GCaMP3-Derived Genetically Encoded Probes as Rapid Intracellular Signals for Calcium Dynamics

Abstract

In intracellular calcium signaling studies genetically encoded probes offer greater intracellular domain specificity than traditional fluorescent signaling molecules, with the ability to be targeted to specific organelles and micro-domains. To this end we evaluated the ability of seven novel mutagenic derivatives of GCaMP3 calcium probe (EF-3, EF-4, EF-34, RS-1, RS-1 EF-3, RS-1 EF-4, and RS-1 EF-34) to rapidly detect intracellular calcium compared to the chemical calcium probe Fura-2 AM. GCaMP3 derived probes were transiently transfected into HEK293T cells which were subsequently incubated in 1 µM Fura-2 AM for 20 minutes prior to experimentation. Fura-2 and GCaMP3-derived probe fluorescence was measured at 380 nm and 488 nm respectively as HEK293T cells were rapidly exposed to 100 µM ATP for 5 s. While mutated probes did not exhibit a greater change in fluorescence than GCaMP3 precursor, mutants EF-3 and EF-4 fluorescence changes were 128.4 and 58.2 % ΔF/Fo respectively, significantly greater than Fura-2 response in the same cells. Additionally, many probes demonstrated faster rise and decay kinetics, with rise time and decay time values up to 150 % faster than Fura-2. Our results suggest that mutations in GCaMP3 can produce genetically encoded probes capable of measuring calcium with equal or larger fluorescence responses as traditional chemical dyes. Additionally, several probes such as GCaMP3 EF-4 demonstrate not only increased fluorescence but dramatically improved kinetic responses - facilitating increasingly sensitive measurements of intracellular calcium signaling.