STIM1 and Orai1 mediate thrombin-induced Ca2+ influx in rat cortical astrocytes

In astrocytes, thrombin leads to cytoplasmic Ca2+ elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca2+ increase generated by an interplay between ER-Ca2+ release and store-operated Ca2+ entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca2+ depletion and binds Orai1 to activate Ca2+ influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca2+ response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca2+ influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca2+ influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca2+ response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca2+ release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca2+ influx.