Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors

Abstract

Improved versions of inositol-1,4,5-trisphosphate (InsP<inf>3</inf>) sensors were created to follow intracellular InsP<inf>3</inf> changes in single living cells and in cell populations. Similar to previous InsP<inf>3</inf> sensors the new sensors are based on the ligand binding domain of the human type-I InsP<inf>3</inf> receptor (InsP<inf>3</inf>R-LBD), but contain a mutation of either R265K or R269K to lower their InsP<inf>3</inf> binding affinity. Tagging the InsP<inf>3</inf>R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of Ins P<inf>3</inf> 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<inf>3</inf> 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<inf>3</inf> 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<inf>3</inf> after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP<inf>3</inf> 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<inf>2</inf> resulted in the fall of InsP<inf>3</inf> 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<inf>3</inf> level indicating an InsP<inf>3</inf>-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP<inf>3</inf> sensor can be used to monitor both the increase and decrease of InsP<inf>3</inf> levels in live cells suitable for high-throughput BRET applications. © 2015, Public Library of Science. All rights reserved.