Differential Roles for STIM1 and STIM2 in Store-Operated Calcium Entry in Rat Neurons

Joanna Gruszczynska-Biegala,Marta B Wisniewska,Pawel Pomorski,Jacek Kuznicki,David Holowka

doi:10.1371/journal.pone.0019285

Joanna Gruszczynska-Biegala, Marta B Wisniewska + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0019285

Copy DOI

Abstract

The interaction between Ca2+ sensors STIM1 and STIM2 and Ca2+ channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. We addressed this issue by establishing the presence and function of STIM proteins. Real-time polymerase chain reaction from cortical neurons showed that these cells contain significant amounts of Stim1 and Stim2 mRNA. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. The number of YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1 complexes was also enhanced by such treatment. The differences observed in the number of STIM1 and STIM2 complexes under SOCE conditions and the differential sensitivity to SOCE inhibitors suggest their distinct roles. Endoplasmic reticulum (ER) store depletion by TG enhanced intracellular Ca2+ levels in loaded with Fura-2 neurons transfected with YFP-STIM1 and ORAI1, but not with YFP-STIM2 and ORAI1, which correlated well with the number of complexes formed. Moreover, the SOCE inhibitors ML-9 and 2-APB reduced Ca2+ influx in neurons expressing YFP-STIM1/ORAI1 but produced no effect in cells transfected with YFP-STIM2/ORAI1. Moreover, in neurons transfected with YFP-STIM2/ORAI1, the increase in constitutive calcium entry was greater than with YFP-STIM1/ORAI1. Our data indicate that both STIM proteins are involved in calcium homeostasis in neurons. STIM1 mainly activates SOCE, whereas STIM2 regulates resting Ca2+ levels in the ER and Ca2+ leakage with the additional involvement of STIM1.

Highlights

Store-operated calcium entry (SOCE), referred to as capacitative calcium entry (CCE), is a phenomenon that has been well characterized in non-excitable cells
We employed quantitative real-time polymerase chain reaction (PCR) with TaqMan primers and probes to analyze the levels of Stim1 and Stim2 expression in primary cultures of cortical and hippocampal neurons and in cortical astrocytes
We examined the functions of STIM1 and STIM2 in the process of SOCE

Summary

Introduction

Store-operated calcium entry (SOCE), referred to as capacitative calcium entry (CCE), is a phenomenon that has been well characterized in non-excitable cells In these cells, the Ca2+ signal usually originates from the induction of metabotropic receptors, leading to the production of IP3 by plasma membranelocated phospholipase and release of Ca2+ from intracellular stores by activity of IP3 receptors. The Ca2+ signal usually originates from the induction of metabotropic receptors, leading to the production of IP3 by plasma membranelocated phospholipase and release of Ca2+ from intracellular stores by activity of IP3 receptors This early stage is followed by SOCE, which relies on extracellular Ca2+ influx through the SOC channels present in the plasma membrane (PM) and is tightly regulated by Ca2+ concentration in the endoplasmic reticulum (ER) [1,2]. Calcium entry into the cytoplasm is replenished in the ER by the activity of the Ca2+ adenosine triphosphatase (ATPase) of sarco/endoplasmic reticulum (SERCA) pump, which refills emptied ER stores [11,12,13]

Methods

Results

Conclusion