BACULOVIRUS EXPRESSION PROVIDES DIRECT EVIDENCE FOR HETEROMERIC ASSEMBLY OF P2X(2) AND P2X(3) RECEPTORS

P2X(2) and P2X(3) are subunits of P2X receptors, cation channels opene d by binding extracellular ATP. cDNAs encoding P2X(2) and P2X(3) recep tor subunits, each with one of two C-terminal epitope tags, were clone d into baculovirus. Virally infected insect cells (Spodoptera frugiper da) expressed moderate to high levels of the corresponding proteins, a s detected by Western blotting, by the specific binding of [S-35]ATP a nd by whole-cell recordings of membrane current evoked by ATP or alpha beta methylene-ATP. In cells infected at the same time with two virus es encoding P2X(2) arid P2X(3) receptors, the two proteins could be cr oss-immunoprecipitated with antibodies specific for either of the epit ope tags. Whole-cell recordings from these cells showed that ATP and a lpha beta methylene-ATP evoked currents with agonist sensitivity and d esensitization quite distinct from those observed when P2X(2) or P2X(3 ) receptors were expressed alone. The results offer a method to expres s large amounts of P2X receptor protein, and they provide direct evide nce that P2X(2) and P2X(3) subunits assemble to form heteromeric chann els having distinct properties from those formed as nets homomers.