NMDA receptors – the new partners of STIM proteins in rat primary cortical neurons

Stromal interaction molecule 1 and 2 (STIM1 and STIM2) are Ca2+ sensors in endoplasmic reticulum (ER) and the key components of store-operated calcium entry (SOCE), which is responsible for refilling the ER with Ca2+ after release into cytosol. The cooperation of STIMs with the calcium channel ORAI1 is crucial for the functioning of SOCE in non-excitable cells. It has been shown that STIMs participate in SOCE also in neurons. However, STIMs seem to play different functions. STIM1 is a major activator of SOCE, while STIM2 regulates the resting Ca2+ level in the ER and constitutive Ca2+ influx into the cell. Recent research has shown that in neurons STIM proteins can affect also activation of voltage-gated channels or ionotropic AMPA receptors (our work). We expected that, in addition to them, there are other receptors activated or inhibited by STIMs, such as NMDA receptors (NMDARs). The aim of this study was to determine if STIM proteins interact with NMDAR and influence the Ca2+ influx through this receptor. In cultured rat cortical neurons we recorded single-cell Ca2+ levels using Fura-2AM. To determine the effects of STIMs on NMDA-induced Ca2+ entry we overexpressed or knocked-down STIM1 or STIM2 using shRNA and lentiviruses. The overexpression of STIMs lowers the Ca2+ influx via NMDAR. This process is also inhibited in neurons isolated from transgenic mice that overexpress STIM1. In turn, lowering expression of STIMs results in an increase in NMDA-induced Ca2+ influx. By co-immunoprecipitation assay we found a physical association of endogenous STIMs with NMDARs and by immunofluorescence staining we observed co-localization of these proteins. The interaction between endogenous proteins was confirmed by in situ Proximity Ligation Assay. In conclusion, our data suggest participation of STIM proteins in neuronal signaling by the interaction with NMDAR.