This study confirms that SNPs in the P2RX7 gene are responsible for GOF and LOF phenotypes of the P2X7 receptor. Both channel and pore functions were affected in the same direction for all the SNPs studied. From a mechanistic point, the functional effects of the mutants on Emax, and the results from the Western blotting assays, point to an increase in the number of receptors being expressed in the presence of the two GOF SNPs and a reduction in the presence of the LOF SNP. Additionally, our Western blotting assays demonstrated that when GOF and LOF SNPs were co-expressed in the same construct, the LOF effect was dominant. This may have relevance in some patients, as more than one SNP per P2RX7 genotype has been described in humans
. Our results are different from earlier studies
 but consistent with a recent publication
 where both 155Tyr and 348Thr function and expression were studied using different techniques. Our results are very similar to those of Bradley et al.
, with respect to 155Tyr, showing that an increased expression is driving the GOF phenotype, but not with respect to 348Thr. In our hands, like 155Tyr, 348Thr also drives more P2X7 protein to be expressed. Our own western blotting data are consistent with radioligand binding assays performed in our laboratory utilising the recently described radioligand [3H]A-804598
. By executing binding assays on intact transfected cells, we indeed found a very similar trend in up- and down-regulation of P2X7 expression with the GOF and LOF variants (A. Mogg, preliminary data). Regardless of the exact cellular mechanisms underlying the process, both our data and those from
 confirm that 348Thr also drives a GOF phenotype.
From a therapeutic point of view, understanding if these highly prevalent human P2X7 variants exhibit a differential sensitivity to small molecule P2X7 antagonists is critical. Profiling three known P2X7 antagonists, we did not identify any differences in their ability to block either the channel or pore functions of the variant receptors. The limited literature on this subject is still inconclusive, with one paper suggesting no changes with one specific P2X7 antagonist (A438079) tested at a single concentration
 and a second paper suggesting a rightward shift in potency, albeit small, with a different P2X7 antagonist, GSK1370319A
. While the reported shift was not dramatic (~6 fold) it nevertheless highlights the absolute need to profile new and emerging drug candidates for their ability to differentially block P2X7 receptors in the wider human population. This preclinical translational work can raise confidence in the ability of new P2X7 directed drugs to be effective in the wider pain patient population or, alternatively, help in tailoring the treatment to the best responders and avoid treating non-responders.
Moreover, we provide new data from an additional independent pain cohort which support recent findings by other groups of a phenotypic relevance of distinct P2X7 GOF SNPs and extend previous findings on OA and PMP to a novel neuropathic condition, DPNP.
Two GOF SNPs rs1718119 (Ala348Thr) and rs208294 (His155Tyr), were nominally associated with a GOF pain phenotype in females but not males. Homozygous female patients with either of these SNPs had higher pain ratings across pain scales, consistent with other recently reported clinical pain conditions such as PMP and OA
. The same study
 also identified a LOF SNP, Arg270His, associated with decreased pain that was not replicated in our study.
The association between genotype and pain sensitivity in the context of gender here described here for the GOF P2X7 SNP rs1718119 (Ala348Thr) are intriguing. A large body of evidence points to gender differences in the prevalence of pain disorders, with less robust data pointing to actual differences in pain intensity in a given disorder, as we are describing (for reviews see
[33, 34]). Earlier studies support our present evidence of a stronger correlation between particular SNPs and pain intensity in the female patient population, while the genetic-pain-gender links in additional relevant receptors and mediators are increasingly reported in the literature. For example, the melanocortin-1 gene has been shown to affect analgesic sensitivity in a gender-dependent manner
 and SNPs in the mu-opiod receptor are associated with higher pain sensitivity in men but not women
. The issue of when these mutations occur and why they persist also merit consideration. Clinically, certain polymorphic variants of P2RX7 may identify patients, especially women, who are at greater risk of developing osteoporosis
. Some P2X7 SNPs eg Glu496Ala, have been associated with a higher prevalence of osteoporosis in Dutch women
. Conversely, other SNPs, eg A348 were reported to be link to less bone resorption and potentially be protective against the development of osteoporosis, in Danish women
[39, 40]. At the preclinical level, P2X7 KO mice have been shown to express a gender-dependent phenotype when exocrine secretion
 or bone remodelling
 have been evaluated. In a transgenic model of ALS, mice have been shown to respond in a gender-dependent manner to a P2X7 blocker
. Overall, the previous studies and our present work support the idea that P2X7 polymorphisms play an important role in determining individual predisposition to pain sensitivity. More work is needed to understand the molecular and physiological nature and overall consequences of these associations.
While our study confirmed an association with increased pain sensitivity with the GOF Ala348Thr SNP as examined in McHugh et al. (2012), we did not detect a differential effect of GOF or LOF on a particular mode of P2X7 function, with both channel and pore activities being affected in the same way. We did not explore all possible SNPs, and our assay conditions could be slightly different from others. The findings in
 suggested that a selective LOF of the pore function, and not the channel function, is linked to reduced pain sensitivity, based on mouse models. This may also be the case in humans, but still has to be demonstrated.
There seems to be good translation between results obtained studying recombinant P2X7 receptors expressed in heterologous expression systems and in vivo findings. Cellular receptor expression levels may impact the downstream pathways activated that consequently result in the increased or decreased pain phenotypes. Indeed, important mechanistic bridging studies at the cellular level have shown that gain of function P2X7 receptors are associated with an increased release of cytokines, like IL-1β, from human peripheral blood cells challenged with ATP analogues
[23, 44]. Increased IL-1β levels are reported in the CSF of patients suffering from different types of pain conditions, including neuropathies and chronic back pain
. Thus the P2X7 polymorphisms may act through neurogenic or inflammatory mediators to enhance or dampen the processes that converge and translate into pain sensitivities.