Ultrafast Genetically Encoded Calcium Indicators for Visualizing Calcium Flux and Action Potentials

Abstract

Genetically encoded calmodulin-based calcium probes (GCaMPs) have become the reporters of choice for visualising the calcium flux associated with action potentials in vivo. A major limitation of currently available GCaMPs is the slow kinetics of fluorescence changes induced by calcium association and dissociation. We have addressed this issue by generating a series of mutants of GCaMP3 in the calcium binding sites of calmodulin alone1 and in combination with mutations in the RS20 target peptide sequence2 with the view of lowering the affinity for calcium and accelerating the calcium response kinetics. The calcium association kinetics for the resulting GCaMP3 EF-hand and peptide mutants were highly cooperative and characterized by a rate limiting conformational change. Fluorescence changes on calcium association were up to 7-fold faster compared to GCaMP3. Calcium dissociation rates were up to 60-fold faster than GCaMP3 and 25-fold faster than the newly developed GCaMP6 fast (GCaMP6f). Dissociation constants (Kd) for calcium were in the μM range with Hill coefficients from 2 to 5. Two-photon cross-sections of mutants were comparable to GCaMP3. Fluorescence responses of mutated GCaMP3s to calcium transients in endothelial cells were similar to those seen with small molecule indicators. The principles employed proved to accelerate the calcium kinetics of GCaMP3 and can be applied to the new generations of GCaMPs to generate low affinity probes.1Jama A et al. JBC, 2011, 286:12308-12316.2Torok K and Trentham DR. Biochemistry, 1994, 33:12807-12820.This work is funded by the Wellcome Trust grant number 094385/Z/10/Z to KT.