Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics

Abstract

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators.

GenEPi: Piezo1-based fluorescent reporter for visualizing mechanical stimuli with high spatiotemporal resolution

“…Among the candidates tested, we identified one GCaMP-Piezo1 fusion variant that satisfied our requirements, Piezo1-1xGSGG-GCaMP-G4 (containing the GCaMP6s RS-1 EF-4 variant 27 ), hereby referred to as GenEPi (Fig. 1c).…”

“…In a systematic screen, we generated a library of reporters by fusing five different low-affinity GCaMPs 26,27 (here denoted as GCaMP-G1 - GCaMP-G5) (with K d -s in the 0.6 to 6 µM range) to the C-terminus of human Piezo1 (Fig. 1a).…”

In vivo microstimulation with cathodic and anodic asymmetric waveforms modulates spatiotemporal calcium dynamics in cortical neuropil and pyramidal neurons of male mice

“…Importantly, it is not clear whether the increased fluorescence intensity is a result of greater 1:1 entrainment, or due to other activation properties such as bursting. Newer developments in genetically encoded voltage (GEVI) and calcium indicators (GECI) with faster kinetics may be able to more comprehensively address these questions [93][94][95]. However, it should be noted that current GEVIs do not provide comparable spatial resolution and suffer from lower signal to noise ratio [96].…”

Accurate action potential inference from a calcium sensor protein through biophysical modeling:

“…At the same time, some calcium ions bind reversibly to GCaMP6s, which contains a calmodulin-derived protein domain with 4 binding sites [90]. Calcium binding to GCaMP6s increases its fluorescence [18], with proposed mechanisms based on conformational changes that alter fluorophore protonation [133,2,59,122,7]. We reasoned that since the individual parts of this system have been extensively studied and quantified, by modeling them together we could quantitatively predict fluorescence from APs and infer APs from fluorescence (Figure 2a).…”