Central P2X₄and P2X₆Channel Subunits Coassemble into a Novel Heteromeric ATP Receptor

Abstract

Ionotropic ATP receptors are widely expressed in mammalian CNS. Despite extensive functional characterization of neuronal homomeric P2X receptors in heterologous expression systems, the subunit composition of native central P2X ATP-gated channels remains to be elucidated. P2X4 and P2X6 are major central subunits with highly overlapping mRNA distribution at both regional and cellular levels. When expressed alone in Xenopus oocytes, P2X6 subunits do not assemble into surface receptors responsive to ATP applications. On the other hand, P2X4 subunits assemble into bona fide ATP-gated channels, slowly desensitizing and weakly sensitive to the partial agonist alpha,beta-methylene ATP and to noncompetitive antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. We demonstrate here that the coexpression of P2X4 and P2X6 subunits in Xenopus oocytes leads to the generation of a novel pharmacological phenotype of ionotropic ATP receptors. Heteromeric P2X4+6 receptors are activated by low-micromolar alpha, beta-methylene ATP (EC50 = 12 microM) and are blocked by suramin and by Reactive Blue 2, which has the property, at low concentrations, to potentiate homomeric P2X4 receptors. The assembly of P2X4 with P2X6 subunits results from subunit-dependent interactions, as shown by their specific copurification from HEK-293 cells transiently transfected with various epitope-tagged P2X channel subunits. Our data strongly suggest that the numerous cases of neuronal colocalizations of P2X4 and P2X6 subunits observed in mammalian CNS reflect the native expression of heteromeric P2X4+6 channels with unique functional properties.

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

“…However, the existence of a large pool of P2X4/5 heteromers in the present study is unlikely, as we failed to identify mRNA for P2X5 in C8-B4 cells. Similarly, we found no evidence of P2X6 proteins in C8-B4 cells, which together with the low potency of ,-methylene ATP strongly argues against P2X4/6 receptors in activated C8-B4 cells (Lê et al, 1998a). A recent expression study concluded that P2X4 and P2X7 subunits may also form heteromeric receptors that are blocked by micromolar concentrations of BBG (Guo et al, 2007), whereas subsequent studies indicated that P2X4 and Figure 14.…”

“…Pharmacological experiments also revealed some distinctive features of P2X4 receptors: ivermectin (IVM) was an allosteric modulator of P2X4 receptors (Khakh et al, 1999b;Priel and Silberberg, 2004), they were relatively resistant to P2X antagonists (Buell et al, 1996;Jones et al, 2000), and antidepressants reduced P2X4 responses (Nagata et al, 2009). Additionally, P2X4 receptors form functional heteromers with P2X6 subunits (Lê et al, 1998a;Ormond et al, 2006) and undergo interactions with P2X7 receptors (Guo et al, 2007;Nicke, 2008;Alqallaf et al, 2009;Boumechache et al, 2009;Casas-Pruneda et al, 2009;MurrellLagnado, 2009). Progress has also been made in understanding the cell biology of P2X4 receptors, with evidence to suggest that they undergo endocytosis and traffic to lysosomes Royle et al, 2002;Toulmé et al, 2006;Qureshi et al, 2007;Stokes and Surprenant, 2009).…”

“…work suggested that recombinant P2X4 receptors formed heteromers with P2X5 and P2X6 subunits (Lê et al, 1998a;Torres et al, 1999). However, the existence of a large pool of P2X4/5 heteromers in the present study is unlikely, as we failed to identify mRNA for P2X5 in C8-B4 cells.…”

Action Potential-Independent Release of Glutamate by Ca2+Entry through Presynaptic P2X Receptors Elicits Postsynaptic Firing in the Brainstem Autonomic Network

“…BzATP (100 M) did not exert an effect similar to that by ␣␤mATP and ATP (our unpublished observation), excluding an involvement of homomeric P2X 7 receptors (Deuchars et al, 2001). Our unpublished observation that ␣␤mATP was much more potent than ATP in increasing mEPSC frequency rules out an involvement of heteromeric P2X 4/6 receptors (Lê et al, 1998) (however, ATP is more prone to extracellular breakdown than ␣␤mATP). We therefore suggest the possibility that P2X receptors of unidentified subunit composition, or under postexpressional modification, underlie the release facilitation with P2X agonists in this study (Patel et al, 2001).…”

Go It Alone No More—P2X7 Joins the Society of Heteromeric ATP-Gated Receptor Channels: Fig. 1.

“…The unique properties of natively expressed ATP-gated channels in certain tissues can be only explained by the heteromeric pairing of particular P2XR subtypes. Current genetic, biochemical, and/or physiological evidence indicates that the extended family of functional P2X receptors includes six homomeric channels composed of P2X1, P2X2, P2X3, P2X4, P2X5, or P2X7 subunits (North, 2002) and six heteromeric channels that involve subunit pairings of P2X1/P2X2 (Brown et al, 2002), P2X1/P2X4 (Nicke et al, 2005), P2X1/P2X5 (Torres et al, 1998), P2X2/P2X3 (Lewis et al, 1995), P2X2/P2X6 (King et al, 2000), or P2X4/P2X6 (Lê et al, 1998). Thus, all P2XR subtypes-with the salient exception of P2X7R-have previously been implicated in the assembly of heteromeric ATPgated ion channels that can comprise unique pharmacological targets in different tissues.…”