Abstract
Genetically encoded Ca(2+) sensors promise sustained in vivo detection of Ca(2+) signals. However, these sensors are sometimes challenged by inconsistent performance and slow/uncertain kinetic responsiveness. The former challenge may arise because most sensors employ calmodulin (CaM) as the Ca(2+)-sensing module, such that interference via endogenous CaM may result. One class of sensors that could minimize this concern utilizes troponin C as the Ca(2+) sensor. Here, we therefore probed the reliability and kinetics of one representative of this class (cyan fluorescence protein/yellow fluorescent protein-fluorescence resonance energy transfer (FRET) sensor TN-L15) within cardiac ventricular myocytes. These cells furnished a pertinent live-cell test environment, given substantial endogenous CaM levels and fast reproducible Ca(2+) transients for testing sensor kinetics. TN-L15 was virally expressed within myocytes, and Indo-1 acutely loaded to monitor "true" Ca(2+) transients. This configuration permitted independent and simultaneous detection of TN-L15 and Indo-1 signals within individual cells. The relation between TN-L15 FRET responses and Indo-1 Ca(2+) transients appeared reproducible, though FRET signals were delayed compared to Ca(2+) transients. Nonetheless, a three-state mechanism sufficed to map between measured Ca(2+) transients and actual TN-L15 outputs. Overall, reproducibility of TN-L15 dynamics, coupled with algorithmic transforms between FRET and Ca(2+) signals, renders these sensors promising for quantitative estimation of Ca(2+) dynamics in vivo.