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

Abstract

SummaryP2X7 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.

The P2X7 Receptor Channel: Recent Developments and the Use of P2X7 Antagonists in Models of Disease

“…In this study, T cells were found to preferentially express P2X7K, whereas macrophages preferentially express P2X7A. This may account for differences in P2X7 signaling between these cell types in rodents (Hong 648 et al, 2009;Xu et al, 2012) and suggests that P2X7 variants may mediate the differential sensitivity of macrophages and lymphocytes to NAD. However, the relative contribution of different P2X7 splice variants on NAD-mediated P2X7 activation in other cell types, such as astrocytes, has not been investigated.…”

Evaluation of the expression and function of the P2X7 receptor and ART1 in human regulatory T-cell subsets

“…These results suggest that, the mechanism of P2X7 receptor activation by ART1-mediated ribosylation does not occur in human immune cells as reported in murine models (Aswad & Dennert, 2006;Hong et al, 2009). This conclusion is supported by results observed with patch-clamp assays in which NAD did not activate the P2X7 receptor and behaved like a partial agonist of the P2X7 receptor (Xu et al, 2012). CD39+ Treg cells may be less sensitive to ATP or NAD because they exhibited a lower level of P2X7 and ART1 expression; however the results with CD39-Treg cells, which expressed higher levels of P2X7 when compared with CD39+ Treg cells, do not support this latter hypothesis.…”

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

“…Both types of resting CD4 ϩ T-lymphocytes failed to respond to either 100 M BzATP or 1 mM ATP; at these concentrations, both agonists fully activate P2X7kRs (57,89). Likewise, activated conventional CD4 ϩ T cells did not respond to either agonist with inward current.…”