Constitutive recycling of ionotropic receptors between the plasma membrane to intracellular compartments allows cells to regulate the surface number of receptors by changing the relative rates of internalisation or insertion. Classically, membrane proteins that are to be endocytosed are recruited to clathrin-coated pits via interactions between YXXF (single amino acid code where F is an amino acid with a bulky, hydrophobic side chain and X is any amino acid) endocytic motifs and the medium (m2) subunit of the AP-2 complex. The molecular details of how recycling ionotropic receptors link to the endocytic pathway, however, are poorly understood.

Previously we have shown that P2X₄, but not P2X₂ receptors are constitutively recycled in HEK293 cells and cultured neurones (Bobanovic et al. 2002; neonatal rats were humanely killed according to UK guidelines). Here, using chimeric P2X₄ÐP2X₂ receptors and deletion mutants of the P2X₄ receptor we found that the C-terminus of P2X₄ is important for trafficking. To identify which residues were involved we generated mutant P2X₄ receptors with alanine substitutions at various positions. Mutation of Y378, G381 and L382 inhibited internalisation of P2X₄ receptors expressed in cultured neurones. Surprisingly, a more conservative phenylanine substitution (Y378F) was equally as disruptive as an alanine substitution (Y378A) at position 378. Residues Y378, G381 and L382 therefore represent a non-canonical tyrosine-based endocytic motif (YXXGL) for the P2X₄ receptor.

Mutant receptors that were internalisation-deficient accumulated at the cell surface as assessed by antibody-labelling of surface receptors in living neurones. In addition, there was a concomitant increase in the size of the functional pool of receptors as assessed by measuring peak current densities in response to application of ATP (100 mM). Since the m2 subunit of the AP-2 complex recognises canonical YXXF endocytic motifs, we generated a cell line that stably expressed a dominant-negative m2 subunit deficient in binding of tyrosine-based endocytic motifs. In these cells there was a strong inhibition of P2X₄ receptor endocytosis and an increase in surface P2X₄ receptor expression. The non-canonical YXXGL motif described here is present in other ion channels and receptors and may be important in their recycling and maintenance of surface expression.

All procedures accord with current UK legislation.