Baculovirus Expression Provides Direct Evidence for Heteromeric Assembly of P2X2and P2X3Receptors

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Abstract
P2X2and P2X3are subunits of P2X receptors, cation channels opened by binding extracellular ATP. cDNAs encoding P2X2and P2X3receptor subunits, each with one of two C-terminal epitope tags, were cloned into baculovirus. Virally infected insect cells (Spodoptera frugiperda) expressed moderate to high levels of the corresponding proteins, as detected by Western blotting, by the specific binding of [35S]ATP and by whole-cell recordings of membrane current evoked by ATP or αβmethylene-ATP. In cells infected at the same time with two viruses encoding P2X2and P2X3receptors, the two proteins could be cross-immunoprecipitated with antibodies specific for either of the epitope tags. Whole-cell recordings from these cells showed that ATP and αβmethylene-ATP evoked currents with agonist sensitivity and desensitization quite distinct from those observed when P2X2or P2X3receptors were expressed alone. The results offer a method to express large amounts of P2X receptor protein, and they provide direct evidence that P2X2and P2X3subunits assemble to form heteromeric channels having distinct properties from those formed as homomers.