Interactions between subunits in agonist activation of heteromeric P2X2/3 receptors

William J. Wilkinson1, Lin-Hua Jiang2, R. Alan North1. 1Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK. 2School of Biomedical Sciences, University of Leeds, Leeds, LS2 9JT, UK

P2X receptors are ligand-gated ion channels gated by extracellular ATP. Seven genes encode P2X subunits, which can form homo- and heteromeric channels. P2X3 homomeric and P2X2/3 heteromeric channels are predominantly found in a subset of primary sensory neurons, and inhibition of P2X 3-containing receptors has been shown to reduce nociceptive behaviour in animal models of chronic inflammatory and neuropathic pain.

Two conserved lysine residues have been identified in the P2X receptor ectodomain that may be involved in ATP binding (Jiang et al., 2000; Roberts et al., 2004). In this study we have further investigated these residues (Lys 69, Lys 308; rat P2X 2 numbering). We expressed these receptors in HEK293 cells, and measured currents evoked by ATP using whole-cell patch clamp recording.

Rat P2X2 receptors containing the single point mutations K69A or K308A gave no current in response to up to 1mM ATP. However, co-expression of these mutants caused a significant recovery in function (13 ± 2.7 pA/pF to 1 mM ATP (n=9)) compared to each mutant alone. Co-expression of either of the P2X2 mutants with P2X3 wild type subunits produced currents with a characteristic P2X2/3 phenotype. EC50 values to αβ-methylene ATP were: 27 ± 3.3 μM (P2X2/3 wild type (n=5)) 22 ± 2.6 μM ( P2X2K69A + P2X3 (n=5)) and 44 ± 2.6 m M (P2X2K308A + P2X3 (n=8)). Conversely, co-expression of wild type P2X2 subunits with P2X3 subunits carrying equivalent point mutations (K63A, K299A) did not produce functional heteromeric channels. Surprisingly, the doubly mutated P2X2 receptor (K69A, K308A) could not be “rescued” into a functional heteromer by co-expression with wild type P2X3 subunits.

The P2X2/3 receptor activated by αβ-methylene ATP contains one P2X2 subunit and two P2X3 subunits (Jiang et al., 2003). Therefore, the simplest interpretation of the rescue of a “dead” P2X2 subunit (i.e. K69A or K308A) by wild type P2X3 subunits is that only two (P2X3) subunits are required to bind the agonist (αβ-methylene ATP) for the channel to open. The finding that the double mutant P2X­2K69A, K308A cannot be rescued by P2X3 may indicate that the agonist binding sites are formed by contributions from lysines on two different subunits. In this case, mutating both the P2X2 lysines to alanine would produce a receptor with only one binding site (i.e. carrying two lysines) located in between the two P2X3 subunits, which would be insufficient for channel function.

Jinag L-H. et al., (2000) J. Biol. Chem. 275(44): 34190-6
Jiang L-H. et al., (2003) J. Neurosci. 23 (26): 8903-10
Roberts J.A. et al., (2004) J. Biol. Chem. 279 (10): 9043-55

This work was funded by the Medical Research Council and the Wellcome Trust.