Development of novel indicators and molecular systems for calcium sensing through protein engineering

Intracellular calcium (Ca²⁺) is involved in a plethora of cell signalling processes and physiological functions. Increases in Ca²⁺ concentration are bona fide biomarkers of neuronal activity, reflecting the spike count, timing, frequency, and the intensity of synaptic input. The development of genetically encoded calcium indicators (GECIs) was a significant advancement in modern neuroscience that enabled real-time visualisation of neuronal activity at single-cell resolution. These indicators leverage the conformational changes induced by calcium-binding proteins, such as calmodulin (CaM) or troponin C (TnC). Harnessing protein engineering approaches such as directed evolution yielded new GECIs with enhanced sensitivity, kinetics, and brightness. Notably, the development of calcium-based integrators, such as scFLARE (single-chain fast light- and activity-regulated expression), convert transient raises in cytosolic Ca²⁺ into a transcriptional readout rather than an optical signal. This review summarises the latest efforts in protein engineering to develop new indicators and molecular systems to sense changes in Ca²⁺ concentrations.