**Facilitation of Neurotransmitter and Hormone Release by P2X Purinergic Receptors**

Vojtech Vavra, Anirban Bhattacharya, Marie Jindrichova and Hana Zemkova *Institute of Physiology of the Academy of Sciences of the Czech Republic, Czech Republic* 

#### **1. Introduction**

60 Neuroscience – Dealing with Frontiers

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Nucleotides are emerging as ubiquitous family of extracellular signaling molecules. Their effects are mediated through a specific class of plasma membrane receptors called purinergic P2 receptors that, according to the molecular structure, are further subdivided into two subfamilies: P2Y and P2X. Specifically, P2X receptors (P2XRs) are ligand-gated ion channels, whereas P2Y receptors (P2YRs) belong to the superfamily of G-protein-coupled receptors.

Purinergic P2XRs are expressed in a wide range of organisms from amoeba to humans (Fountain et al., 2007). In mammals, seven P2X subunits (termed P2X1-7) have been found (North, 2002). These receptors appeared early in evolution and have a widespread distribution on many neurons and non-neuronal cells. The P2XRs comprise the family of trimeric channels that use the energy of extracellular ATP binding to initiate a depolarizing flux of cations, including calcium, through the pore of channels. The extracellular actions of ATP are terminated by ectonucleotidases, leading to the generation of ADP, a primary agonist for some P2YRs, and adenosine, the common agonist for adenosine subtypes of receptor (Ralevic and Burnstock, 1998). Substantial progress has been made in elucidating the roles these receptors play under physiological and pathological conditions and in our understanding of the functional, structural, and pharmacological properties of seven P2X receptor subtypes. Purinergic signaling is involved in several basic physiological responses such as embryonic and stem cell development, pain sensation, regulation of renal blood flow, inflammatory responses, auditory neurotransmission etc., whereas pathophysiology of purinergic signalling includes stroke, thrombosis, osteoporosis, kidney failure, bladder incontinence, cystic fibrosis, dry eye, cancer and brain disorders (Khakh and North, 2006; Surprenant and North, 2009; Burnstock, 2011). In excitable cells, P2XR activation causes an increase in the cytosolic Ca2+ concentration via two distinct mechanisms: by membrane depolarization resulting in voltage-dependent Ca2+ entry and by Ca2+ entry through the P2XR itself. The role of P2XR involves fast synaptic transmission mediated by ATP in both the peripheral (Evans et al., 1992) and central nervous systems (Edwards et al., 1992), modulation of neuronal excitability (Khakh and Henderson, 1998), long-term potentiation (Sim et al., 2006), and stimulation of hormone secretion (luteinizing hormone, prolactin, oxytocin and vasopressin) (Kapoor and Sladek, 2000; Stojilkovic, 2009; Stojilkovic et al.,

Facilitation of Neurotransmitter and Hormone Release by P2X Purinergic Receptors 63

amino]-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide dihydrochloride (AZ 10606120) (Michel et al., 2007) and 3-[[5-(2,3-dichlorophenyl)-1*H*-tetrazol-1-yl]methyl] pyridine

The P2XR subtypes are functionally differentiated also by comparisons in their calcium permeability which is relatively high, in the range from 2.7 % (P2X3R) to 12.4 % (P2X1R) of

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

Functional studies predict that both transmembrane helices move during gating (Li et al., 2004; Silberberg et al., 2005) and the P2XR apparently forms a parallel six-helix bundle, in the center of which is an aqueous cavity (Duckwitz et al., 2006; Li et al., 2011). While TM2 plays a key role in the formation of the ion pore and selectivity filter during receptor activation (Rassendren et al., 1997; Egan et al., 1998; Haines et al., 2001b; Haines et al., 2001a; Jiang et al., 2001; Migita et al., 2001; Li et al., 2004; Khakh and Egan, 2005; Silberberg et al., 2005; Kawate et al., 2009; Kracun et al., 2010) and is also critical as a hydrophobic anchor by which the receptor is fixed in the membrane (Torres et al., 1999), a contribution from TM1 to channel gating has also been suggested (Haines et al., 2001b; Haines et al., 2001a; Jiang et al., 2001; Samways et al., 2008; Jindrichova et al., 2009). In particular, TM2 residues Thr336, Thr339 and Ser340 (P2X2R numbering) contribute to formation of the selective filter, the narrow region in the channel pore, (Migita et al., 2001; Egan and Khakh, 2004). Conserved TM2 residue Asp355 (P2X5 numbering, human receptor form) has also been shown to be important for this function and it initiates oligomerization of subunits in the membrane (Duckwitz et al., 2006). It is clear that different residues are involved in the formation of selectivity filter of the other P2XR subtypes because their transmembrane helices are only 39-55% identical with the P2X2 subunit (North, 2002). For example, residues Gly340 and

hydrochloride (A 438079) (Nelson et al., 2006).

**2.1 Molecular structure of P2X receptors** 

channel opening thus remains to be elucidated.

**2.2 Role of TM2 in receptor function** 

total ATP-induced current (Egan and Khakh, 2004).

2010b). ATP is also the dominant messenger for neuron-glia communication (Guthrie et al., 1999; Newman, 2003; Fields and Burnstock, 2006).

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.

### **2. P2X receptors**

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

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] amino]-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide dihydrochloride (AZ 10606120) (Michel et al., 2007) and 3-[[5-(2,3-dichlorophenyl)-1*H*-tetrazol-1-yl]methyl] pyridine hydrochloride (A 438079) (Nelson et al., 2006).

The P2XR subtypes are functionally differentiated also by comparisons in their calcium permeability which is relatively high, in the range from 2.7 % (P2X3R) to 12.4 % (P2X1R) of total ATP-induced current (Egan and Khakh, 2004).
