Functional regulation of P2X6 receptors by N-linked glycosylation:: Identification of a novel αβ-methylene ATP-sensitive phenotype

Investigation of rat recombinant P2X(6) receptors has been limited because of the difficulty in obtaining functional expression in heterologous systems. In this study, we demonstrate glycosylation-dependent regulation of recombinant P2X(6) receptor function and associated conferral of a novel phenotype that is sensitive to the P2X(1) and P2X(3) receptor agonist, alphabeta-methylene ATP. In cells functionally expressing P2X(6) receptors, ATP and alphabeta-methylene ATP evoked slowly desensitizing inward currents (EC50 values, 0.5 and 0.6 muM, respectively) with slow kinetics of current decay on agonist washout. 2',3'-O-(2,4,6-trinitrophenyl ATP) and iso-pyridoxalphosphate-6-azophenyl-2'-5'-disulfonate were effective antagonists (IC50 values, 0.8 and 22 muM, respectively); however, suramin was relatively ineffective. Reverse transcription-polymerase chain reaction analysis confirmed the absence of other P2X receptor subunits. Western analysis of membrane fractions from functional and nonfunctional clones confirmed the presence of P2X(6) at the cell membrane but revealed a difference in apparent molecular mass of immunoreactive products (similar to70 and similar to60 kDa, respectively). N-glycosidase F treatment of both functional and nonfunctional receptor cell membranes increased the electrophoretic mobilities of immunoreactive products, with both proteins migrating at similar to55 kDa, demonstrating an increased level of glycosylation of the P2X(6) receptor in functional compared with nonfunctional cells. This study demonstrates that nonfunctional rat recombinant P2X(6) receptors 1) are expressed on the membrane surface of human embryonic kidney cells and 2) are glycosylated. Expression of the novel functional receptor phenotype is associated with further glycosylation, resulting in an apparently larger molecular mass. These results suggest that P2X(6) receptor subunits contribute to alphabeta-methylene ATP sensitivity.