More cross-talk between purinergic receptors

How does a simple molecule found in every cell exert a variety of specific actions throughout the body? The answer of course is that its actions are mediated via different receptors on different cells. A striking example is provided by ATP, which provides the energy source for all cells but also acts as an extracellular signalling molecule. It is a neurotransmitter in the central and peripheral nervous systems, and a regulator of afferent signalling, blood flow, inflammatory responses, cell proliferation and cell death (Surprenant & North, 2009). It acts upon a large number of different receptors, which can be classified into the metabotropic P2Y and ionotropic P2X receptors. The P2X receptors are multimeric proteins which can coassemble to make homotrimeric and heterotrimeric complexes with different functions. Given that the different P2X subtypes are frequently coexpressed in the same cell, the potential for mixing and matching of subunits is a physiologically important mechanism for increasing the diversity of responses mediated via this class of receptors.

The paper by Casas-Pruneda et al. (2009) in this issue of The Journal of Physiology provides evidence for a functional interaction between P2X₄ and P2X₇ receptors, both when heterologously coexpressed and for the endogenous receptors in mouse salivary acinar cells. P2X₄ receptors have a high affinity for ATP and P2X₇ receptors, a low affinity, and they are coexpressed in many different cells including immune cells, epithelia and endothelia. As homomeric receptors they differ in their signalling properties as well as their pharmacology, and there has been much speculation about whether or not they interact with each other. There is also considerable interest in these receptors as potential drug targets for a wide variety of disorders including chronic inflammatory disease, neurodegenerative disease and cancer. An understanding of the subunit composition of the native P2X₄ and P2X₇ receptors is therefore required.

Casas-Pruneda et al. (2009) examined the relative contributions of P2X₄ and P2X₇ receptors to the acinar cell currents evoked by a high concentration of ATP (5 mm). With Na⁺ as the charge-carrying ion, the acinar cell currents had a time course resembling recombinant mP2X₇ receptor currents in HEK293 cells with no obvious P2X₄ receptor-mediated component. This is not altogether unexpected because endogenous P2X₄ receptors in other cells have been shown to have a predominantly intracellular distribution (Qureshi et al. 2007). When Na⁺ was replaced with TEA⁺ in the recording solutions, however, the time course of the acinar cell currents changed; they decayed much faster in the continued presence of ATP and no longer resembled the kinetics of the recombinant mP2X₇ receptor currents recorded under similar conditions. Neither did the current resemble mP2X₄ receptor currents, which, surprisingly, were abolished in TEA⁺ solutions. This is surprising in so far as the rat isoform of P2X₄ is reported to undergo pore dilatation in the presence of ATP (Khakh et al. 1999) and the dilated I₂ state is permeable to NMDG⁺ and therefore would be expected to also be permeable to TEA⁺. Coexpression of P2X₄ with P2X₇ in HEK293 cells altered the time course of the ATP-evoked TEA⁺ currents; they decayed more rapidly than mP2X₇ receptor currents, they could not be described by the sum of currents carried by two independent populations of P2X₄ and P2X₇ receptors, and they were similar in time course to the acinar cell currents. In further experiments, Casas-Pruneda et al. (2009) showed that when mP2X₄ and mP2X₇ were coexpressed in HEK293 cells, Na⁺ currents evoked by a high concentration of ATP (5 mm) were insensitive to the P2X₄-selective positive modulator, ivermectin (IVM, 3 μm). In contrast, when mP2X₄ was expressed alone the currents were enhanced 3-fold by IVM and there was also a small potentiation of the mP2X₇ receptor currents. The lack of IVM sensitivity seen at high agonist concentration for the coexpressed recombinant receptors was reproduced in acinar cells, when currents were measured under similar conditions.

This demonstration of a functional interaction between P2X₄ and P2X₇ for both heterologously expressed and native receptors is in agreement with a recent study by Guo et al. (2007). Neither study elucidated the nature of the interaction between P2X₄ and P2X₇, which could occur within a heterotrimeric complex or between homotrimers. Coassembly of subunits to form heterotrimers is subtype specific but the molecular determinants that govern this have not yet been established. One argument against P2X₄ and P2X₇ preferentially assembling as heterotrimers is that where the distribution of these receptors has been examined, they differ in their subcellular localization; P2X₇ is predominantly at the plasma membrane whereas P2X₄ is predominantly intracellular (Qureshi et al. 2007; Boumechache et al. 2009). There is, however, some overlap in their distribution; P2X₄ receptors have been shown to traffic to and from the surface of microglia (Boumechache et al. 2009), and both receptors are found in phagosomes (Qureshi et al. 2007; Kuehnel et al. 2009).

Two recent studies investigated the subunit composition of native P2X₄ and P2X₇ receptor complexes by taking advantage of differences in their molecular mass to discriminate between homo- and heterotrimers (Nicke, 2008; Boumechache et al. 2009). Both studies conclude that the preferred assembly pathway for P2X₄ and P2X₇ is formation of homotrimers. It therefore seems likely that currents with novel characteristics are produced because of an interaction between the P2X₄ and P2X₇ homotrimers rather than because of the generation of heterotrimers. With the recent report of an ATP-signalling mechanism within phagosomes (Kuehnel et al. 2009) and the demonstration of P2X₄ and P2X₇ existing here (Qureshi et al. 2007; Kuehnel et al. 2009), it will be important to determine how these two receptors co-ordinate responses to luminal as well as extracellular ATP.