Colin W. Taylor

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are high-conductance channels that allow the regulated redistribution of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and, at specialized membrane contact sites (MCSs), to other organelles. Only a subset of IP3Rs release Ca2+ to the cytosol in response to IP3. These ‘licensed’ IP3Rs are associated with Kras-induced actin-interacting protein (KRAP, also known as ITPRID2) beneath the plasma membrane. It is unclear whether KRAP regulates IP3Rs at MCSs. We show, using simultaneous measurements of Ca2+ concentration in the cytosol and mitochondrial matrix, that KRAP also licenses IP3Rs to release Ca2+ to mitochondria. Loss of KRAP abolishes cytosolic and mitochondrial Ca2+ signals evoked by stimulation of IP3Rs via endogenous receptors. KRAP is located at ER–mitochondrial membrane contact sites (ERMCSs) populated by IP3R clusters. Using a proximity ligation assay between IP3R and voltage-dependent anion channel 1 (VDAC1), we show that loss of KRAP reduces the number of ERMCSs. We conclude that KRAP regulates Ca2+ transfer from IP3Rs to mitochondria by both licensing IP3R activity and stabilizing ERMCSs.

Loss of endoplasmic reticular (ER) Ca²⁺ activates store-operated Ca²⁺ entry (SOCE) by causing the ER localized Ca²⁺ sensor STIM to unfurl domains that activate Orai channels in the plasma membrane at membrane contact sites (MCS). Here, we demonstrate a novel mechanism by which the inositol 1,4,5 trisphosphate receptor (IP₃R), an ER-localized IP₃-gated Ca²⁺ channel, regulates neuronal SOCE. In human neurons, SOCE evoked by pharmacological depletion of ER-Ca²⁺ is attenuated by loss of IP₃Rs, and restored by expression of IP₃Rs even when they cannot release Ca²⁺, but only if the IP₃Rs can bind IP₃. Imaging studies demonstrate that IP₃Rs enhance association of STIM1 with Orai1 in neuronal cells with empty stores; this requires an IP₃-binding site, but not a pore. Convergent regulation by IP₃Rs, may tune neuronal SOCE to respond selectively to receptors that generate IP₃.

Loss of endoplasmic reticular (ER) Ca ²⁺ activates store-operated Ca ²⁺ entry (SOCE) by causing the ER localized Ca ²⁺ sensor STIM to unfurl domains that activate Orai channels in the plasma membrane at membrane contact sites (MCS). Here we demonstrate a novel mechanism by which the inositol 1,4,5 trisphosphate receptor (IP ₃ R), an ER-localized IP ₃ -gated Ca ²⁺ channel, regulates neuronal SOCE. In human neurons, SOCE evoked by pharmacological depletion of ER-Ca ²⁺ is attenuated by loss of IP ₃ Rs, and restored by expression of IP ₃ Rs even when they cannot release Ca ²⁺ , but only if the IP ₃ Rs can bind IP ₃ . Imaging studies demonstrate that IP ₃ Rs enhance association of STIM1 with Orai1 in neuronal cells with empty stores; this requires an IP ₃ -binding site, but not a pore. Convergent regulation by IP ₃ Rs, may tune neuronal SOCE to respond selectively to receptors that generate IP ₃ .

The folding capacity of membrane and secretory proteins in the endoplasmic reticulum (ER) can be challenged by physiological and pathological perturbations, causing ER stress. If unresolved, this leads to cell death. We report a role for iRhom pseudoproteases in controlling apoptosis due to persistent ER stress. Loss of iRhoms causes cells to be resistant to ER stress-induced apoptosis. iRhom1 and iRhom2 interact with IP₃receptors, critical mediators of intracellular Ca²⁺signalling, and regulate ER stress-induced transport of Ca²⁺into mitochondria, a primary trigger of mitochondrial membrane depolarisation and cell death. iRhoms also bind to the anti-apoptotic regulator BCL-2, attenuating the inhibitory interaction between BCL-2 and IP₃receptors, which promotes ER Ca²⁺release. The discovery of the participation of iRhoms in the control of ER stress-induced cell death further extends their potential pathological significance to include diseases dependent on protein misfolding and aggregation.

The P2X4 purinergic receptor is targeted to endolysosomes, where it mediates an inward current dependent on luminal ATP and pH. Activation of P2X4 receptors was previously shown to trigger lysosome fusion, but the regulation of P2X4 receptors and their role in lysosomal Ca²⁺ signaling are poorly understood. We show that lysosomal P2X4 receptors are activated downstream of plasma membrane P2X7 and H₁ histamine receptor stimulation. When P2X4 receptors are expressed, the increase in near-lysosome cytosolic [Ca²⁺] is exaggerated, as detected with a low-affinity targeted Ca²⁺ sensor. P2X4-dependent changes in lysosome properties were triggered downstream of P2X7 receptor activation, including an enlargement of lysosomes indicative of homotypic fusion and a redistribution of lysosomes towards the periphery of the cell. Lysosomal P2X4 receptors, therefore, have a role in regulating lysosomal Ca²⁺ release and the regulation of lysosomal membrane trafficking.

Regulation of IP₃receptors (IP₃Rs) by IP₃and Ca²⁺allows regenerative Ca²⁺signals, the smallest being Ca²⁺puffs, which arise from coordinated openings of a few clustered IP₃Rs. Cells express thousands of mostly mobile IP₃Rs, yet Ca²⁺puffs occur at a few immobile IP₃R clusters. By imaging cells with endogenous IP₃Rs tagged with EGFP, we show that KRas-induced actin-interacting protein (KRAP) tethers IP₃Rs to actin beneath the plasma membrane. Loss of KRAP abolishes Ca²⁺puffs and the global increases in cytosolic Ca²⁺concentration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional IP₃R clusters and results in more Ca²⁺puffs and larger global Ca²⁺signals. Endogenous KRAP determines which IP₃Rs will respond: it tethers IP₃R clusters to actin alongside sites where store-operated Ca²⁺entry occurs, licenses IP₃Rs to evoke Ca²⁺puffs and global cytosolic Ca²⁺signals, implicates the actin cytoskeleton in IP₃R regulation and may allow local activation of Ca²⁺entry.

Significance: Store-operated Ca ²⁺ entry (SOCE) links Ca ²⁺ release from endoplasmic reticulum (ER) to Ca ²⁺ entry across the plasma membrane (PM). SOCE is unusual in requiring interaction between proteins in different membranes. STIM1, when it senses loss of ER Ca ²⁺ , unfurls domains that interact with Orai1 PM Ca ²⁺ channels. The stoichiometry of the SOCE complex is contentious, but it determines the regulation and functional consequences of SOCE. We show that native complexes are likely to comprise a single Orai1 channel and a few STIM1 dimers, too few to cluster Orai1 channels. We suggest that SOCE may be digitally regulated by local ER depletion, and that local SOCE-evoked Ca ²⁺ fluxes are small enough to allow substantial intracellular redistribution of Ca ²⁺ through ER tunnels.