Abstract
Ca2+ influx by store-operated Ca2+ channels is a major mechanism for intracellular Ca2+ homeostasis and cellular function. Here we present evidence for the dynamic interaction between the SOCE-associated regulatory factor (SARAF), STIM1 and Orai1. SARAF overexpression attenuated SOCE and the STIM1-Orai1 interaction in cells endogenously expressing STIM1 and Orai1 while RNAi-mediated SARAF silencing induced opposite effects. SARAF impaired the association between Orai1 and the Orai1-activating small fragment of STIM1 co-expressed in the STIM1-deficient NG115-401L cells. Cell treatment with thapsigargin or physiological agonists results in direct association of SARAF with Orai1. STIM1-independent interaction of SARAF with Orai1 leads to activation of this channel. In cells endogenously expressing STIM1 and Orai1, Ca2+ store depletion leads to dissociation of SARAF with STIM1 approximately 30s after treatment with thapsigargin, which paralleled the increase in SARAF-Orai1 interaction, followed by reinteraction with STIM1 and dissociation from Orai1. Co-expression of SARAF and either Orai1 or various N-terminal deletion Orai1 mutants did not alter SARAF-Orai1 interaction; however, expression of C-terminal deletion Orai1 mutants or blockade of the C-terminus of Orai1 impair the interaction with SARAF. These observations suggest that SARAF exerts an initial positive role in the activation of SOCE followed by the facilitation of SCDI of Orai1.
Highlights
Store operated Ca2+ entry (SOCE) is a major pathway of calcium influx in non-excitable cells, and is essential for the activation of many cellular processes
Our results suggest that SOCEassociated regulatory factor (SARAF) dissociates from STIM1 upon Ca2+ store discharge probably to interact with Orai[1] and cooperate with STIM1 in the activation of Orai[1] and, SARAF rapidly re-associates with STIM1 to participate in the inactivation of SOCE
Our results indicate that SARAF interacts and activates Orai[1] channels in a scenario that allows direct association of both proteins independently of STIM1, which has been reported to be negative regulated by SARAF17,18
Summary
Store operated Ca2+ entry (SOCE) is a major pathway of calcium influx in non-excitable cells, and is essential for the activation of many cellular processes This mechanism is initiated by the depletion of the intracellular Ca2+ stores, mainly the endoplasmic reticulum (ER). STIM1 N-terminus exhibits an EF-hand motif, which, upon Ca2+ dissociation, leads to oligomerization and clustering of STIM1 into puncta located at the ER–plasma membrane junctions[10] This transition is accompanied by a conformational reorganization of its cytosolic region from a closed to an extended state leading to the exposition of the SOAR domain (amino acids 344–44211; known as OASF (233–450/474)[12], CAD (342–448)[13] or Ccb[9] (339–444)14), which results in full activation of Orai[115]. Results are recorded as arbitrary optical density units, expressed as mean ± S.E.M. and presented as percentage of control. * and *** represent p < 0.05 and p < 0.001, as compared to their respective controls
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
