The P2X receptor subtypes P2X₂, P2X₄ and P2X₆ have been shown to have an overlapping distribution in the CNS (Rubio & Soto, 2001). Both P2X₂ and P2X₄ can function as homomeric receptors unlike P2X₆ which is non-functional on its own but can form functional heteromers with both P2X₂ and P2X₄. When expressed individually in mammalian cell lines the P2X receptor subtypes have very different distributions. P2X₂ is stably expressed at the plasma membrane, P2X₄ has a punctate, endosomal-like distribution within the cell and P2X₆ is retained in the ER. We have used both immunocytochemistry and biotinylation to compare the trafficking of the heteromeric receptors with that of the homomeric.

We introduced extracellular epitopes into all three subtypes to allow detection of surface receptors in HEK293 cells. We found that when P2X₆ was expressed alone most of the receptors were retained within the ER. There was a small amount of surface expression in some cells suggesting that overexpression caused saturation of the retention pathway. When P2X₆ was co-expressed with either P2X₂ or P2X₄ its subcellular distribution changed. Most notably there was a dramatic increase in the number of P2X₆ receptors at the surface. When P2X₆ was expressed with P2X₄, it was internalised and when it was expressed with P2X₂ it was stable at the cell surface similar to the P2X₂ and P2X₄ homomers.

P2X₆ expressed alone in HEK293 cells gave two products of sizes 40 kDa and 45 kDa. The lower band corresponded to the weight of the unglycosylated receptor and the higher band corresponded to a glycosylated form of the receptor; it disappeared when it was incubated with N-glycosidase. Interestingly, when P2X₆ was expressed with either P2X₂ or P2X₄ an additional product appeared at 46 kDa. This suggests that co-expression with P2X₂ or P2X₄ allowed the carbohydrate groups to be further processed in the Golgi on the way to the cell surface.