal activity in vivo at the cellular level, genetically-encoded indicators have been developed which track calcium flux that accompanies membrane depolarisation and action potentials. Green Fluorescent-Calmodulin Protein (GCaMP) type Ca2+ indicators are based on a circularly permutated EGFP molecule (cpEGFP) flanked at the N and C termini by the smooth muscle myosin light chain kinase derived RS20 peptide and calmodulin (CaM), respectively, which form a tight complex upon calcium binding. This interaction stabilises the intrinsically fluorescent deprotonated form of cpEGFP inducing a fluorescence enhancement. While the stability and brightness of GCaMP type indicators have increased significantly, their slow response kinetics to action potentials have posed a limitation to their application. To address the issue of slow association and dissociation kinetics of GCaMPs, our hypothesis was that the calcium response kinetics will be accelerated if the binding affinity of Ca2+.CaM.RS20 complex is weakened. To achieve that, point mutations were introduced in the EF-hands of CaM1 (mutants EF-1 to EF-4) and in the RS20 target peptide sequence2 (mutant RS-1) of GCaMP3 and GCaMP6.Methods: Following site-directed mutagenesis based on our rational design, GCaMP proteins were expressed in E.coli and purified by a single-step affinity chromatography. All in vitro experiments were performed at physiological ionic strength at 20°C. Equilibrium binding constants were measured by single photon fluorescence. The calcium dissociation and association kinetics of the GCaMP mutants were obtained by stopped-flow fluorimetry and the two-photon fluorescence properties were measured by spectroscopy. Calcium responses were tested in endothelial cells (HUVEC) stimulated by ionomycin.Results: Dissociation constants (Kd) for calcium obtained from the equilibrium calcium binding experiments were in the μM range (0.5-5.6 μM) with Hill coefficients from 2 to 5. Calcium dissociation rates were up to 60-fold faster than GCaMP3 with an off-rate of 102±1 (S.E.M) s-1 (n=9). Fluorescence changes on calcium association were highly cooperative and characterized by a rate limiting conformational change. The limiting rates were up to 7-fold faster compared to GCaMP3, with an observed association rate of 145±2 (S.E.M) s-1 (n=20). Two-photon cross-sections and fluorescence responses to calcium influx in endothelial cells of mutated GCaMPs were comparable to those of GCaMP3. Conclusion: The principles employed proved to accelerate the calcium response kinetics of GCaMPs and can be applied to the new generations of GCaMPs to generate low affinity probes.