P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current–voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs undergo exocytosis. Finally, the use of P2X4-pHluorin123 showed that the modulator ivermectin did not increase the PM fraction of P2X4 receptors and acted allosterically to potentiate P2X4 receptor responses. Collectively, our data suggest that P2X4-pHluorin123 represents a useful optical probe to quantitatively explore P2X4 receptor distribution, trafficking, and up-regulation.

Intracellular Ca 2+ transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca 2+ has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca 2+ indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca 2+ signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca 2+ microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca 2+ signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2–6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by ∼3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking.

Brain astrocytes signal to each other and neurons. They use changes in their intracellular calcium levels to trigger release of transmitters into the extracellular space. These can then activate receptors on other nearby astrocytes and trigger a propagated calcium wave that can travel several hundred micrometers over a timescale of seconds. A role for endogenous ATP in calcium wave propagation in hippocampal astrocytes has been suggested, but the mechanisms remain incompletely understood. Here we explored how calcium waves arise and directly tested whether endogenously released ATP contributes to astrocyte calcium wave propagation in hippocampal astrocytes. We find that vesicular ATP is the major, if not the sole, determinant of astrocyte calcium wave propagation over distances between ∼100 and 250 μm, and ∼15 s from the point of wave initiation. These actions of ATP are mediated by P2Y1 receptors. In contrast, metabotropic glutamate receptors and gap junctions do not contribute significantly to calcium wave propagation. Our data suggest that endogenous extracellular astrocytic ATP can signal over broad spatiotemporal scales.

ATP-gated P2X channels are the simplest of the three families of transmitter-gated ion channels. Some P2X channels display a time- and activation-dependent change in permeability as they undergo the transition from the relatively Na + -selective I 1 state to the I 2 state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X 2 (rP2X 2 ) channels does not occur at mouse P2X 2 (mP2X 2 ) channels expressed in oocytes. Domain swaps, species chimeras, and point mutations were employed to determine that two specific amino acid residues in the cytosolic tail domain govern this difference in behavior between the two orthologous channels. The change in pore diameter was characterized using reversal potential measurements and excluded field theory for several organic ions; both rP2X 2 and mP2X 2 channels have a pore diameter of ∼11 Å in the I 1 state, but the transition to the I 2 state increases the rP2X 2 diameter by at least 3 Å. The I 1 to I 2 transition occurs with a rate constant of ∼0.5 s −1 . The data focus attention on specific residues of P2X 2 channel cytoplasmic domains as determinants of permeation in a state-specific manner.