Cross-talk between stim proteins and ionotropic receptors in neurons

INTRODUCTION: Depletion of Ca2+ in endoplasmic reticulum (ER) is sensed by STIM proteins, which then activate influx of these ions via Orai channels from the extracellular environment. This process is called the Store-Operated Calcium Entry (SOCE), and its role in non-excitatory cells is to refill ER with Ca2+. In neurons, however, SOCE is also used for signaling and seems to involve several types of channels. AIM(S): The aim of this study is to determine which ionotropic receptors (IR) react with STIM proteins and are involved in calcium influx during SOCE. METHOD(S): In cultured cortical neurons we recorded single-cell Ca2+ levels using Fura-2AM. SOCE was measured after depletion of intracellular Ca2+ stores by thapsigargin and subsequent incubation of cells in 2 mM Ca2+ media. To investigate the involvement of IR in SOCE, we applied antagonists of these receptors such as CNQX and NBQX (AMPAR), MK‑801, memantine and D-AP5 (NMDAR). To determine the effects of SOCE on IR agonist-induced Ca2+ entry we applied SOCE inhibitors (ML9, SKF96365). The co-immunoprecipitation assay was used to detect the interaction between STIMs and IR. Electrophysiology experiments are also being performed. RESULTS: We report that ML-9 and SKF reduced AMPAand NMDA‑induced Ca2+ influx. In addition, SOCE was decreased by CNQX, NBQX, D‑AP5, memantine, but no significant effect was observed in the presence of MK801. Physical association of endogenous STIM proteins with endogenous GluA1 or GluA2 subunits of AMPAR and N2B subunit of NMDAR we detected by immunoprecipitation. Application of SOCE inhibitor, SKF96365, had no effect on the amplitude of AMPA-mediated miniature excitatory postsynaptic currents (mEPSCs). CONCLUSIONS: Ca2+ measurements using specific inhibitors and immunoprecipitation experiments indicate that STIM proteins might participate in neuronal signaling by the interaction with ionotropic receptors such as AMPA and NMDA. FINANCIAL SUPPORT: Supported by funds from a National Science Centre (2011/01/D/NZ3/02051, JGB).