The ATP-gated P2X7 receptor is a low affinty cation channel which mediates a range of responses, from cell proliferation to cell death, and is a key regulator of the inflammatory response. Emerging evidence supports an important role for lipid rafts in the regulation of P2X7 receptor signalling, but the mechanism of this regulation is not yet understood (Garcia-Marcos et al., 2006; Barth et al., 2007; Gonnord et al., 2009). Lipid rafts are regions of the plasma membrane enriched in cholesterol and sphingolipids which provide specialised platforms for the organisation of signalling pathways, and by dynamic remodelling can directly modulate signal transduction. We have evidence that disrupting lipid rafts enhances P2X7 receptor-mediated large pore formation, as measured by dye uptake. Dye uptake, measured using confocal microscopy, is predominantly mediated via the P2X7 receptor but following cholesterol depletion, the enhanced kinetics of dye uptake appears to involve the pannexin-1 hemichannel. Inhibitors of pannexin-1 can block this increase and so can the non-specific caspase inhibitor, Z-VAD-FMK. To investigate targeting of the P2X7 receptor and pannexin-1 to lipid rafts we have separated cellular components on a discontinuous sucrose gradient by ultracentrifugation following their heterologous expression in either HEK293 cells or TSA 201 cells. Both proteins appear in the light lipid raft fractions although not exclusively. This targeting can be manipulated by the co-expression of either wild type or a dominant negative mutant of caveolin-1 and by stomatin. Interactions between these proteins are being investigated by co-immunoprecipitation experiments and FRET as are the effects on P2X7 receptor—pannexin-1 signalling.