Endolysosomal targeting and function of mammalian P2X4 receptors

P2X4 receptors have a widespread distribution throughout the body. In primary cells where we have examined their subcellular distribution, including macrophages, microglia and endothelial cells, the receptors are localized to late endosomes and lysosomes (LEL). Mutational analysis of heterologously expressed receptors has shown that a C-terminal tyrosine based motif and N-terminal dileucine-like motif are both involved in targeting the receptor to LELs. Within lysosomes, P2X4 receptors resist degradation by virtue of N-linked glycans, and they can be delivered from lysosomes to the surface to increase the expression of functional receptors. In activated microglia in culture, a proportion of receptors traffic to and from the cell surface in a dynamic manner and further activation of microglia by LPS was shown to increase the number at the cell surface. In cultured bone marrow derived macrophages, however, receptors were localized exclusively to LELs and no expression was detected at the surface unless lysosome exocytosis was stimulated by incubation with a weak base. We are interested in whether or not P2X4 receptors can function within the acidic environment of the lysosome or phagosome, as well as at the plasma membrane. Intracellular P2X-like receptors have been identified in Dictyostelium and are thought to function within the acidic environment of the contractile vacuole. With the extracellular solution at lysosomal pH (4.6), P2X4 receptors at the cell surface of HEK293 cells can be activated by millimolar extracellular ATP. We are examining the effects of wild type and mutant P2X4 receptor expression on LEL function and calcium signalling.