Measurement of Inositol 145-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

摘要

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 (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP 3 binding affinity. Tagging the InsP 3R-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 Ca²⁺ levels in BRET experiments, the Cameleon D3 Ca²⁺ 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 Ca²⁺-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 Ca²⁺-signal preceded the fall of InsP 3 level indicating an InsP 3-, independent, direct regulation of capacitative Ca²⁺ 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 InsP₃ levels in live cells suitable for high-throughput BRET applications.