Received for publication October 27, 2005.
Revised February 1, 2006.
Accepted for publication February 1, 2006.

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

Abstract

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 the other P2X subunits, the P2X₆ subunit cannot form homomeric receptors, and when expressed alone is retained in the endoplasmic reticulum (ER) in monomeric form (Barrera et al., 2005). However, other studies have shown that P2X₆ can form functional heteromeric receptors with P2X₂ and P2X₄ subunits. Here, we have used a combination of immunocytochemistry, surface biotinylation, and atomic force microscopy (AFM) to investigate the assembly and trafficking of the P2X₆ 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 upon its partner. Through the use of targeted mutation, we demonstrate that an uncharged region at the N-terminus of P2X₆ exerts an inhibitory effect on its assembly and export from the ER. When this region is removed, or when charge is added to it, P2X₆ forms homotrimeric assemblies, undergoes complex glycosylation and is delivered to the plasma membrane, albeit less efficiently than the P2X₂ receptor. The N-terminal mutants were, however, non-functional. Substituting the uncharged 14-amino acid N-terminal region for the equivalent region of P2X₂ increased ER retention but was not sufficient to prevent formation of functional homomeric receptors. We propose that the N-terminus of the P2X₆ subunit contributes to a mechanism that prevents the inappropriate export and plasma membrane expression of non-functional P2X receptors.

Key words: Purinergic, Purinergic, Func. analysis receptor/ion channel mutants, Fluorescence techniques, Immunocytochemistry, Mutagenesis/Chimeric approaches

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