N-LINKED GLYCOSYLATION IS ESSENTIAL FOR THE FUNCTIONAL EXPRESSION OF THE RECOMBINANT P2X(2) RECEPTOR

P2X receptors are integral membrane proteins that belong to the growin g family of transmitter-gated ion channels. The extracellular domain o f these receptors contains several consensus sequences for N-Linked gl ycosylation that may contribute to the functional expression of the ch annel. We have previously reported the extracellular orientation of as paragine residues 182, 239, and 298 of the P2X(2) receptor subunit by showing that the protein is glycosylated at each site [Torres, G. E., et al. (1998) FEES Lett. 425, 19-23 (1)]. In this study, we focused on the consequences of removing N-linked glycosylation from the P2X(2) r eceptor by using two different approaches, tunicamycin treatment or si te-directed mutagenesis. HEK-293 cells stably transfected with the P2X (2) receptor subunit showed little or no response to ATP after tunicam ycin treatment. In addition, loss of function was observed with the el imination of all three N-Linked glycosylation sites from P2X(2). Cell surface labeling with biotin or indirect immunofluorescence revealed t hat the expression of the nonglycosylated receptors produced by either tunicamycin or site-directed mutagenesis is greatly reduced at the ce ll surface, indicating that the nonglycosylated P2X(2) receptors are r etained inside the cell. These data provide the first direct evidence for a critical role of N-linked glycosylation in the cell surface expr ession of a P2X receptor subunit.