Topological analysis of the ATP-gated ionotropic [correction of ionotrophic] P2X2 receptor subunit.

We investigated the transmembrane topology of the P2X2 receptor subunit expressed in HEK 293 cells. Initial studies using two P2X subunits expressed in tandem indicated that the amino- and carboxy-termini are on the same side of the membrane. Immunofluorescence studies showed the cytoplasmic orientation of the amino- and carboxy-termini. Finally, N-glycosylation scanning mutagenesis revealed that reporter sites inserted into the central loop, but not those in the amino- or carboxy-terminal regions, were glycosylated, thus suggesting an extracellular placement for that domain. Our results support a two-transmembrane arrangement for P2X receptors with intracellular amino- and carboxy-termini.