Orai1 internalization and STIM1 clustering inhibition modulate SOCE inactivation during meiosis

Abstract

Store-operated Ca 2؉ entry (SOCE) is a ubiquitous Ca 2؉ influx pathway activated in response to depletion of intracellular Ca 2؉ stores. SOCE is a primary modulator of intracellular Ca 2؉ dynamics, which specify cellular responses. Interestingly, SOCE inactivates during M phase but the mechanisms involved remain unclear. SOCE is mediated by clustering of the ER Ca 2؉ sensor STIM1 in response to Ca 2؉ store depletion, leading to gating of the plasma membrane SOCE channel Orai1. Here we show that SOCE inactivation in meiosis is the result of internalization of Orai1 into an intracellular vesicular compartment and to the inability of STIM1 to cluster in response to store depletion. At rest, Orai1 continuously recycles between the cell membrane and an endosomal compartment. We further show that STIM1-STIM1 interactions are inhibited during meiosis, which appears to mediate the inability of STIM1 to form puncta following store depletion. In contrast, STIM1-Orai1 interactions remain functional during meiosis. Combined, the removal of Orai1 from the cell membrane and STIM1 clustering inhibition effectively uncouple store depletion from SOCE activation in meiosis. Although STIM1 is phosphorylated during meiosis, phosphomimetic and alanine substitution mutations do not modulate STIM1 clustering, arguing that phosphorylation does not mediate STIM1 clustering inhibition during meiosis.phosphorylation ͉ store-operated calcium entry ͉ trafficking

Mid-range Ca2+ signalling mediated by functional coupling between store-operated Ca2+ entry and IP3-dependent Ca2+ release

“…IP 3 -mediated store depletion results in the translocation of STIM1 to the cortical ER domain just below the cell membrane, where it co-localizes in clusters with Orai1 in a single focal plane as visualized in cells co-expressing STIM1 and Orai1 ( Supplementary Fig. 5B) 45,47 . In contrast to Orai1, which localizes to the STIM1 clusters following store depletion, CaCCs remain diffusely distributed at the cell membrane, but are excluded from the STIM1 puncta (Fig.…”

“…These results argue that store depletion with IP 3 enhances the ability of CaCC to respond to Ca 2 þ flowing into the cell through SOCE. To confirm that this effect is specific to IP 3 and SOCE, we activated SOCE with the low-affinity Ca 2 þ chelator, TPEN, which is known to buffer free intraluminal ER Ca 2 þ thus simulating store depletion 44,45 . In a similar manner to thapsigargin and ionomycin, TPEN failed to generate the large I ClT observed following IP 3 df treatment (Fig.…”

“…To test this possibility, we injected oocytes with high concentration of IP 3 (B2.5 mM), which results in rapid Ca 2 þ release similar to what is observed with ionomycin, and leads to a large I ClT following store depletion ( Supplementary Fig. 1E) 44,45 . We further treated cells with lysophosphatidic acid (LPA), which is known to activate G-protein coupled receptors and stimulate phospholipase C (PLC) to generate IP 3 in Xenopus oocytes 46 .…”

Store-Operated Calcium Channels: New Perspectives on Mechanism and Function

“…In platelets there is evidence for Orai1 insertion into the PM, but in this case in response to elevated [Ca 2þ ] i following activation of SOCE (Woodard et al 2008). A powerful example of how regulation of Orai1 trafficking can modulate SOCE occurs during meiosis, when internalization of Orai1 helps inactivate SOCE (Yu et al 2009 Park et al showed that the binding is direct, based on GST pulldowns and copurification of Orai1 and CAD. Constitutive activation of I CRAC by CAD binding to Orai1 offers the strongest evidence to date in favor of a conformational coupling mechanism involving direct proteinprotein interactions, and argues against a diffusible messenger model (though it does not rule out the possibility that a diffusible messenger could modulate CRAC or provide a parallel activation pathway).…”

STIM1/Orai1-mediated SOCE: current perspectives and potential roles in cardiac function and pathology

“…The K domain is predicted to contain a flexible central amphipathic ␣-helix that is flanked by globular positively charged regions in a dumbbell-like structure (148). The region between the ID and K domains appears to be the major regulatory region and contains most of the currently identified phosphorylation sites (62,92,109,143). Although it is now well recognized that STIM1 is a phosphoprotein (31,62), the molecular pathways responsible for controlling STIM1 phosphorylation are not well defined and the physiological function of many of the phosphorylation sites remain to be identified.…”