An uncharged region within the N terminus of the P2X6 receptor inhibits its assembly and exit from the endoplasmic reticulum

ATP-gated P2X receptors are trimeric complexes formed by the homomeric or heteromeric assembly of seven different subunits. We have shown previously that, unlike all of the other P2X subunits, the P2X 6 subunit cannot form homomeric receptors and when expressed alone is retained in the endoplasmic reticulum (ER) in monomeric form (J Biol Chem 280: 107591 10765, 2005). However, other studies have shown that P2X 6 can form functional heteromeric receptors with P2X 2 and P2X 4 subunits. In this study, we used a combination of immunocytochemistry, surface biotinylation, and atomic force microscopy to investigate the assembly and trafficking of the P2X 6 subunit, both alone and as part of a heteromer. We show that as a heteromer, it exits the ER and is either stably expressed at the cell surface or constitutively internalized, depending on its partner. Through the use of targeted mutation, we demonstrate that an uncharged region at the N terminus of P2X 6 exerts an inhibitory effect on its assembly and export from the ER. When this region is removed, or when charge is added to it, P2X 6 forms homotrimeric assemblies, undergoes complex glycosylation and is delivered to the plasma membrane, albeit less efficiently than the P2X 2 receptor. The N-terminal mutants were, however, nonfunctional. Substituting the uncharged 14-amino acid N-terminal region for the equivalent region of P2X 2 increased ER retention but was not sufficient to prevent the formation of functional homomeric receptors. We propose that the N terminus of the P2X 6 subunit contributes to a mechanism that prevents the inappropriate export and plasma membrane expression of nonfunctional P2X receptors.