Gain and loss of function of P2X7 receptors: mechanisms, pharmacology and relevance to diabetic neuropathic pain

Ursu et al. Molecular Pain 2014, 10:37 http://www.molecularpain.com/content/10/1/37 MOLECULAR PAIN RESEARCH Open Access Gain and loss of function of P2X7 receptors: mechanisms, pharmacology and relevance to diabetic neuropathic pain Daniel Ursu1†, Philip Ebert2†, Emily Langron1, Cara Ruble2, Leanne Munsie2, Wei Zou3, Bonnie Fijal2, Yue-Wei Qian2, Terry A McNearney2, Adrian Mogg1, Olivera Grubisha1, Kalpana Merchant2 and Emanuele Sher1* Abstract Background: Genetic causes of exaggerated or reduced pain sensitivity in humans are well known. Recently, single nucleotide polymorphisms (SNPs) in the gene P2RX7, coding for the ATP-gated ion channel P2X7, have been described that cause gain-of-function (GOF) and loss-of-function (LOF), respectively of this channel. Importantly, P2RX7 SNPs have been associated with more or less severe pain scores in patient suffering of post-mastectomy pain and osteoarthritis. Results: The functional consequences of some P2RX7 SNPs (rs208294 (His155Tyr), rs1718119 (Ala348Thr) and rs3751143 (Glu496Ala)) were studied in recombinant cells in vitro. Our findings suggest a correlation between GOF and LOF of P2X7 and actual channel protein expression. Both channel and pore function for these mutant P2X7 receptors changed in parallel to protein levels. On the other hand, the mutant receptors did not differ in their sensitivity to known P2X7 agonists and antagonists. We further demonstrated that in patients with diabetic peripheral neuropathic pain (DPNP), the presence of the GOF SNPs rs208294 (His155Tyr) and rs1718119 (Ala348Thr) is associated, in females, with higher pain intensity scores. Conclusions: Our present results confirm the physiological relevance of some of the SNPs in the P2RX7 gene and show that the presence of these genetic variants correlates with pain sensitivity also in a diabetic neuropathic pain patient population. Keywords: P2X receptors, Single nucleotide polymorphism, Gain-of-function, Pain Background The P2X7 receptor, coded by the P2RX7 gene, plays a critical role in mediating disparate physiological functions of extracellular ATP, including the regulation of immune responses, inflammation, bone metabolism, cell proliferation and cancer, as well as neuronal-glial cross-talk in both the peripheral and the central nervous systems [1]. More specifically, in recent years, strong evidence has been accumulating on the involvement of P2X7 receptors in various pathological neurological conditions, including inflammatory and neuropathic pain, neuroinflammation, and neurodegeneration [2-6]. * Correspondence: † Equal contributors 1 Lilly Research Centre, Eli Lilly & Co. Ltd., Sunninghill Road, GU20 6PH Windlesham, Surrey, UK Full list of author information is available at the end of the article P2X7 is a member of a family of cationic channels (P2X1-P2X7), having a homo- or hetero-trimeric stoichiometry [7]. Each of the three subunits contains two transmembrane domains (TM1 and TM2), a large extracellular loop, and intracellular N and C termini. The seven subunits comprising the P2XR family share approximately 30-40% homology in their primary sequence, but differ vastly in the length of their carboxy termini [1,8]. Several splice variants and SNPs are known for these subunits [9]. The whole P2X family is increasingly recognized as an important opportunity for novel drug discovery [10]. P2X7 receptors are activated by relatively high ATP concentrations, in the mM range, normally achieved only in the vicinity of damaged cells, in synaptic clefts, or in the context of paracrine-like cell-cell interactions. A typical, albeit not unique, property of P2X7 receptors is the ability, © 2014 Ursu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ursu et al. Molecular Pain 2014, 10:37 http://www.molecularpain.com/content/10/1/37 upon prolonged activation by ATP, to transition from a channel function, which allows the passage of only small ions such as Ca2+ or K+, to a pore function, which allows the passage of larger molecules, up to ~ 900 Da. This transition to a pore function, which can be mediated by protein-protein interactions between P2X7 and pannexin1 subunits [11], as well as changes in permeability of P2X7 itself [12], triggers a series of intracellular events and, in particular, maturation of the “inflammasome” [13]. As a consequence, activation of P2X7 receptors has been shown to trigger the maturation and/or release of very important inflammatory mediators, most notably IL1-β, TNFα and PGE2 [14-20]. P2RX7 is known to be a highly polymorphic gene [9]. P2RX7 SNPs have been recently studied also in the context of nociception. An association was found between specific SNPs in the P2RX7 gene and pain sensitivity in both mice and humans [21]. Significant variability in allodynia scores was found by analysing a large number of mouse strains subjected to the Spared Nerve Injury (SNI) model. Genetic analysis revealed that the haplotype block with the strongest correlation genome wide was within the P2RX7 gene. Further experimental work in mice demonstrated that a loss-of-function (LOF) of the mouse P2X7 receptor, specifically in its pore function, was responsible for the relative insensitivity in nociceptive testing. Similarly, pain sensitivity was linked to P2RX7 gene polymorphisms in women with post-mastectomy pain (PMP) and osteoarthritis (OA), with those carrying the gain-of-function (GOF) Tyr155 allele at rs208294 (H155Y) [22] reporting more pain than carriers of the His155 allele. Carriers of the LOF His270 allele at rs7958311 (R270H), reported less pain intensity than carriers of the Arg270 allele. In this paper we present additional functional studies of the rs208294 (H155Y) GOF variant, and extend these studies to include two additional reported SNPs of interest, the GOF rs1718119 (Ala348Thr) [23] and the LOF rs3751143 (Glu496Ala) [24]. We also generated two additional P2X7 expressing clones containing double mutations where the Ala348Thr and Glu496Ala changes were added to the Tyr155 SNP background. Our data support the idea that the functional consequences of some of these SNPs are related to changes in P2X7 cellular expression levels, with the receptors expressed maintaining similar function and pharmacology. Furthermore, we share preliminary findings of an association between GOF P2X7 SNP variants and reported pain intensity scores in a cohort of patients with diabetic peripheral neuropathic pain (DPNP). Results P2RX7 SNPs cause gain- and loss-of function phenotypes Three previousl (...truncated)