N-Linked Glycosylation Is Essential for the Functional Expression of the Recombinant P2X2Receptor

Abstract

P2X receptors are integral membrane proteins that belong to the growing family of transmitter-gated ion channels. The extracellular domain of these receptors contains several consensus sequences for N-linked glycosylation that may contribute to the functional expression of the channel. We have previously reported the extracellular orientation of asparagine residues 182, 239, and 298 of the P2X₂ receptor subunit by showing that the protein is glycosylated at each site [Torres, G. E., et al. (1998) FEBS Lett. 425, 19−23 (1)]. In this study, we focused on the consequences of removing N-linked glycosylation from the P2X₂ receptor by using two different approaches, tunicamycin treatment or site-directed mutagenesis. HEK-293 cells stably transfected with the P2X₂ receptor subunit showed little or no response to ATP after tunicamycin treatment. In addition, loss of function was observed with the elimination of all three N-linked glycosylation sites from P2X₂. Cell surface labeling with biotin or indirect immunofluorescence revealed that the expression of the nonglycosylated receptors produced by either tunicamycin or site-directed mutagenesis is greatly reduced at the cell surface, indicating that the nonglycosylated P2X₂ receptors are retained inside the cell. These data provide the first direct evidence for a critical role of N-linked glycosylation in the cell surface expression of a P2X receptor subunit.