**2.2 Role of TM2 in receptor function**

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

Facilitation of Neurotransmitter and Hormone Release by P2X Purinergic Receptors 65

network of hydrophobic interactions B, The structural model shows Tyr42 side chain interactions with residues in TM2 helix of neighboring subunit (blue). The distance between OH:Tyr42 and TM2 atoms Ile333, Ile337 and Met336 are indicated using dashed lines. The figure was made using PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre,

Fig. 1. Homology modeling of TM domains of P2X4 receptor.

**3. Modulatory effect of P2X receptors on synaptic transmission** 

Following the discovery of purinergic neurotransmission in 1972 in non-adrenergic, noncholinergic inhibitory nerves in guinea-pig taenia coli, ATP was identified as a cotransmitter in both sympathetic and parasympathetic peripheral nerves and latter also in the central nervous systems (Burnstock, 2011). Of all tissues investigated, the mammalian brain has the highest levels of purines and the greatest variety of the ATP-binding P2XRs (Buell et al., 1996; Collo et al., 1996; Seguela et al., 1996). Both neurons and glial cells release ATP and express P2XRs (Fields and Stevens, 2000; Raivich, 2005; Inoue et al., 2007) and it is common that several subtypes of P2XRs are expressed in the plasma membrane of one cell (Abbracchio et al., 2009). Neurons release ATP by exocytosis together with other neurotransmitters, such as GABA, glycine, glutamate and noradrenaline (Jo and Schlichter, 1999; Robertson et al., 2001; Sokolova et al., 2001; Jo and Role, 2002; Day et al., 1993). Glia have been shown to release ATP in response to mechanical and electrical stimulation (Newman, 2003; Burnstock, 2004), although the precise mechanisms have not been identified. The most frequent receptor forms in the brain are P2X2, P2X4, and P2X6 as well as heteromers composed of P2X2+X6, P2X4+X6, and perhaps P2X1+X4 receptors (Buell et al., 1996; Collo et al., 1996). Activation of P2X receptors by extracellular ATP acts mainly as a short-term signal but has also several long-term effects. The short-term effects involve 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) and long-term potentiation (Sim et al., 2006). Long-term (trophic) role comprise cell proliferation, differentiation and death, growth of axons during development and regeneration (Heine et al., 2006; Burnstock, 2011). Like other transmitters, ATP can

Schrödinger, LLC.).

Leu343 are important for P2X4R, in addition to Ser341 (position Thr336 in P2X2) and Ala344 (Thr339 in P2X2) (Jelinkova et al., 2008). The gating properties of P2X channels are affected by alanine mutation of conserved TM2 residue Gly342 that exhibited reduced sensitivity to ATP in the P2X2R (Li et al., 2004) and P2X4R (Jelinkova et al., 2008). This residue has been suggested to play a role in helix motion as a point of local flexibility, acting like a hinge between the lower and the upper part of TM2 (Khakh and Egan, 2005). The region between P2X2R residues Gly342 and Asp349 most probably contributes to formation of the channel pore gate (Egan et al., 1998). As mentioned above, the P2X2 and P2X7 receptors display a time- and activation-dependent increase in large cation permeability (Virginio et al., 1999). Dilation of the pore could proceed due to channel rearrangements that occur at the interface between TM1 and TM2 of neighboring subunits (Jiang et al., 2003; Khakh and Egan, 2005). Functional studies identified three TM1 residues (Phe31, Arg33 and Gln37) and six TM2 residues (Ile328, Ile332, Ser340, Gly342, Trp350 and Leu352) that might be involved in the increase of pore diameter at P2X2R (Khakh and Egan, 2005).

#### **2.3 Role of TM1 and molecular basis of calcium conductivity**

A study performed on several subtypes of P2X receptors revealed a key role for aromatic residues in the upper part of TM1 in sensitivity to agonist (Jindrichova et al., 2009). Out of several aromatic residues of TM1, Tyr42 (P2X4 numbering) is the only residue that is fully conserved among all species examined thus far (Bavan et al., 2009). Alanine or cysteine substitution of conserved TM1 tyrosine generated a constitutively active channel that exhibited enhanced ATP sensitivity in P2X2R (Haines et al., 2001a; Li et al., 2004), P2X3R (Jindrichova et al., 2009; Jindrichova et al., 2011) and P2X4R (Jelinkova et al., 2008; Jindrichova et al., 2009). This residue is important also for other receptor functions: it has been suggested to control Ca2+ permeability as an inter-pore binding site for Ca2+ in P2X2R (Samways and Egan, 2007), to link TM1 with TM2 of adjacent subunit to control P2X4R deactivation (Stojilkovic et al., 2010a) (Fig.1) or to stabilize desensitized states in P2X3R (Jindrichova et al., 2011). High Ca2+ permeability of P2X1R and P2X4R has been ascribed to negatively charged ectodomain residues glutamate and aspartate, localized near the membrane at the end of TM1 and at the beginning of TM2 (Samways and Egan, 2007). However, negatively charged residues are also present at the same positions in P2X3R and P2X7R which exhibit relatively low Ca2+ permeability (Samways and Egan, 2007) indicating that other residues are also involved in calcium conductivity of P2XRs. The dilatation of P2X2R and P2X7R channel pore is also accompanied by abnormal calcium influx.

These properties, particularly the high throughput for Ca2+ ions, account for numerous physiological functions stimulated by ATP and P2XR in the central nervous system. These involve an increase in neuronal activity (Khakh et al., 2003), potentiation of neurotransmitter release (Sperlagh et al., 2007) and stimulation of hormone secretion (luteinizing hormone, prolactin, oxytocin and vasopressin) (Kapoor and Sladek, 2000; Stojilkovic, 2009; Stojilkovic et al., 2010b).

A, Three-dimensional model and positions of the first transmembrane domains (C) and the second transmebrane domains (N) of the rat P2X4R as viewed from the extracellular side. Individual subunits are differentiated by color (gray, green and blue). The Tyr42 residue (red) is between TM1 and TM2 helices of adjacent subunits and its side chain is in close proximity to residues Ile333, I337 and Met336 (orange) from the adjacent subunit, forming a

Leu343 are important for P2X4R, in addition to Ser341 (position Thr336 in P2X2) and Ala344 (Thr339 in P2X2) (Jelinkova et al., 2008). The gating properties of P2X channels are affected by alanine mutation of conserved TM2 residue Gly342 that exhibited reduced sensitivity to ATP in the P2X2R (Li et al., 2004) and P2X4R (Jelinkova et al., 2008). This residue has been suggested to play a role in helix motion as a point of local flexibility, acting like a hinge between the lower and the upper part of TM2 (Khakh and Egan, 2005). The region between P2X2R residues Gly342 and Asp349 most probably contributes to formation of the channel pore gate (Egan et al., 1998). As mentioned above, the P2X2 and P2X7 receptors display a time- and activation-dependent increase in large cation permeability (Virginio et al., 1999). Dilation of the pore could proceed due to channel rearrangements that occur at the interface between TM1 and TM2 of neighboring subunits (Jiang et al., 2003; Khakh and Egan, 2005). Functional studies identified three TM1 residues (Phe31, Arg33 and Gln37) and six TM2 residues (Ile328, Ile332, Ser340, Gly342, Trp350 and Leu352) that might be involved in the

A study performed on several subtypes of P2X receptors revealed a key role for aromatic residues in the upper part of TM1 in sensitivity to agonist (Jindrichova et al., 2009). Out of several aromatic residues of TM1, Tyr42 (P2X4 numbering) is the only residue that is fully conserved among all species examined thus far (Bavan et al., 2009). Alanine or cysteine substitution of conserved TM1 tyrosine generated a constitutively active channel that exhibited enhanced ATP sensitivity in P2X2R (Haines et al., 2001a; Li et al., 2004), P2X3R (Jindrichova et al., 2009; Jindrichova et al., 2011) and P2X4R (Jelinkova et al., 2008; Jindrichova et al., 2009). This residue is important also for other receptor functions: it has been suggested to control Ca2+ permeability as an inter-pore binding site for Ca2+ in P2X2R (Samways and Egan, 2007), to link TM1 with TM2 of adjacent subunit to control P2X4R deactivation (Stojilkovic et al., 2010a) (Fig.1) or to stabilize desensitized states in P2X3R (Jindrichova et al., 2011). High Ca2+ permeability of P2X1R and P2X4R has been ascribed to negatively charged ectodomain residues glutamate and aspartate, localized near the membrane at the end of TM1 and at the beginning of TM2 (Samways and Egan, 2007). However, negatively charged residues are also present at the same positions in P2X3R and P2X7R which exhibit relatively low Ca2+ permeability (Samways and Egan, 2007) indicating that other residues are also involved in calcium conductivity of P2XRs. The dilatation of

P2X2R and P2X7R channel pore is also accompanied by abnormal calcium influx.

et al., 2010b).

These properties, particularly the high throughput for Ca2+ ions, account for numerous physiological functions stimulated by ATP and P2XR in the central nervous system. These involve an increase in neuronal activity (Khakh et al., 2003), potentiation of neurotransmitter release (Sperlagh et al., 2007) and stimulation of hormone secretion (luteinizing hormone, prolactin, oxytocin and vasopressin) (Kapoor and Sladek, 2000; Stojilkovic, 2009; Stojilkovic

A, Three-dimensional model and positions of the first transmembrane domains (C) and the second transmebrane domains (N) of the rat P2X4R as viewed from the extracellular side. Individual subunits are differentiated by color (gray, green and blue). The Tyr42 residue (red) is between TM1 and TM2 helices of adjacent subunits and its side chain is in close proximity to residues Ile333, I337 and Met336 (orange) from the adjacent subunit, forming a

increase of pore diameter at P2X2R (Khakh and Egan, 2005).

**2.3 Role of TM1 and molecular basis of calcium conductivity** 

network of hydrophobic interactions B, The structural model shows Tyr42 side chain interactions with residues in TM2 helix of neighboring subunit (blue). The distance between OH:Tyr42 and TM2 atoms Ile333, Ile337 and Met336 are indicated using dashed lines. The figure was made using PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.).

Fig. 1. Homology modeling of TM domains of P2X4 receptor.
