We present evidence that residue R578 in human P2X7
is important for processing newly synthesized P2X7
in the ER where N-linked glycosylation addition/trimming and oligomerization occurs 
. Although we and other groups have demonstrated that mutations in the distal C-terminus of P2X7
result in attenuated activity and cell surface presentation 
, this is the first report to our knowledge that describes a possible mechanism by which amino acid changes in this region result in reduced function. Our data indicate there is an apparent size difference between wild-type P2X7
and the R578Q variant, and this mass difference is attributed in part to the presence of increased oligosaccharides on the R578Q mutant. This result supports the idea that mutation of R578 prevents the proper addition and/or trimming of N-linked glycosylation modifications, due to defective P2X7
processing in the ER 
contains the sequence encoding an Arg-based ER retention signal beginning at amino acid W575 that conforms to the sequence Φ/Ψ/R-R-X-R where Φ/Ψ represents an aromatic or bulky hydrophobic residue and X is any amino acid ( and 
). Our data demonstrating that mutation of R578 results in reduced activity and impaired oligomerization and modification of N-linked glycosylation is supportive of the idea that P2X7
R578Q has an ER protein processing defect. One potential explanation for why mutation of R578 attenuates P2X7
activity is that disruption of the R-X-R sequence restricts ER retention, causing P2X7
to traffic through the ER before it is properly oligomerized and its N-linked glycans are processed by N-glycan-modifying enzymes. We present several data that indicate P2X7
oligomerizes in the secretory pathway and is trafficked to the plasma membrane as a trimer: 1) P2X7
trimers are detected on the plasma membrane even in the absence of ligand stimulation () and 2) cell-permeable DSS cross-links P2X7
monomers ( and ). There are numerous reports of plasma membrane-bound receptors oligomerizing in the ER 
. Thus, we propose that both the oligomerization and N-linked glycosylation defects observed for P2X7
are attributed to inefficient processing in the ER (). As shown in , oligomerization of P2X7
appears to be independent of N-linked glycosylation, thus the defect in N-linked glycosylation addition/trimming likely does not contribute to the defect in the ability of P2X7
R578Q to trimerize. We postulate that the rate by which P2X7
R578Q travels through the ER is altered, potentially through a retention defect, contributing to both the oligomerization and glycosylation problems ().
Model of wild-type and mutant P2X7 assembly.
In contrast to the idea that P2X7 contains an R-X-R ER retention signal, mutation of R576 within the Φ/Ψ/R-R-X-R sequence did not result in reduced activity while mutation of R574 attenuated P2X7 signaling and pore formation (). These data indicate that if P2X7 contains an Arg-based ER retention signal, it does not conform to the Φ/Ψ/R-R-X-R sequence. An alternative explanation to ER retention is that R574 and R578 belong to an ER exit motif. Mutation of R578 to glutamine may prevent P2X7 from entering the golgi, allowing for the excessive addition of N-glycans that cannot be trimmed by N-glycan modifying enzymes found later in the secretory pathway. Based on the data presented, we cannot rule out the possibility that oligomerization occurs in the golgi and thus R578 may belong to an ER exit motif.
Because a large portion of P2X7 wild-type and R578Q are localized in the ER, as evident by their sensitivity to Endo H (), it is difficult to identify the stage in the secretory pathway where the R578Q defect is occurring. More studies are required to determine whether P2X7 R578Q exhibits an ER retention and/or ER exit defect and if its trafficking kinetics through the secretory pathway are indeed altered. A detailed analysis will be needed to identify the specific stage in the pathway where the R578Q variant exhibits its defect. Nonetheless, this investigation demonstrates that the distal C-terminus is critical for normal P2X7 activity and that mutation of this region causes decreased receptor function through defective N-linked glycosylation processing, oligomerization, and trafficking to and/or from the plasma membrane.
Our tunicamycin studies revealed the interesting finding that the basal state levels of P2X7
monomers are regulated by an N-linked glycosylation-dependent mechanism. As observed in , treatment with tunicamycin reduces P2X7
protein levels although the cell viability values are similar for cells treated with vehicle, and tunicamycin does not appear to reduce the levels of the loading control vinculin. Thus, it is possible that P2X7
synthesis and/or stability are regulated by a protein(s) that is N-linked glycosylated. It would be interesting to characterize the signaling networks that regulate P2X7
transcription and protein turnover as this has been largely unexplored. A recent report has shown that the Specificity protein 1 (Sp1) transcription factor regulates P2X7
levels in primary cortical neurons and astrocytes 
In summary, we provide evidence that human P2X7 residues R574 and R578 are required for BzATP-stimulated signaling and pore formation, and that the mutation of R578 to glutamine results in impaired oligomerization and the improper addition and/or trimming of N-linked glycosylation modifications, indicative of a potential processing defect in the ER. To our knowledge, these data are the first to help define the mechanism by which the P2X7 distal C-terminus contributes to normal receptor function.