Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides

Marko A Popovic,Peter Varnai,Stanko S Stojilkovic,Marek K Korzeniowski,Tamas Balla,Zsofia Szentpetery

doi:10.1074/jbc.m109.012252

Abstract

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca2+ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca2+ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P(2) depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I(CRAC) currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

Highlights

Store-operated Ca2ϩ entry (SOCE)3 is a ubiquitous Ca2ϩ entry pathway that is regulated by the Ca2ϩ content of the endoplasmic reticulum (ER) [1]
Cells were transfected with the plasma membrane (PM)-targeted FRB construct and the mRFPFKBP12-fused 5-phosphatase domain, which is cytosolic under basal conditions but becomes recruited to the PM after the addition of rapamycin causing rapid depletion of PtdIns[4,5]P2 from the membrane
The ERlocalized STIM1 protein makes a contact with the PM upon ER Ca2ϩ store depletion, and it contains a polybasic domain at its C terminus that is perfectly suited to serve as a phosphoinositidebinding module, especially in the multimerized form in which STIM1 exists in the ER Ca2ϩ-depleted state [18]

Summary

Introduction

Store-operated Ca2ϩ entry (SOCE)3 is a ubiquitous Ca2ϩ entry pathway that is regulated by the Ca2ϩ content of the endoplasmic reticulum (ER) [1]. Rapid Dephosphorylation of PtdIns[4,5]P2 to PtdIns4P Does Not Inhibit SOCE and Has Only Minor Effects on STIM1 Translocation from the Tubular to PM-adjacent ER—First we wanted to determine whether isolated changes in PtdIns[4,5]P2 could alter STIM1 movements or the activation of Orai1-mediated Ca2ϩ influx in intact cells.

Results

Conclusion