Extracellular nucleotide di-and triphosphates such as ATP and ADP mediate their effects through purinergic P2 receptors belonging to either the metabotropic P2Y or the ionotropic P2X receptor family. The P2X 7 R is a unique member of the P2X family, which forms a pore in response to ligand stimulation, regulating cell permeability, cytokine release, and/or apoptosis. This receptor is also unique in that its affinity for the ligand benzoyl-benzoyl ATP (BzATP) is at least 10-fold greater than that of ATP. Primary human fetal astrocytes in culture express low-levels of P2X 7 R mRNA and protein, and BzATP induces only a slight influx in intracellular calcium [Ca 2+ ] i , with little demonstrable effect on gene expression or pore formation in these cells. We now show that, following treatment with the proinflammatory cytokine IL-1β, BzATP induces a robust rise in [Ca 2+ ] i with agonist and antagonist profiles indicative of the P2X 7 R. IL-1β also induced the formation of membrane pores as evidenced by the uptake of YO-PRO-1 (375 Da). Quantitative real-time PCR demonstrated transient upregulation of P2X 7 R mRNA in IL-1β-treated cells, while FACS analysis indicated a similar upregulation of P2X 7 R protein at the cell membrane. In multiple sclerosis lesions, immunoreactivity for the P2X 7 R was demonstrated on reactive astrocytes in autopsy brain tissues. In turn, P2X 7 R stimulation increased the production of IL-1-induced nitric oxide synthase activity by astrocytes in culture. These studies suggest that signaling via the P2X 7 R may modulate the astrocytic response to inflammation in the human central nervous system.

Members of the mammalian transient receptor potential (TRP) family form cation-permeable channels at the plasma membrane implicated in capacitative calcium influx after activation by either second-messenger-mediated pathways or store depletion, or both. This study shows that with the use of RT-PCR, Western blotting, and immunohistochemistry, resting astrocytes express TRPC4 at the cell membrane, particularly at sites of cell-to-cell contact. By confocal imaging and immunoelectron microscopy, we detected co-localization of TRPC4 with the scaffolding protein zonula occludens 1 (ZO-1), and demonstrated that immunoprecipitation with antibodies to ZO-1 brought down TRPC4, and vice-versa. It has been proposed that the targeting of TRPC4 to the cell membrane is dependent on the interaction of the C-terminal TRL motif with PDZ domains. Using transfection of astrocytes with myc-tagged TRPC4 or TRL-motif truncated TRPC4 (deltaTRL), we found that deltaTRL localized predominantly to a juxtanuclear compartment, whereas the wild-type protein showed cell surface distribution. Deletion of the TRL motif also reduced plasma membrane expression as assessed by cell surface biotinylation experiments. Using GST fusion proteins, we found that TRPC4 interacted with the PDZ1 domain of ZO-1 and that this was also dependent on the TRL motif. Thus, our data demonstrate that the PDZ-interacting domain of TRPC4 controls its cell surface localization. These data implicate TRPC4 in the regulation of calcium homeostasis in astrocytes, particularly as part of a signaling complex that forms at junctional sites between astrocytes.