Mutant IP3 receptors attenuate store-operated Ca2+ entry by destabilizing STIM–Orai interactions in Drosophila neurons

Abstract

Store-operated Ca2+ entry (SOCE) occurs when loss of Ca2+ from the endoplasmic reticulum (ER) stimulates the Ca2+ sensor, STIM, to cluster and activate the plasma membrane Ca2+ channel Orai (encoded by Olf186-F in flies). Inositol 1,4,5-trisphosphate receptors (IP3Rs, which are encoded by a single gene in flies) are assumed to regulate SOCE solely by mediating ER Ca2+ release. We show that in Drosophila neurons, mutant IP3R attenuates SOCE evoked by depleting Ca2+ stores with thapsigargin. In normal neurons, store depletion caused STIM and the IP3R to accumulate near the plasma membrane, association of STIM with Orai, clustering of STIM and Orai at ER–plasma-membrane junctions and activation of SOCE. These responses were attenuated in neurons with mutant IP3Rs and were rescued by overexpression of STIM with Orai. We conclude that, after depletion of Ca2+ stores in Drosophila, translocation of the IP3R to ER–plasma-membrane junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE.

Structure and Function of IP3Receptors

“…We need also to consider whether the scaffolding of signaling proteins by IP 3 Rs serves only to funnel information toward an IP 3evoked Ca 2+ signal, or might these scaffolds fulfil additional, and possibly unrelated, roles. There is, for example, evidence that IP 3 Rs, independent of their ability to release Ca 2+ from the ER, can modulate SOCE (Chakraborty et al 2016) and, since the mechanisms are not yet clear (Thillaiappan et al 2019), they may arise through scaffolding of proteins by IP 3 Rs.…”

“ER-Ca2+ sensor STIM regulates neuropeptides required for development under nutrient restriction in Drosophila”

“…As reduction of IP 3 R resulted in a weak phenotype (Fig. S1A), the focus was shifted to dSTIM, as both proteins are known to positively regulate SOCE in Drosophila neurons (7,12). Knockdown of dSTIM (dSTIM IR ) with either sNPF-GAL4 or Crz-GAL4, resulted in reduced pupariation under NR to the same extent, with no effect on development on normal food ( Fig.…”

“…neurons (12), suggesting that reduced neuronal output of NPs can be overcome by enhancing intracellular Ca 2+ signaling.…”

“…In flies (Drosophila melanogaster; Dmel), dSTIM regulates functioning of neurons (7)(8)(9)(10)(11)(12), fat body cells (13) and intestinal stem cells (14). dSTIM-mediated SOCE has been demonstrated in Dmel neurons (7), with functional consequences in certain neuronal subtypes: regulation of flight in dopaminergic neurons (8,15) and development in proteindeprived media in glutamatergic neurons (10).…”

“…Further, dSTIM over-expression in insulinproducing neuropeptidergic neurons could restore Ca 2+ homeostasis in a non-autonomous manner in other neurons of an IP 3 R mutant (16), indicating an important role for dSTIM in NE cell output as well as compensatory interplay between SOCE regulators IP 3 R and dSTIM. At least in Dmel, all three proteins -IP 3 R, dSTIM and Orai -interact to regulate SOCE in neurons (7,12). NE cells are secretory neurons that produce NPs, the most diverse group of neuronal signaling agents.…”