Calcium Indicators with Fluorescence Lifetime-Based Signal Readout: A Structure-Function Study

Abstract

The calcium cation is a crucial signaling molecule involved in numerous cellular pathways. Beyond its role as a messenger or modulator in intracellular cascades, calcium's function in excitable cells, including nerve impulse transmission, is remarkable. The central role of calcium in nervous activity has driven the rapid development of fluorescent techniques for monitoring this cation in living cells. Specifically, genetically encoded calcium indicators (GECIs) are the most in-demand molecular tools in their class. In this work, we address two issues of calcium imaging by designing indicators based on the successful GCaMP6 backbone and the fluorescent protein BrUSLEE. The first indicator variant (GCaMP6s-BrUS), with a reduced, calcium-insensitive fluorescence lifetime, has potential in monitoring calcium dynamics with a high temporal resolution in combination with advanced microscopy techniques, such as light beads microscopy, where the fluorescence lifetime limits acquisition speed. Conversely, the second variant (GCaMP6s-BrUS-145), with a flexible, calcium-sensitive fluorescence lifetime, is relevant for static measurements, particularly for determining absolute calcium concentration values using fluorescence lifetime imaging microscopy (FLIM). To identify the structural determinants of calcium sensitivity in these indicator variants, we determine their spatial structures. A comparative structural analysis allowed the optimization of the GCaMP6s-BrUS construct, resulting in an indicator variant combining calcium-sensitive behavior in the time domain and enhanced molecular brightness. Our data may serve as a starting point for further engineering efforts towards improved GECI variants with fine-tuned fluorescence lifetimes.

Keywords: FLIM; fluorescence lifetime; genetically encoded indicators; quantitative calcium imaging; structural analysis.

Figure 1

Schematics showing the design of chimeric proteins used in the study. At the top, the spatial structure of GCaMP6 is shown, with the mutations required for the EGFP modification. In the center, there is a linear scheme of the GCaMP-type backbone. The numbering of amino acid positions corresponds to those of the unmodified proteins (EGFP and calmodulin). At the bottom (left and right), the modified variants, GCaMP6s-BrUS and GCaMP6s-BrUS-145, are displayed, with the introduced modifications displayed below them.

Figure 2

Calcium sensitivity of purified GCaMP6s, GCaMP6s-BrUS, and GCaMP6s-BrUS-145 measured in the intensiometric mode. (A) The dependence of fluorescence intensity at 510 nm (λ_ex = 475 nm) on calcium concentration, expressed as [Ca²⁺]_free (see Supplementary Table S1 for details on the correspondence between [Ca²⁺]_free and [CaEGTA]). (B) Column histogram displaying the relative fluorescence intensity changes observed within the [Ca²⁺]_free range of 0–39 μM (corresponds to the [CaEGTA] range of 0–10 mM). Standard errors of the mean (S.E.M.) are shown for each data point (n = 3).

Figure 3

The graph describing the dependence of the amplitude-weighted mean fluorescence lifetime of indicator variants on calcium concentration ([CaEGTA] and [Ca²⁺]_free; see Supplementary Table S1 for details). λ_ex = 450 nm, repetition rate is 20 MHz. Standard errors of the mean (S.E.M.) are shown for each data point (n = 3).

Figure 4

Comparison of GCaMP6s-BrUS and GCaMP6s-BrUS-145 structures. (A,B) Superposition of GCaMP6s-BrUS and GCaMP6s-BrUS-145 (magenta) structures from two views. Color scheme for GCaMP6s-BrUS is the following: EFGP domain—green, CaM domain—blue, M13 helix—orange, and the linker 314–319—light gray. Chromophore and calcium ions are shown in light and dark gray color for GCaMP6s-BrUS and GCaMP6s-BrUS-145, respectively. Red arrows point to the shift of the linker 314–319 (A) and the C-lobe of the CaM domain (B). (C–F) Differences in the conformation of residues surrounding the chromophore. Panels (C,E) represent GCaMP6s-BrUS structure and (D,F) GCaMP6s-BrUS-145. Solvent molecules are shown as red spheres. Hydrogen bonds are depicted as dashed blue lines.

Figure 5

Calcium sensitivity of GCaMP6s-BrUS-389K fluorescence. (A) The dependence of fluorescence intensity at 510 nm (λ_ex = 475 nm) on calcium concentration, expressed as [Ca²⁺]_free. (B) The graph describing the dependence of the amplitude-weighted mean fluorescence lifetime on calcium concentration ([CaEGTA] and [Ca²⁺]_free). λ_ex = 450 nm, repetition rate is 20 MHz. Standard errors of the mean (S.E.M.) are shown for each data point (n = 3).

Figure 6

Calcium sensitivity of GCaMP6s-BrUS-389K/398G fluorescence. (A) The dependence of fluorescence intensity at 510 nm (λ_ex = 475 nm) on calcium concentration, expressed as [Ca²⁺]_free. (B) The graph describing the dependence of the amplitude-weighted mean fluorescence lifetime on calcium concentration ([CaEGTA] and [Ca²⁺]_free). λ_ex = 450 nm, repetition rate is 20 MHz. Standard errors of the mean (S.E.M.) are shown for each data point (n = 3).