Orai1 and Orai3 in Combination with Stim1 Mediate the Majority of Store-operated Calcium Entry in Astrocytes

Abstract

Astrocytes are non-excitable cells in the brain and their activity largely depends on the intracellular calcium (Ca2+) level. Therefore, maintaining the intracellular Ca2+ homeostasis is critical for proper functioning of astrocytes. One of the key regulatory mechanisms of Ca2+ homeostasis in astrocytes is the store-operated Ca2+ entry (SOCE). This process is mediated by a combination of the Ca2+-store-depletion-sensor, Stim, and the store-operated Ca2+-channels, Orai and TrpC families. Despite the existence of all those families in astrocytes, previous studies have provided conflicting results on the molecular identification of astrocytic SOCE. Here, using the shRNA-based gene-silencing approach and Ca2+-imaging from cultured mouse astrocytes, we report that Stim1 in combination with Orai1 and Orai3 contribute to the major portion of astrocytic SOCE. Gene-silencing of Stim1 showed a 79.2% reduction of SOCE, indicating that Stim1 is the major Ca2+-store-depletion-sensor. Further gene-silencing showed that Orai1, Orai2, Orai3, and TrpC1 contribute to SOCE by 35.7%, 20.3%, 26.8% and 12.2%, respectively. Simultaneous gene-silencing of all three Orai subtypes exhibited a 67.6% reduction of SOCE. Based on the detailed population analysis, we predict that Orai1 and Orai3 are expressed in astrocytes with a large SOCE, whereas TrpC1 is exclusively expressed in astrocytes with a small SOCE. This analytical approach allows us to identify the store operated channel (SOC) subtype in each cell by the degree of SOCE. Our results propose that Stim1 in combination with Orai1 and Orai3 are the major molecular components of astrocytic SOCE under various physiological and pathological conditions.

Stim and Orai mediate constitutive Ca2+ entry and control endoplasmic reticulum Ca2+ refilling in primary cultures of colorectal carcinoma cells

“…Nevertheless, a recent investigation clearly showed that heteromeric Orai1/Orai3 channels mediate Stim1- and Stim2-dependent SOCE induced by 5α-dihydrotestosterone (DHT) in LNCaP cells [ 33 ]. In addition, Stim1, Stim2, Orai1 and Orai3 were found to mediate SOCE in mouse dorsal root ganglion neurons [ 74 ] and mouse astrocytes [ 75 ]. Finally, Orai1 and Orai3 were shown to mediate Ca 2+ entry in response to store depletion in mouse pulmonary artery smooth muscle cells [ 76 ].…”

Astrocytic glycogen metabolism in the healthy and diseased brain

“…1), which is present in astrocytes (48,53). AC8 can be activated by SOCE in the absence of GPCR activation, perhaps in part due to a physical association with Orai1 channels (55)(56)(57), although the relative roles of TRPC vs. Orai channels for mediating SOCE remains controversial (58,59). Clearly, SOCE represents a non-receptor-mediated way of initiating Ca 2+ /cAMPdependent glycogen breakdown; however, in situ SOCE would only occur following an initial cytosolic Ca 2+ signal depleting intracellular Ca 2+ stores such as a GPCR-Gαq-IP3-mediated store depletion.…”

Spontaneous calcium signaling of cartilage cells: from spatiotemporal features to biophysical modeling

“…If the [Ca 2+ ] i activities are controlled by multiple parallel or independent processes, the spatiotemporal parameters of the [Ca 2+ ] i fluctuations should follow a standard gaussian normal distribution according to the central limit theorem (21). However, the spatiotemporal characteristics of [Ca 2+ ] i peaks (e.g., number for multiple peaks from a single cell, time it takes to reach a peak from the baseline) of the same cell population were found to follow a lognormal distribution rather than a normal distribution (22,23). Little knowledge is available to interpret this statistical distribution profile of the [Ca 2+ ] i peak parameters.…”