Abstract
Regulation of critical cellular functions, including Ca2+-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca2+ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca2+]i due to intracellular Ca2+ release and Ca2+ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca2+]i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca2+]i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca2+ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca2+ store depletion in these cells, it is not clear how the cells decode individual Ca2+ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca2+]i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca2+ entry generates [Ca2+]i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca2+ entry generates sustained [Ca2+]i elevation at high [CCh] and contributes to frequency of [Ca2+]i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca2+ dependent gene expression pathways, consistent with the different patterns of [Ca2+]i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca2+]i generated by Orai1, independent of global [Ca2+]i changes or TRPC1-mediated Ca2+ entry. In contrast, Ca2+ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca2+ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca2+-dependent gene expression pathways.
Highlights
Stimulation of cells with physiologically relevant agonists that target G protein- or tyrosine kinase-coupled receptors leads to increases in cytosolic [Ca2+] ([Ca2+]i) as a result of inositol 1,4,5triphosphate (IP3)-induced Ca2+ release from intracellular Ca2+ stores via the IP3 receptors (IP3Rs) and Ca2+ influx via plasma membrane Ca2+ channels
Following CCh stimulation of human submandibular gland (HSG) cells, a fairly sustained [Ca2+]i elevation was seen, with an initial rapid increase followed by a sustained elevation above baseline that slowly declined over time (Figure 1A shows the average response in a cell population)
The data discussed above suggest that inclusion of functional TRPC1 channels leads to modulation of the [Ca2+]i signals generated by Orai1 channels
Summary
Stimulation of cells with physiologically relevant agonists that target G protein- or tyrosine kinase-coupled receptors leads to increases in cytosolic [Ca2+] ([Ca2+]i) as a result of inositol 1,4,5triphosphate (IP3)-induced Ca2+ release from intracellular Ca2+ stores via the IP3 receptors (IP3Rs) and Ca2+ influx via plasma membrane Ca2+ channels. Two types of oscillations are seen; baseline oscillations that are usually seen at very low [agonist] or oscillations over a sustained elevation in baseline [Ca2+]i at relatively higher [agonist] Such oscillatory responses have been proposed to represent the physiological mode of signaling in many cell types and have been observed in almost all cell types, including cell lines as well as primary cell preparations from various tissues [3,5,6,7,8]. These oscillations primarily reflect repetitive cycles of
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