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

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.

Neuronal SOCE: Myth or Reality?

“…Figure 1D), as now documented by several groups in different types of neurons [4][5][6][7][8]. In fact, depletion of ER and cytoplasmic Ca 2+ is so fast that it partially occludes or masks thapsigargin-mediated ER Ca 2+ release ( Figure 1C).…”

“…SOCE responses in both cortical and hippocampal neurons (arguably the best studied cell types for SOCE) vary from negligible [5,11], to relatively weak [4,9,12], to levels approaching SOCE activity in non-neuronal cells [6,13,14]. The caveats associated with SOCE detection undoubtedly contribute to these discrepancies, but it is also possible that this variability arises from cell-tocell differences in SOCE activity.…”

The role of Ca2+ signaling on the self-renewal and neural differentiation of embryonic stem cells (ESCs)

“…Knockdown of STIM2 resulted in even more severe neural differentiation defects than STIM1 knockdown. Notably, both STIM1 and STIM2 contribute to the regulation of Ca 2+ homeostasis in rat neurons but they exhibit distinct functions since STIM2 is mainly involved in regulating the resting level of Ca 2+ in the ER whereas STIM1 prominently activates SOCE [81]. In addition to activating the Orai1 channel, STIM1 can also activate TRPCs and inhibit VGCCs via direct interaction with these proteins [82] [83] [84].…”

STIM Proteins As Regulators of Neuronal Store-Operated Calcium Influx

“…The EF hand domain of STIM2 proteins has significantly lower affinity for Ca 2+ than STIM1, and it has been established that STIM2 proteins primarily control steady-state ER and cytosolic Ca 2+ homeostasis [4]. Both STIM1 and STIM2 proteins are expressed in hippocampal neurons [5], and both of these proteins have been implicated in control of nSOC. Klejman et al observed that STIM1 is colocalized with Orai1 upon depletion of Ca 2+ stores in dendrites and soma [6].…”

“…However, deletion of both Stim genes resulted in a pronounced impairment in spatial learning and memory and in enhanced LTP [14]. STIM2 is a predominant isoform in the hippocampus [5,15]. Genetic deletion of STIM2 protein impaired SOC and confers protection from neurological damage in a model of focal cerebral ischemia [16].…”