Sphingosine 1 Phosphate Regulates Store‐Operated Calcium Entry through binding to STIM1

Abstract

A growing body of evidence suggests that sphingosine kinase 1 (SK1)/ sphingosine 1‐phosphate (S1P) pathway contributes to vascular dysfunction through dysregulation of Ca2+ mobilization. We demonstrated recently that intracellular S1P triggers store‐operated Ca2+ (SOC) entry and independent of G protein‐coupled S1P receptors suggesting SOC as intracellular target for S1P. However, the mechanism by which S1P modulates SOC entry is not well defined. SOC is composed of two molecular components; stromal interaction molecules (STIM), and Orai proteins. While STIM function as Ca2+ sensors within the ER, the Orai proteins constitute the pore‐forming unit of SOC in PM. We first investigated the role of SK1/S1P on STIM1/Orai1 assembly by confocal microscopy. Pretreating transiently transfected HEK293 cells with SK1 specific inhibitor inhibited angiotensin‐induced colocalization of GFP.STIM1 and mCherry Orai1. Similarly, using BRET assay, inhibition of SK1 decreased NET BRET values of STIM1‐FlAsH and Orai1‐Rluc in transiently transfected HEK293 cells stimulated with GPCR agonists. Our molecular modeling simulation predicted direct binding of S1P to EF/SAM1 domain of STIM1 molecule. This is initially assessed by in vitro binding assay revealed binding of biotinylated S1P to STIM1 in the membrane fractions of vascular smooth muscle cell lysates. We then used circular dichroism spectroscopy (CD) to measure the thermostability of the EF/SAM1 protein in the presence or absence of S1P. Addition of S1P to EF/SAM1 induced a consistent left shift indicative of lower melting temperature (Tm) compared to vehicle control. To further validate direct binding, we prepared 15N‐labeled EF/SAM1 for the NMR spectroscopy. A five point titration series with S1P caused perturbations of residue specific chemical shift due to binding interactions between EF/SAM1 and S1P using 2D TROSY (Transverse Relaxation Optimized Spectroscopy). Collectively, our data provide evidences of intracellular role for S1P in regulation of SOC through direct binding to STIM1.Support or Funding InformationAmerican Heart Association 17GRNT33700222 (HME)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.