Measurement of Inositol 1,4,5-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP (3)) sensors were created to follow intracellular InsP (3) changes in single living cells and in cell populations. Similar to previous InsP (3) sensors the new sensors are based on the ligand binding domain of the human type-I InsP (3) receptor (InsP(3)R-LBD), but contain a mutation of either R265K or R269K to lower their InsP (3) binding affinity. Tagging the InsP (3)R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP (3) in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP (3) concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP (3) sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP (3) after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP (3) and Ca(2+) levels in BRET experiments, the Cameleon D3 Ca(2+) sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P (2) resulted in the fall of InsP (3) level, followed by the decrease of the Ca(2+)-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca(2+)-signal preceded the fall of InsP (3) level indicating an InsP (3)-, independent, direct regulation of capacitative Ca(2+) influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP (3) sensor can be used to monitor both the increase and decrease of InsP (3) levels in live cells suitable for high-throughput BRET applications.