Live-cell transforms between Ca2+ transients and FRET responses for a troponin-C-based Ca2+ sensor

Hock Tay Lai, Oliver Griesbeck, David T. Yue

Research output: Contribution to journalArticle

Abstract

Genetically encoded Ca2+ sensors promise sustained in vivo detection of Ca2+ 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 Ca2+-sensing module, such that interference via endogenous CaM may result. One class of sensors that could minimize this concern utilizes troponin C as the Ca2+ 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 Ca2+ transients for testing sensor kinetics. TN-L15 was virally expressed within myocytes, and Indo-1 acutely loaded to monitor "true" Ca2+ 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 Ca2+ transients appeared reproducible, though FRET signals were delayed compared to Ca2+ transients. Nonetheless, a three-state mechanism sufficed to map between measured Ca2+ transients and actual TN-L15 outputs. Overall, reproducibility of TN-L15 dynamics, coupled with algorithmic transforms between FRET and Ca2+ signals, renders these sensors promising for quantitative estimation of Ca 2+ dynamics in vivo.

Original languageEnglish (US)
Pages (from-to)4031-4040
Number of pages10
JournalBiophysical journal
Volume93
Issue number11
DOIs
StatePublished - Dec 1 2007

ASJC Scopus subject areas

  • Biophysics

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