Association of the IP3R to STIM1 provides a reduced intraluminal calcium microenvironment, resulting in enhanced store-operated calcium entry

Alicia Sampieri,Karla Santoyo,Alexander Asanov,Luis Vaca

doi:10.1038/s41598-018-31621-0

Abstract

The involvement of inositol trisphosphate receptor (IP3R) in modulating store-operated calcium entry (SOCE) was established many years ago. Nevertheless, the molecular mechanism responsible for this observation has not been elucidated to this date. In the present study we show that IP3R associates to STIM1 upon depletion of the endoplasmic reticulum (ER) by activation of the inositol trisphosphate signaling cascade via G-protein coupled receptors. IP3R-STIM1 association results in enhanced STIM1 puncta formation and larger Orai-mediated whole-cell currents as well as increased calcium influx. Depleting the ER with a calcium ATPase inhibitor (thapsigargin, TG) does not induce IP3R-STIM1 association, indicating that this association requires an active IP3R. The IP3R-STIM1 association is only observed after IP3R activation, as evidenced by FRET experiments and co-immunoprecipitation assays. ER intraluminal calcium measurements using Mag-Fluo-4 showed enhanced calcium depletion when IP3R is overexpressed. A STIM1-GCaMP fusion protein indicates that STIM1 detects lower calcium concentrations near its EF-hand domain when IP3R is overexpressed when compared with the fluorescence reported by a GCaMP homogenously distributed in the ER lumen (ER-GCaMP). All these data together strongly suggest that activation of inositol trisphosphate signaling cascade induces the formation of the IP3R-STIM1 complex. The activated IP3R provides a reduced intraluminal calcium microenvironment near STIM1, resulting in enhanced activation of Orai currents and SOCE.

Highlights

Store-operated calcium entry (SOCE) is a conserved mechanism by which the depletion of the endoplasmic reticulum (ER) is conveyed to calcium-permeable channels at the plasma membrane (PM), triggering calcium influx from the extracellular space and into the cell cytosol[1]
In the present study we show that IP3 receptor (IP3R) is recruited to STIM1 puncta upon depletion of the ER via activation of G-protein coupled receptors associated to the inositol-triphosphate (IP3) and phospholipase C signaling cascade
IP3R are recruited to the puncta and strong co-localization is observed between YFP-IP3R and STIM1-CFP after bradykinin stimulation (Fig. 1B, Supplementary Video 1)

Summary

Introduction

Store-operated calcium entry (SOCE) is a conserved mechanism by which the depletion of the endoplasmic reticulum (ER) is conveyed to calcium-permeable channels at the plasma membrane (PM), triggering calcium influx from the extracellular space and into the cell cytosol[1]. A physiological mechanism responsible for the activation of SOCE results from the stimulation of G-protein coupled receptors associated to the inositol-triphosphate (IP3) and phospholipase C cascade, resulting in the release of calcium from ER, via the IP3 receptor (IP3R)[2]. The emptying of the ER initiates the dissociation of ER luminal calcium from the EF-hand of the stromal interacting molecule (STIM1) This protein is a sensor of the calcium content in the ER3. Initial studies indicated that a direct coupling between IP3R and calcium influx channels was responsible for the activation of SOCE10. In the present study we show that IP3R is recruited to STIM1 puncta upon depletion of the ER via activation of G-protein coupled receptors associated to the inositol-triphosphate (IP3) and phospholipase C signaling cascade. The association of STIM1 and IP3R at the puncta is supported by co-localization confocal microscopy studies, Förster resonance energy transfer (FRET), total internal reflection microscopy (TIRFM) and co-immuniprecipitation assays

Methods

Results

Conclusion