Role of Sphingosine Kinase 1 in Regulation of Transmembrane Calcium Flux

Presentation Number: MON 512
Date of Presentation: April 3rd, 2017

Hesham Mokhtar El-Shewy*1 and Yuri K. Peterson2
1Medical University of South Carolina, Charleston, SC, 2College of Pharmacy, Medical University of South Carolina, Charleston, SC

Abstract

Sphingosine kinase 1 (SK1)/ sphingosine 1-phosphate (S1P) pathway has been reported to play a role in regulation of vascular tone by mobilization of intracellular Ca2+. We demonstrated recently that SK1/S1P mediates transmembrane Ca2+ flux through store-operated Ca2+ channel (SOC). However, the exact mechanism by which SK1 mediates regulation of SOC is not well defined. 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 triggers SOC-dependent Ca2+ influx indicating that SK1/S1P axis regulates SOC via a novel intracellular mechanism and independent of G protein-coupled S1P receptors. To investigate the role of SK1/S1P on STIM1/Orai1 assembly, we employed confocal microscopy and by BRET assay. Inhibition of SK1 inhibited colocalization of GFP.STIM1 and mCherry Orai1, and decreased NET BRET values of STIM1-FlAsH and Orai1-Rluc in transiently transfected HEK293 cells stimulated with angiotensin II (AngII). Our molecular modeling simulation predicted direct binding of S1P to STIM protein. In vitro binding assay revealed binding of S1P to STIM1 in the membrane fractions of vascular smooth muscle cell lysates. Site mutagenesis of the predicted S1P binding sites to STIM1 inhibited both AngII and intracellular S1P-dependent Ca2+ flux without affecting Ca2+ release from intracellular stores. Circular dichroism study revealed binding of S1P to STIM1 and not to its mutant protein suggesting a direct binding of S1P and STIM1. Collectively, these findings reveal a mechanistic insight into regulation of SOC-dependent transmembrane Ca2+ flux via modulation of STIM1 by intracellular SK1/S1P axis.

 

Nothing to Disclose: HME, YKP