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Pro-inflammatory and pro-apoptotic P2X7 receptor

2.5 Purinergic mechanisms involved in pain

2.5.3 Pro-inflammatory and pro-apoptotic P2X7 receptor

In the past, most treatments for chronic pain have been directed towards silencing of neuronal signaling in nociceptive sensory nerve fibers. Nevertheless, recent discoveries in pain research have demonstrated the major role of glial and immune cells in these pathological conditions. It is now obvious that pain cannot be fully combated if neuroinflammatory pro-nociceptive signals from glia and immune cells continue to stimulate the sensory neurons. This has now become one of the important novel principles in the search for better pain-controlling therapeutics.

The primary role in pain associated neuroinflammation belongs to P2X7 receptors, which are predominantly expressed in immune and glial cells (Collo et al., 1997; Rassendren et al., 1997). Chronic activation of P2X7 receptors underlies many types of pathologic pain. One of key mechanisms implicating P2X7 receptors in neuroinflammation is the release of cytokines.

ATP activation of P2X7 receptors promotes the release of several cytokines including IL-1β (Solle et al., 2001; Chessell et al., 2005; Di Virgilio et al., 2009; Skaper et al., 2010; Weisman et al., 2012).

P2X7 receptors are widely distributed in bone tissues and have been reported to play an important role in bone signaling, in particular, in bone formation/lesion (Gartland et al., 2003;

Ke et al., 2003; Grol et al., 2012). ATP release has also been demonstrated to be critical for the induction of multinucleated macrophages by the inflammatory cytokine GM-CSF, a process that is crucial for the development of healthy osteoclasts (Lemaire et al., 2011). P2X7 receptors have been implicated in the modulation and pathophysiology of chronic pain (S McGaraughty et al., 2007) including malignant and non-malignant bone pain (Yang et al., 2015). Therefore, targeting the P2X7 receptor in bone cancers and osteoporosis, may represent a new therapeutic approach to combat uncontrolled pain. Over the last decade, a major effort

has been made to devise new specific P2X7 antagonists, some of which have been demonstrated to improve chronic pain treatments in animal models (Honore et al., 2006; S McGaraughty et al., 2007). Unfortunately, the mechanisms of action for these drugs are still poorly understood, requiring further studies in the search for effective therapeutics (Bhattacharya and Biber, 2016).

Figure 11. Chanel pore structure. (A) The drug-binding pocket narrows during P2X7 activation. Cartoon representation of the turret formed by β13 and β14 shown as the top and the side views. (B) Dot representations of the internal space through the center of the apo closed artificially truncated version of the panda P2X7 receptor (pdP2X7, top), the apo closed zfP2X4 (4DW0; middle), and the ATP-bound zfP2X4 (4DW1; bottom). Purple: > 2.3 Å; green: 1.15–2.3 Å; red: < 1.15 Å. (C) Central pore radii of the three P2X crystal structures shown in (B). Adopted from

Karasawa and Kawate, 2016

(eLife2016;5:e22153 DOI:10.7554/eLife.22153).

Previously, the term “desensitization”

was infrequently used for P2X7 receptors despite the clear evidence that there is a reversible reduction in their responses during sustained agonist application, which fits with the definition of desensitization. However, recent studies revealed a new kinetic model of P2X7 receptor activated by its specific agonist BzATP and demonstrated, that P2X7 receptors can desensitize (Khadra et al.,

2013; Yan et al., 2008a). Along with desensitization, the term ‘sensitization’ was also introduced by these authors. Sensitization is a phenomenon associated with the generation of the secondary peak (also interpreted as a large pore opening). It is important to appreciate that these two functionally opposing processes i.e. desensitization and pore opening, can mask each other (Khadra et al., 2013). In particular, P2X7 receptor desensitization has been demonstrated to appear at high agonist concentrations, but desensitization is usually masked by receptor sensitization (Khadra et al., 2013). The nature and the probability of P2X7 pore dilation was considered as one of the most intriguing functions of this receptor. This hypothesis has been developed and studied during last 20 years; however currently it is a subject of an ongoing debate triggered by the recent studies of Li et al. and Harkat et al. (Li et al., 2015; Harkat et al., 2017). This new research suggested an alternative explanation for the secondary peak generation, i.e. an ion depletion mechanism (Li et al., 2015). Moreover, it

was suggested that the P2X7 receptor can be selective to small ions and large cations simultaneously.

Very recently, the structures of hP2X3 and giant panda P2X7 (pgP2X7), tick P2X and zebrafish P2X4 (zfP2X4) receptors were published (Karasawa and Kawate, 2016; Kasuya et al., 2016, 2017; Mansoor et al., 2016) further extending our knowledge of how the structural changes occur upon agonist binding and receptor activation. These new studies described P2X receptors in various states and in the presence of different ligands, such as ATP and its analogues, providing a more detailed picture of the structural changes occurring during the receptor functional cycle. In general, a single P2X7 subunit for example of pdP2X7 receptor resembles the “dolphin-like” shape of zfP2X4 (45% identical) (Kawate et al., 2009; Hattori and Gouaux, 2012a; Karasawa and Kawate, 2016) and hP2X3 (38% identical) (Mansoor et al., 2016). However, channel opening of the P2X7 receptor, in contrast to P2X3 and P2X4 receptors, involves an additional unique conformational step: both the 'left flipper' and the turret narrow need to be present for efficient ATP binding (Karasawa and Kawate, 2016). In addition, it was found in the P2X7 receptor that there was a connection between the 'left flipper' movement after ATP binding and the pore-lining transmembrane helix through the turret, which supports movements of the upper body domain and is involved in coordinating the channel opening. Notably, the inter-subunit cavity in the upper body domain of the P2X7 receptor (formed by β13 and β14) is much wider than in zfP2X4 or in hP2X3 (Fig 8, 10 and 11) (Karasawa and Kawate, 2016; Mansoor et al., 2016). Furthermore, the turret and the cleft corresponding to the P2X7 drug-binding pocket remain relatively occluded in zfP2X4 and in hP2X3 after activation by ATP (Fig 11).

Nevertheless, subunit-specific functional properties of P2X7 receptors underlying such fundamental phenomena as desensitization and sensitization (previously referred as large pore opening) remain to be clarified. Thus, there is a clear need of further investigations of the functions mediated by the P2X7 receptor taking into account the newly developed view introduced by Li et al., and Harkat et al., however, without totally discarding the old models.

3 Aims of the Study

The main objectives of this project were to explore the basic properties of P2X3 and P2X7 receptors and the anti-nociceptive action of novel ATP-analogues.

To accomplish these objectives, five goals were established:

 To investigate agonist and antagonist properties of two novel stable synthetic ATP-analogues (AppCH2ppA and AppNHppA) on P2X3 receptors.

 To explore their potential anti-nociceptive effects in vitro and in vivo.

 To elucidate agonist and antagonist properties at P2X3 and P2X7 receptors of the ApppI, ATP-analogue, generated by NBPs.

 To test the role of extracellular Ca2 in the anti-nociceptive action of ApppI To clarify the role of the left-flipper domain of the P2X7 receptor in basic properties of this

receptor type.

4 Materials and Methods

4.1 ANIMALS AND BEHAVIORAL TESTING (STUDIES I-III)