The short isoform of extended synaptotagmin-2 controls Ca2+ dynamics in T cells via interaction with STIM1

Abstract

ca 2+ release-activated Ca 2+ (CRAC) channels elevate cytoplasmic Ca 2+ concentration, which is essential for T cell activation, differentiation and effector functions. T cell receptor stimulation induces depletion of the endoplasmic reticulum (ER) Ca 2+ stores, which is sensed by stromal interaction molecule 1 (STIM1). STIM1 translocates to the ER-plasma membrane (PM) junctions to interact with ORAI1, the pore subunit of the CRAC channels. Here, we show that two members of the extended synaptotagmin (E-Syt) family, E-Syt1, and the short isoform of E-Syt2 (E-Syt2S), contribute to activation of CRAC channels in T cells. Knockdown or deletion of both ESYT1 and ESYT2 reduced store-operated ca 2+ entry (SOCE) and ORAI1-STIM1 clustering in Jurkat T cells. Further, depletion of E-Syts in primary T cells decreased Ca 2+ entry and cytokine production. While the ER-PM junctions were reduced in both HeLa and Jurkat T cells deleted for ESYT1 and ESYT2, SOCE was impaired only in Jurkat T cells, suggesting that the membrane-tethering function of E-Syts is distinct from their role in SOCE. Mechanistically, E-Syt2S, the predominant isoform of E-Syt2 in T cells, recruited STIM1 to the junctions via a direct interaction. This study demonstrates a membrane-tethering-independent role of E-Syts in activation of CRAC channels in T cells. Ca 2+ release-activated Ca 2+ (CRAC) channels mediate a sustained increase in cytoplasmic Ca 2+ concentration that is essential for T cell activation. T cell receptor stimulation induces the depletion of the endoplasmic reticulum (ER) Ca 2+ stores that activates CRAC channels via a process called store-operated Ca 2+ entry (SOCE). STIM1 and ORAI1 are two essential components of CRAC channels. STIM1 is an ER-resident regulatory subunit that senses depletion of the ER Ca 2+ stores, and the plasma membrane (PM)-resident ORAI1 is the pore subunit of CRAC channels. Upon store depletion, STIM1 multimerizes and translocates from the ER to the ER-PM junctions (a space of 10-15 nm) via passive diffusion and interacts with ORAI1 to induce its opening 1-5. The C-terminal poly(K) residues of STIM1 interact with phosphatidylinositol 4,5-bisphosphate (PIP 2) in the PM for its recruitment to the ER-PM junctions. Further, protein interactions also play a crucial role for STIM1 recruitment into the junctions. We recently identified junctional proteins, including junctate and junctophilins, as important components of the ER-PM junctions in T cells 6,7. Junctate-junctophilin complex is localized at the ER-PM junctions, and after sensing ER Ca 2+ depletion via its ER-luminal Ca 2+-binding motif, junctate recruits STIM1 into the junctions. Other molecules such as TMEM110 (alternatively, STIM-activating enhancer) and SARAF (SOCE-associated regulatory factor) have also been identified as components of the ER-PM junctions in T cells 8-10. All these molecules interact with STIM1 to modulate its function, including translocation into the junctions (e.g., junctate and junctophilin-4) and induction of conformational changes (e....

Enforced tethering elongates the cortical endoplasmic reticulum and limits store-operated Ca2+ entry

“…Here, we show that the only apparent ultrastructural change occurring during SOCE in non-treated HEK-293 T cells is a cER elongation that is likely mediated by endogenous STIM proteins, indicating that the cER gap distance is not dynamically regulated by the activity of endogenous E-Syt1. An earlier report showed that depletion of both E-Syt1 and E-Syt2 inhibits SOCE in Jurkat T cells but not in HeLa cells ( Woo et al, 2020 ) and our siRNAs targeting either isoform did not inhibit SOCE in HEK-293 T cells (data not shown). This might reflect the expression of distinct isoforms in immune and non-immune cells, with the activating isoform E-Syt2b possibly accounting for the inhibitory effects of E-Syt1 and E-Syt2 silencing in Jurkat T cells.…”

The Role of Lipids in CRAC Channel Function

“…Although fragments of the STIM1/Orai1 interplay with accessory proteins are often wellknown, the complete molecular picture of all interactions is still lacking. Furthermore, it remains unclear whether all these proteins interplay with each other in the same cell at the ER-PM junction or whether this diversity of available ER-PM proteins act in a cell type-specific manner, as it was identified for E-Syts [136,143]. Some proteins may also form ER-PM junctions independently of STIM proteins, opening up the intriguing possibility that different types of junctions form in a cell in response to stimulation by varying amounts of agonist.…”

“…In fact, while deletion of the STIM1 PIP 2 binding site, specifically the lysine-rich region, at the very end of the STIM1 C-terminus (STIM1 ∆K) was unable to translocate into puncta in the absence of Orai1, only co-expression with E-Syt2S triggered STIM1 ∆K oligomerization. The long isoform of the E-Syt2 protein, E-Syt2L, is assumed to act inhibitory on STIM1 recruitment to the PM [143] (Table 1).…”

Calcium Signaling in T Cells and Chronic Inflammatory Disorders of the Oral Cavity

“…STIM proteins are anchored to the ER membrane and function as Ca 2+ sensors that detect ER Ca 2+ depletion. After sensing ER Ca 2+ depletion, STIM1 and STIM2 translocate to the ER-plasma membrane (PM) junctions defined by junctional proteins (Woo et al 2020). Interaction between STIM1 and Orai1 at the ER-PM junction results in the activation and opening of the CRAC channels, thereupon producing a sustained increase in intracellular calcium levels (Fig.…”