El modelo de negocio y la oposición de las empresas tradicionales

In this thesis, I have described the successful cloning of an ATP-gated channel, P2Xg, and three proton-gated channels, ASIC a , p, and y. The corresponding ligands of these channels, ATP and protons, are major chemical activators of nociceptors and can evoke pain in humans. With respect to their tissue-specific expression, P2Xg and ASIC p may be very good targets for analgesic drug development. Below, I summarise the results of this study.

The primary sequence of P2Xg shows about 40% homology to other P2X receptors (P2X1.7) which represent a new family of ligand-gated channels with two transmembrane domains, intracellular N- and C-terminals, a large extracellular loop, and conserved cysteine residues for forming similar secondary structure. Most P2X receptors are widely expressed in both neural and non-neural tissues, except P2Xg which is exclusively expressed in sensory neurones (Table 1-3). The expression of P2X3 is further defined in a subset of sensory neurones which are mainly peripherin- positive neurones with small diameter cell bodies and slow conducting axons. Recent studies suggest that the GDNF responsive neurones which express the glycolipid marker isolectin B4 are the major sites of expression of P2Xg (S.B. McMahon, personal communication)

The most potent ligands of P2Xg are 2-methylthio-ATP» ATP> a,p-methylene-ATP which are able to activate P2Xg receptors expressing in oocytes, and evoke inward current in msec; the ligand activation can be reversibly inhibited by suramin which is an analgesic drug. Electrophysiological analysis characterised the channel as an non- selective cation channel with permeability to calcium and fast desensitisation. This desensitisation is calcium-dependent and regulated by the type II phosphatase calcineurin in oocytes.

Developmental analysis showed P2X3 transcripts are expressed as early as the period of sensory neurogenesis (El 1.5 in the rat). In E12.5 rat, P2X3 transcripts are found in

somatovisceral primary sensory neuomes which are involved in nociception, including neurones of trigeminal, facial, petrsal, nodose, and dorsal root ganglia, but not in other kinds of primary sensory neurones, such as olfactory epithelia, retina, and trigeminal mesencephalic nuclei. The embryonic expression of P2Xg transcripts is in most somatovisceral primary sensory neurones rather than a subset of neurones in adult.

The cloning of P2Xg has suggested a connection between ATP and nociception because of its exclusive expression in small diameter neurones of DRG. This idea was later confirmed by another group who showed that P2Xc;-immunostaining and ATP-induced inward current indeed appear in fluorescence-traced nociceptors of rat tooth-pulp (Cook

et al., 1997). ATP being a painful signal molecule is a very attracting idea, because of its abundant and constant storage inside cells. In humans, the painful role of ATP was demonstrated in blister-base preparation (Bleenhen & Keele, 1997). As many purinoceptors, both P2X and P2Y receptors, are found in sensory neurones, the signal transduction of the proposed ATP-induced nociception or pain may be quite complicated. P2Yj was claimed to be an ATP receptor responding to light mechanical stimulation (Nakamura & Strimatter, 1996). The rapid hydrolysis of ATP to adenosine is another complication since adenosine was also shown to have a painful role in humans and null mutant mice for the adenosine receptor Aja have recently been shown to have increased nociceptive thresholds (Ledent et al., 1997). The exclusive expression of P2Xg in sensory neurones may give a hint of its role in nociception. However, direct evidence for the hypothesis: “the activation of P2Xg by ATP results in nociception” should be carried out by constructing a null mutant of P2Xg. Only in this way, can one clearly see the role of P2Xg in nociception by behavioural tests.

Another DRG-specific ligand-gated channel is ASIC p, which is a splice variant of acid-sensing ionic channel (ASIC) (Waldmann et al., 1997a). Primary sequence of ASIC p shows that it belongs to the DEG/ENaC superfamily, whose structure is two transmembrane-spanning, with intracellular N- and C-terminals, and a larger

extracellular loop (like P2X receptors but bigger). The different cysteine composition between DEG/ENaC and P2X suggests they are structurally different receptor families (North, 1996b).

The expression of ASIC P is exclusive to sensory neurones, and in a small (about 15%) population of DRG neurones. When expressed in COS cells, ASIC p responds to extracellular low pH (pH< 6.0) by evoking an inward current with fast desensitisation. Electrophysiological analysis showed ASIC P is a proton-gated, non-selective cation channel, with calcium permeability.

Tissue acidosis has been found to play a dominant role in inflammatory pain (Steen et al., 1996). Protons can activate and sensitise nociceptors in ischemia and inflamed tissues where pH levels go down to 5.4 (Steen et al., 1995; Reeh & Steen, 1996). The proton-gated channel involved in nociception is described as a non-selective cation channel in which low pH can evoke a sustained inward current (Bevan & Geppetti, 1994). The cloning of DRG-specific ASIC p may provide a way to convey the acid signal evoking nociception. However, ASIC p alone seems not to present the novel proton-gated channel measured in DRG, according to the difference in electrophysiological properties. Heteromultimerisation of homologous subunits is commonly found with ion channels (Hollmann & Heinemann, 1994). At the moment, four proton-gated channels have been cloned, ASIC a , p, y, and DRASIC, which may be implicated in the novel proton-gated channel as a heteromultimeric channel. Apart from the possible combination amongst these proton-gated channel subunits, their interaction with structurally similar P2Xg receptor may be also worth investigating because low pH has been shown to enhance ATP-induced membrane depolarisation in sensory neurones (Li et al., 1996).

Finally, in the interest of primary targets of nociception, the cloning of the P2X3 receptor and the proton-gated channels, ASIC a , p, y, provides good candidates.

Further studies of these channels may be able to provide useful information for analgesic drug development.

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