Microglial activation is an integral part of neuroinflammation associated with many neurodegenerative conditions. Interestingly, a number of neurodegenerative conditions exhibit enhanced P2X 7 receptor (P2X 7 R) expression in the neuroinflammatory foci where activated microglia are a coexisting feature. Whether P2X 7 R overexpression is driving microglial activation or, conversely, P2X 7 R overexpression is a consequence of microglial activation is not known. We report that overexpression alone of a purinergic P2X 7 R, in the absence of pathological insults, is sufficient to drive the activation and proliferation of microglia in rat primary hippocampal cultures. The trophic responses observed in microglia were found to be P2X 7 R specific as the P2X 7 R antagonist, oxidized ATP (oxATP), was effective in markedly attenuating microgliosis. oxATP treatment of primary hippocampal cultures expressing exogenous P2X 7 Rs resulted in a significant decrease in the number of activated microglia. P2X 7 R is unusual in exhibiting two conductance states, a cation channel and a plasma membrane pore, and there are no pharmacological agents capable of cleanly discriminating between these two states. We used a point mutant of P2X 7 R (P2X7RG345Y) with intact channel function but ablated pore-forming capacity to establish that the trophic effects of increased P2X 7 R expression are exclusively mediated by the pore conductance. Collectively, and contrary to previous reports describing P2X 7 R as a "death receptor," we provide evidence for a novel trophic role for P2X 7 R pore in microglia.

The importance of the cytosolic C-terminal region of the P2X7 receptor (P2X7R) is unquestioned, yet little is known about the functional domains of this region and how they may contribute to the numerous properties ascribed to this receptor. A structure-function analysis of truncated and single-residue-mutated P2X7 receptors was performed in HEK-293 cells and Xenopus oocytes. Cells expressing receptors truncated at residue 581 (of 595) have negligible ethidium ion uptake, whereas those expressing the P2X7R truncated at position 582 give wild type ethidium ion uptake suggesting that pore formation requires over 95% of the C-terminal tail. Channel function was evident even in receptors that were truncated at position 380 indicating that only a small portion of the cytosolic region is required for channel activity. Surprisingly, truncations in the region between residues 551 and 581 resulted in non-functional receptors with no detectable cell surface expression in HEK-293 cells. A more detailed analysis revealed that mutations of single residues within this region could also abolish receptor function and cell surface expression, suggesting that this region may participate in regulating the surface expression of the pore-forming P2X7R.

The P2X 7 receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X 7 receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X 7 function was measured by ATPinduced fluxes of Rb ؉ , Ba 2؉ , and ethidium ؉ into various lymphocyte subsets and was decreased to values of ϳ25% of normal. The expression of the P2X 7 receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X 7 carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-␥ up-regulated P2X 7 function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X 7 receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.The purinergic P2X 7 receptor is a ligand-gated channel, selective for cationic permeants, which has a wide distribution including cells of the immune and hemopoietic system (1, 2). Activation of this receptor by brief exposure to extracellular ATP opens a channel that allows Ca 2ϩ and Na ϩ influx and K ϩ efflux and that initiates a cascade of intracellular downstream events. These include the stimulation of phospholipase D (3, 4), the activation of membrane metalloproteases (5-7), and the stimulation of intracellular caspases, which eventually lead to the apoptotic death of the target cell (8, 9). P2X 7 activation also leads to extensive membrane blebbing (10), which is a typical morphological feature of the apoptotic process. P2X 7 receptors have two transmembrane domains with intracellular amino and carboxyl termini, and the P2X 7 receptor differs from other members of the P2X receptor family in having a long carboxyl terminus of 240 amino acids from the inner membrane face (11). This long carboxyl terminus is necessary for the permeability properties of the P2X 7 receptor because truncation of this tail abolishes ATP-induced uptake of the fluorescent dye Yo-Pro-1 (12). P2X 7 has an oligomeric structure in the membrane based on trimeric or larger complexes of identical subunits (13,14), and there is evidence that P2X 7 interacts with a number of structural and adhesion proteins in a complex at the cell surface (15). Phosphorylation of a tyrosine at amino acid 343 of the P2X 7 primary structure has been proposed as being important for maintaining the full activity of the P2X 7 channel (15). A number of regulatory d...

Residues considered essential for ATP binding to the human P2X 7 receptor (hP2X 7 R) were investigated. HEK293 cells or Xenopus oocytes were transfected with wild-type or sitedirected mutants of hP2X 7 R constructs and channel/pore activity measured in the presence of ATP or 2P P,3P P-O-(4-benzoylbenzoyl)-ATP (BzATP). Barium uptake and ethidium influx into HEK293 cells were abolished in cells expressing K193A and K311A mutants, and were partially reduced in cells expressing mutant P210A. K193A and K311A mutations also completely abolished responses to ATP and BzATP in Xenopus oocytes as measured by electrophysiology. These results indicate that K193 and K311 are essential residues in ATP binding in the hP2X 7 R. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The present study examines whether changes in P2X7 purinergic receptor density precede formation of the cytolytic pore characteristic of this receptor. We fused P2X7 receptors with enhanced green fluorescent protein (EGFP) at the amino or carboxy termini (EGFP-P2X7 and P2X7-EGFP). Electrophysiological characterization in Xenopus oocytes revealed wild-type responses to ATP for GFP-tagged receptors. However, differences in sensitivity to ATP were apparent with the P2X7-EGFP receptor displaying a threefold reduction in ATP sensitivity compared with control. Ethidium ion uptake was used to measure cytolytic pore formation. Comparison of tagged receptors with wild type in HEK-293 and COS-7 cells showed there was no significant difference in ethidium ion uptake, suggesting that fusions with EGFP did not interfere with cytolytic pore formation. Confocal microscopy confirmed that tagged receptors localized to the plasmalemma. Simultaneous monitoring of EGFP and ethidium ion fluorescence revealed that changes in receptor distribution do not precede pore formation. We conclude that it is unlikely that large scale changes in P2X7 receptor density precede pore formation.

Regulatory interactions between the endoplasmic reticulum (ER) and the mitochondria in the control of intracellular free Ca2+ concentration ([Ca2+]I), may be of importance in the control of many cell functions, and particularly those involved in initiating cell death. We used targeted Ca2+ sensors (cameleons) to investigate the movement of Ca2+ between the ER and mitochondria of intact cells and focused on the role of the mitochondrial permeability transition (MPT) in this interaction. We hypothesized that release of Ca2+ from mitochondria in response to a known MPT agonist (atractyloside) would cause release of ER Ca2+, perpetuating cellular Ca2+ overload, and cell death. Targeted cameleons (mitochondria and ER) were imaged with confocal microscopy 2-3 days following transient transfection of human embryonic kidney 293 cells. Opening of the MPT resulted in specific loss of mitochondrial Ca2+ (blocked by cyclosporin A), which was sequestered initially by ER. The ER subsequently released this Ca2+ load, leading to a global Ca2+ elevation, a response that was not observed when ER Ca2+-ATPases were blocked with cyclopiazonic acid. Thus, ER plays an important role in moderating changes in intracellular Ca2+ following MPT and may play a key role in cell death initiated by mitochondrial mechanisms.

1 Angiotensin II has a well described dose-dependent biphasic action on proximal tubule¯uid uptake, although the concentration and eect of endogenous luminal angiotensin II remain controversial. 2 Shrinking split-droplet micropuncture was used to examine the¯uid uptake in response to the luminal application of three AT 1 antagonists (losartan, EXP3174, candesartan). 4 All three antagonists at a low concentration (10 78 M) decreased¯uid uptake. EXP3174 and candesartan at a higher concentration (10 75 M) also decreased¯uid uptake in contrast to the previously reported eect of losartan. 5 We conclude that the endogenous concentration of antiotensin II in the proximal luminal¯uid is low and exerts a stimulatory eect on¯uid absorption. Losartan at concentrations greater than 10 76 M may have a non-selective action on¯uid uptake.

1. Understanding the regulation of calcium (Ca2+), the most common of the mineral ions within the human body, has always been of extreme interest to physiologists. While the importance of Ca2+ in contributing to physiological events through regulation of levels has been significantly established, seldom is consideration given to the intricacies of this ion and its mechanics in producing such diverse physiological responses in different regions of the cell. 2. The present review will summarize new methodologies used in our laboratories for the study of two major intracellular organelles, mitochondria and the nucleus. These techniques are based predominantly on the use of molecular biological approaches to both create and then target protein-based sensor molecules to specific intracellular locations. 3. The regulation of Ca2+ in the mitochondria and nucleus is of particular interest to us because of the central involvement of these organelles in: (i) cardiac cell responses during ischaemia/reperfusion; and (ii) the control of gene expression, respectively.