Involvement of the carboxyl terminal of P2P2 receptors in trafficking and function

Chang Guo, Ruth D. Murrell-Lagnado. University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK.

P2X receptors are plasma membrane ion channels gated by extracellular ATP. They are widespread throughout the peripheral and central nervous systems. Each P2X receptor subunit has two transmembrane (TM) domains, a large cysteine-rich extracellular loop and intracellular amino and carboxyl termini (North RA., 2002 .) These subunits form homomeric (P2X1-5, P2X7) and heteromeric (P2X1/2, P2X1/5, P2X2/3, P2X2/6 and P2X4/6) assemblies. The carboxyl terminal, which differs for the different subtypes, is involved in the trafficking and targeting of these receptors. A 42 amino acid long region in the carboxyl terminal of P2X2 receptors has been shown to be involved in P2X2 binding to βIII tubulin. There is also a functional interaction between P2X2 receptors and other ligand-gated ion channels, such as 5-HT 3 receptors and ρ1/GABA c receptors, at the carboxyl terminal of P2X2 receptors (Gendreau S et al, 2003, Boue-Grabot E et al, 2003, Boue-Grabot E et al, 2004.)

To understand the role of carboxyl terminal in the trafficking and function of P2X2 receptors, P2X2wt and the mutants containing deletions of different parts of the carboxyl terminal (P2X2∆371-4 56, P2X2∆371-4 60, P2X2∆371-4 64 and P2X2∆371-472) were transfected into NRK cells using Lipofectamine™ 2000 (Invitrogen) and the surface expression was compared between them. Total and surface expressions were assayed by biotinylation followed by Western blots and immunofluorescence techniques. Both P2X2 carboxyl terminal spliced variants were expressed at the plasma membrane, as were all the mutants except for the P2X2∆371-472. This mutant with the largest deletion in the carboxyl terminal was mostly retained in the Golgi apparatus, although it was still functional as assessed by whole-cell patch clamp. Co-expression with P2X2wt showed that it could still assemble and traffic to the surface as a heteromeric complex. It also assembled with P2X6, but this heteromer was not expressed at the surface. Thus, the last 8 amino acids of carboxyl terminal, which are conserved in different spliced forms of P2X2 receptors, appear to be involved in the stabilization of P2X2 receptors at the cell surface.

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