**2.1 Molecular structure of P2X receptors**

62 Neuroscience – Dealing with Frontiers

2010b). ATP is also the dominant messenger for neuron-glia communication (Guthrie et al.,

This review summarizes recent investigations and our contribution to the knowledge about the molecular structure and mechanism of function of P2X receptors, and also how do they facilitate neurotransmitter release in the brain and secretion of hormones in pituitary.

The P2XRs are ATP-gated non-selective cation channels which are permeable to Na+, K+, Ca2+ and small organic cations (Valera et al., 1994; Egan and Khakh, 2004). The discovery of P2XRs introduced a third class of ligand-gated ion channels (North, 1996), distinct from the first class, represented by nicotinic acetylcholine receptors, γ-aminobutyric acid (GABA) receptors, glycine receptors and 5-hydroxytryptamine receptors, and the second class, represented by glutamate receptors, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) and N-methyl-D-aspartate receptors (NMDAR). In mammals, seven genes encode the P2XR subunits (North, 2002) which can form functional channels as homo- and heterotrimers (Lewis et al., 1995; Nicke et al., 1998; Stoop et al., 1999). The distinct P2XR subtypes are functionally differentiated by comparisons in their sensitivity to ATP and its analogs, antagonists and allosteric modulators. Prolonged application of ATP causes a decrease in the conductivity of some P2XRs, a process termed receptor desensitization, which is also receptor-specific (North, 2002). For P2X1R and P2X3R the rate of desensitization is very fast - within milliseconds, the P2X4R desensitizes within seconds. The P2X2R and P2X7R do not desensitize, but their pore diameter increases in the continuous presence of agonist (Virginio et al., 1999). Subunit P2X6 does not form functional homomeric channels as it is retained in the endoplasmic reticulum in the monomeric form (Barrera et al., 2005; Ormond et al., 2006), but its heteromeric assemblies are functional (Le et al., 1998; King et al., 2000). So far, three combinations of heteromeric P2X6R channels have been characterized in functional and biochemical studies: P2X1+P2X6 (Nicke et al., 2005),

Various subclasses of P2XRs are activated with different potencies to ATP and its analogs. Low micromolar concentrations of ATP activate most of P2XRs, only the P2X7R requires millimolar concetrations of ATP. The P2X1R and P2X3R, are also potently activated by αβmethylene ATP (αβmeATP) and the P2X7R by 2',3'-0´,3´-O-(4-benzoylbenzoyl)-ATP (BzATP) in micromolar range (Jacobson et al., 2002); even though BzATP is also an agonist for P2X1, P2X3 and P2X4 receptors. P2XRs are inhibited by pyridoxalphosphate-6 azophenyl-2', 4'-disulfonic acid (PPADS) and suramin, which are both non-selective P2X antagonists (Coddou et al., 2011). Pharmacological experiments revealed two distinct features of P2X4R: relative resistance to suramin and PPADS (Buell et al., 1996), and robust sensitivity to positive modulatory effect of ivermectin, a high molecular weight lipophilic compound used as an antiparasitic agent in human and veterinary medicine (Burkhart, 2000), whereas other subtypes of P2XR family are ivermectin-insensitive (Khakh et al., 1999; Jelinkova et al., 2006). Among P2XRs, the P2X7R is unique as it gradually develops permeability to organic cations, causing a sustained current growth accompanied with cell blebbing and death (Surprenant et al., 1996; Di Virgilio et al., 1998; Mackenzie et al., 2005; Adinolfi et al., 2010; Yan et al., 2010). The P2X7Rs are blocked by KN62, and reactive blue-2, and also by a more potent and selective antagonists *N*-[2-[[2-[(2–hydroxyethyl)amino]ethyl]

1999; Newman, 2003; Fields and Burnstock, 2006).

P2X2+P2X6 (King et al., 2000) and P2X4+P2X6 (Le et al., 1998).

**2. P2X receptors** 

Each P2XR subunit consists of a large extracellular domain which binds agonists, two helical transmembrane domains (TM1 and TM2) and cytoplasmic N- and C-termini (Valera et al., 1994; Egan et al., 2004). There are 10 conserved cysteine residues in the ectodomain that form five intrasubunit disulfide bonds (SS1-SS5) (Clyne et al., 2002; Ennion and Evans, 2002; Rokic et al., 2010). Using the baculovirus-Sf9 cell expression system, the P2X2R was expressed and purified, and its structure was observed using electron microscopy. These images showed that the P2X2R protein resembles an inverted three-sided pyramid 215 Å in height and 200 Å in side length (Mio et al., 2005), providing visual evidence of the trimeric composition of the P2XR family. This shape has been in principle confirmed by crystalization of zebrafish P2X4.1R (Kawate et al., 2009). Crystal structure shows that extracellular domain of P2XR trimer, rich in beta-strands, contains three non-canonical, intersubunit ATP-binding sites and three major subunit-to-subunit contacts, the role of which is still unknown. There are no contacts between the subunits at the base of the extracellular domain, proximal to the TM1 and TM2 domains; these lateral fenestration are suggested to provide a possible pathway for ions to enter and exit the channel pore (Kracun et al., 2010; Kawate et al., 2011). Crystal was solved in the absence of ATP and shows P2X4.1R in its closed state; subunit rearrangement after ATP binding and subsequent ion channel opening thus remains to be elucidated.
