P2X receptors are ionotropic receptors gated by ATP and are composed of two transmembrane domains, an extracellular ligand binding site and intracellular N and C-termini. It has been shown that P2X₄ receptors are rapidly cycled between the cell surface and endosomal compartments and that this internalization is dependent on a non-canonical tyrosine-based endocytic motif contained within the C-terminus of P2X₄ (Royle et al. 2002). This endocytic motif mediates the increased internalization that occurs on application of ATP.

CD8 is commonly used as a reporter molecule to study receptor trafficking as it is transported the cell surface and does not internalize. By transplanting the C-terminus of P2X₄ onto CD8 (CD8 4Ct), and following the trafficking of the CD8 4Ct chimera using antibody labelling in live HEK293 cells (from the European Collection of Cell Cultures), we saw that the chimera did internalize. This demonstrates that the C-terminus of P2X₄ contains all the determinants necessary for the internalization of the chimeric protein. Mutation of the endocytic motif within the P2X₄ C-terminus of the chimeric receptor resulted in an increase in surface expression however internalization was not inhibited to the same extent as in the wild-type receptor. Internalization of both P2X₄ and CD8 4Ct was clathrin-dependent and could be inhibited using siRNA directed against clathrin heavy chain.In the P2X₄ receptor C-terminus there are two clusters of lysine residues. Mutation of the first cluster of lysine residues prevented the increased internalization of the wild-type P2X₄ receptor that occurs on ATP application, but trafficking and function appeared to be otherwise unaffected. In contrast, mutation of the lysine residues immediately upstream of the endocytic motif greatly reduced the amount of receptor cycling between the plasma membrane and endocytic compartments. It also made the receptor non-functional, however function does not appear to be a prerequisite for receptor trafficking. The non-functional P2X₄ G347I mutant was constitutively trafficked to and from the plasma membrane similar to the wild-type receptor.

We have used the CD8 4Ct chimera to identify molecular determinants of trafficking. It has the advantage that the effects of mutations can be analysed without concern for changes in receptor conformation or function.