A NOVEL AND EFFICIENT METHOD FOR THE STABLE EXPRESSION OF HETEROMERICION CHANNELS IN MAMMALIAN-CELLS

The 5' untranslated leader sequence from the encephalomyocarditis viru s was used to engineer bicistronic or tricistronic expression vectors encoding two subunits (P2X(2) and P2X(3)) of an ATP-gated cation chann el. Human embryonic kidney (293) and chinese hamster ovary (CHO-K1) ce lls were transfected with the vector, and stable cell lines were gener ated by single cell subcloning. Selection was made in a 96-well format on the basis of a sustained increase in intracellular calcium (fluore scence of Fluo3-loaded cells) evoked by the ATP analog alpha beta meth ylene ATP. A high proportion of transformants expressed heteromeric re ceptors containing both P2X(2) and P2X(3) subunits, as evidenced by a nondesensitizing current in response to alpha beta methylene ATP. The method is fast and simple and could be generally useful for the stable expression of heteromultimeric channel proteins.