Subunit arrangement in P2X receptors

ATP-gated ionotropic receptors (P2X receptors) are distributed widely in the nervous system. For example, a hetero-oligomeric receptor containing both P2X(2) and P2X(3) subunits is involved in primary afferent sensation. Each subunit has two membrane-spanning domains. We have used disulfide bond formation between engineered cysteines to demonstrate close proximity between the outer ends of the first transmembrane domain of one subunit and the second transmembrane domain of another. After expression in HEK 293 cells of such modified P2X(2) or P2X(4) subunits, the disulfide bond formation is evident because an ATP-evoked channel opening requires previous reduction with dithiothreitol. In the hetero-oligomeric P2X(2/3) receptor the coexpression of doubly substituted subunits with wild-type partners allows us to deduce that the hetero-oligomeric channel contains adjacent P2X(3) subunits but does not contain adjacent P2X(2) subunits. The results suggest a "head-to-tail" subunit arrangement in the quaternary structure of P2X receptors and show that a trimeric P2X(2/3) receptor would have the composition P2X(2)(P2X(3))(2).