P2X7 receptor activates multiple selective dye-permeation pathways

ABSTRACT P2X7 receptor has gained an increasing importance as a drug target. One important response to P2X7 receptor stimulation is the uptake of large molecular weight tracers into cells. However, mechanism for this response is not understood clearly, but it is generally believed that a nonselective large pore protein forms this P2X7 receptor-activated permeability pathway. We examined human embryonic kidney (HEK) 293 cells transfected with rat P2X7 receptors (HEK-rP2X7) and a macrophage derived cell line, RAW 264.7, that expresses an endogenous P2X7 receptor. We used confocal microscopy to investigate uptake of different types of dyes into these cells after ATP application. Stimulation of P2X7 receptors in HEK-rP2X7 cells activated two different dye uptake pathways. The first was permeable to the cationic fluorescent dyes YO-PRO-1 and TO-TO-1 but not to the anionic dyes lucifer yellow and calcein and did not require intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) increase to be activated. The second pathway permeated only lucifer yellow and was completely dependent on [Ca 2ϩ ] i for activation. In RAW 264.7 cells, P2X7 receptor stimulation activated uptake of ethidium, YO-PRO-1, TO-TO-1, lucifer yellow, and calcein. Again, two different permeation pathways were discerned in RAW 264.7 cells: one permeated only ethidium and the other one, only lucifer yellow. We did observed no clear [Ca 2ϩ ] i dependence for these permeation pathways. Our results demonstrate that instead of a single nonselective pore, P2X7 receptor seems to activate at least two permeation pathways, one for cationic and one for anionic dyes with different activation properties. The P2X7 receptor is a member of P2X receptor family, which is composed of ligand-gated ion channels. Activated P2X7 receptor causes not only a cationic membrane current, but also permeabilization of the cell membrane to large molecular weight molecules P2X7 receptors are known to be important in the pathophysiology of arthritis and mediation of pain (for review, see Article, publication date, and citation information can be found a

Splice variants of the P2X7 receptor reveal differential agonist dependence and functional coupling with pannexin-1

P2X7 receptors function as ATP-gated cation channels but also interact with other proteins as part of a larger signalling complex to mediate a variety of downstream responses that are dependent upon the cell type in which they are expressed. Receptor-mediated membrane permeabilization to large molecules precedes the induction of cell death, but remains poorly understood. The mechanisms that underlie differential sensitivity to NAD are also unknown. By studying alternative variants of the mouse P2X7 receptor we show that sensitivity to NAD is mediated through the P2X7k variant, which has a much more restricted distribution than the P2X7a receptor, but is expressed in T lymphocytes. The altered N-terminus and TM1 of the P2X7k receptor enhances the stability of the active state of this variant compared with P2X7a, thereby increasing the efficacy of NAD-dependent ADP ribosylation as measured by ethidium uptake, a rise in intracellular Ca(2+) and the activation of inward currents. Co-expression of P2X7k and P2X7a receptors reduced NAD sensitivity. P2X7k-receptor-mediated ethidium uptake was also triggered by much lower BzATP concentrations and was insensitive to the P451L single nucleotide polymorphism. P2X7k-receptor-mediated ethidium uptake occurred independently of pannexin-1 suggesting a pathway intrinsic to the receptor. Only for the P2X7aL451 receptor could we resolve a component of dye uptake dependent upon pannexin-1. Signalling occurred downstream of the activation of caspases rather than involving direct cross talk between the channels. However, an in situ proximity assay showed close association between P2X7 receptors and pannexin-1, which would facilitate ATP efflux through pannexin-1 acting in an autocrine manner

Many ways to dilate the P2X7 receptor pore

The P2X7 receptor is associated with two different membrane permeabilities: a small cation conductance which opens within milliseconds, followed by the appearance of a second channel carrying higher molecular weight compounds (including organic dyes) after prolonged agonist stimulation. This activation profile has also been found in cells expressing P2X2 and P2X4 receptors; however, the P2X7 receptor-dependent pathway has the unique ability to activate pro-inflammatory signalling in macrophages. In this issue of the BJP, Marques-da-Silva et al. demonstrate that colchicine is a potent inhibitor of both P2X7 and P2X2 receptor-dependent dye uptake, without affecting the ion channels. Colchicine also blocked the pro-inflammatory signalling downstream of P2X7 receptor activation, both in vitro and in vivo. This report suggests that the dye uptake associated with activation of P2X7 receptors is distinct from the P2X7 receptor ion channel and could be a therapeutic target for the treatment of chronic inflammation