Regulation of Store‐Operated Calcium Channels by Sphingosine 1 Phosphate

Abstract

Several studies have implicated sphingosine kinase 1 (SK1)/ sphingosine 1‐phosphate (S1P) axis in mobilization of intracellular Ca2+ and defective vascular tone. We and other independent investigators demonstrated recently that SK1/S1P mediates transmembrane Ca2+ flux through store‐operated Ca2+ channel (SOC). SOC is composed of two molecular components; stromal interaction molecules (STIM), and Orai proteins. STIM function as Ca2+ sensors within the ER, and Orai proteins at PM. Depletion of Ca2+ from ER leads to oligomerization and translocation of STIM to bind Orai at the PM to activate Ca2+ flux. We found that S1P regulates SOC through yet undefined intracellular mechanism that does not involve activation of G protein‐coupled S1P receptors. Here, we have studied the mechanism by which SK/S1P mediates activation of SOC. Molecular modeling predicted the binding of S1P with STIM1 of SOC in monomer and dimer forms. Unlike biotin ceramide and biotin controls, only biotinylated S1P bound to endogenous STIM1 in the membrane fraction of cell lysates that was inhibited with competitive cold S1P suggesting specificity of S1P binding to STIM1. Intriguingly, direct mutation of the predicted S1P binding sites to STIM1 markedly inhibited angiotensinII (AngII) and intracellular S1P‐dependent Ca2+ flux without affecting Ca2+ release from intracellular stores. To examine the effect of SK1/S1P on STIM1/Orai1 assembly by BRET assay, we generated STIM1‐FlAsH and Orai1‐Rluc plasmids. We found that NET BRET values of AngII‐dependent stimulated HEK293 cells transiently transfected with STIM1‐FlAsH and Orai1‐Rluc were significantly inhibited by SK1 inhibitor. Functionally, transfecting STIM1 mutants in HEK293 cells inhibited cell proliferation compared to vehicle control. These findings suggest the requirement of intracellular S1P for activation of SOC via modulation of STIM1 and provide novel mechanistic and functional insights into pathophysiology of vascular dysfunction.