ACCELERATED COMMUNICATION Role of Ectodomain Lysines in the Subunits of the Heteromeric P2X2/3 Receptor

Lysine residues near each end of the receptor ectodomain (in rat P2X2 Lys 69 and Lys) have been implicated in ATP binding to P2X receptors. We recorded membrane currents from human embryonic kidney cells expressing P2X subunits and found that lysine-to-alanine substitutions at equivalent positions in the P2X3 receptor (Lys 63 and Lys) also prevented channel function. Heteromeric P2X2/3 receptors are formed when P2X2 and P2X3 subunits are expressed together; they can be distinguished by their relatively sustained response to methylene-ATP. By coexpression of wild-type P2X3 and mutated P2X2 subunit, we found that the heteromeric P2X2/3 channel functioned normally when either lysine in the P2X2 subunit was mutated to alanine (i.e., [K69A] or [K308A]) but not when both lysines were mutated to alanine (i.e., [K69A, K308A]). However, coexpression of wild-type P2X2 with a mutated P2X3 subunit ([K68A] or [K299A]) produced no functional heteromers. The rescue of the single lysine mutant P2X2 subunit by wildtype P2X3 (but not the converse) suggests that the heteromeric channel contains one P2X2 and two P2X3 subunits and that the receptor functions essentially normally as long as two subunits are not mutated. The failure to rescue function in the P2X2 subunit with both lysines mutated by wild-type P2X3 suggests that these residues from two different subunits interact in agonist binding or channel opening. The heteromeric P2X2/3 receptor is of interest because of its predominant expression on a subset of primary afferent nerves involved in the sensation of chronic noxious damage (Jarvis, 2003), visceral distension (Vlaskovska et al., 2001), hypoxia (Gourine, 2005), and taste (Finger et al., 2005). A key role for these receptor subunits is supported by experiments using P2X3 gene knockouts (Cockayne et al., 2000, 2005), P2X3 RNA suppression (Barclay et al., 2002; Honoré et al., 2002), and pharmacological antagonists selective for P2X3 subunit-containing receptors (Jarvis et al., 2002). There is good evidence that P2X subunits form channels as trimers (Nicke et al., 1998; North, 2002; Jiang et al., 2003; Barrera et al., 2005). Like other ligand-gated channels, P2X receptors can form by homoor hetero-oligomeric assembly of subunits (for review, see North, 2002). Although both P2X2 and P2X3 subunits are able to form homomeric channels, the heteromeric P2X2/3 receptor that is also formed can be distinguished by its unique functional properties (Lewis et al., 1995). P2X2 receptors are activated by ATP but not by the analog -methylene-ATP ( meATP), and the current is largely sustained through a 2-s agonist application. P2X3 receptors are activated by both ATP and meATP, and the current desensitizes very rapidly ( 100 ms). The current through heteromeric P2X2/3 channels can be identified as the sustained component when meATP is the agonist (Lewis