Our results demonstrate that a normal healthy tissue such as corneal epithelium expresses one or more P2X7
splice variants. We demonstrate that the diabetic cornea displays significantly enhanced expression of P2X7
receptor and variant transcript compared to control. Our data indicate that the P2X7
receptor in the corneal epithelium is expressed as both full-length and variant forms and displays canonical and non-canonical activity. In Ca2+
mobilization experiments, BzATP induces a canonical response in control cultures in the presence of Mg2+
, or in the absence of extracelluar Ca2+
. We demonstrated that P2X 4, 5, 6, and 7 receptor transcripts are expressed indicating that desensitization of the BzATP response following stimulation with ATP is not a P2X1
receptor mediated response since it is not present 
. In addition, we have shown that as cells transition from monolayer to stratified the relative expression of the forms shift and there is detectable uptake of ToPro-3 only in the most apical cells. These indicate a functional change in phenotype as the confluent monolayer cultures do not take up the ToPro-3 in the presence or absence of BzATP. While previous studies have found variants in inflammatory or cancerous cells, it has not been shown in healthy tissue as a potential means for a single protein to modulate its cellular phenotype.
Previous studies have described polymorphisms of P2X7
and a number of mutations have been found in inflammatory cells 
. To date, eight variants resulting from alternative splicing have been identified 
. In the corneal epithelium all 13 exons of P2X7
from genomic DNA were found to be present along with some single nucleotide polymorphisms. None were synonymous with the known loss of function or gain of function mutations previously observed. As the corneal epithelium has the ability to express a full-length transcript containing all exons, additional PCR was performed using primers that anneal to variants f and j. Since these variants lack exon 8, and the primers that were used span the deleted sequence, a product would not be detected if exon 8 were maintained. Therefore the PCR product of the expected size was amplified and the resulting product was sequenced and confirmed to be P2X7
. The region that was amplified contained a frameshift that is common to both f and j. The splicing of variant f does not lead to a C-terminal truncation but to a truncation of the N-terminus. Since BzATP causes enhanced pERK, a function of the N-terminus, it is unlikely that P2X7f
is the receptor being expressed in HCLE cells 
. This is supported by data showing that the cells do not display cytotoxicity and confluent cells do not display dye uptake, which are all characteristics attributed to the carboxyl terminus. Together these data indicate that epithelial cells express a variant form of P2X7
receptor that we hypothesize is j.
The corneal epithelium is an excellent model tissue for studying the role of the P2X7
receptor and its variants. It is avascular, and renewal of the corneal epithelium occurs via movement of basal cells upward with shedding of the outermost epithelial layer into the tear fluid or in migration after injury. Since there were changes in the expression of truncated and full-length P2X7
receptor in epithelial cells during the transition from confluent monolayers to stratified cultures, we have begun to examine a number of pathologies, specifically epithelium from age matched diabetic and non-diabetic corneas. In diabetic tissue there are often changes in the adhesion of epithelium to the basal lamina and impaired wound repair 
. We have shown altered wound repair and faulty epithelial adhesion in the P2X7−/−
. While some studies indicate that the P2X7
receptor is associated with acceleration of Type I diabetes 
, other laboratories have hypothesized that apoptosis is defective in Type I diabetes 
. Our results demonstrate that the diabetic cornea displays significantly enhanced expression of canonical P2X7
receptor compared to control along with an increase in Ki67 mRNA. These indicate that the variant mediates expression by acting as a dominant negative and may explain why others have found apoptosis to be defective in diabetes. Future studies will evaluate the presence of full-length and truncated forms along with the ability to form hetero- or homotrimers in normal and diseased tissue.
While the P2X7j
was detected in human cancerous cervical epithelial cells 
, it is not unreasonable that it would be expressed in corneal epithelium. Investigators have shown that proteins change their regulation as the epithelium migrates to heal a wound and then again when it stratifies 
. As we detect more of the 42 kDa form compared to the 75 kDa form in sparse cells and the inverse in stratified cells, the expression may depend on the state of epithelial integrity. In other words, the variant may provide the tissue flexibility as it migrates and becomes stratified during the process of wound closure and again as the cells move apically and shed. In addition the diabetic tissue displays a trend toward enhanced expression of the variant j compared to control. It is possible that the corneal epithelium expresses other variants and this will be a subject of future studies.
In other systems such as Xenopus laevis
, the P2X7
receptor lacks the C-terminus and experiments have demonstrated that it can associate in varying ratios with the full-length receptor to form a heterotrimeric receptor with altered activity 
. Still others have shown that P2X7j
hetero-oligomerizes with the full-length receptor resulting in antagonism of canonical activity 
. Our physiological and biochemical data support this premise as our crosslinking studies indicate the presence of 220 kDa homotrimers along with heterotrimers containing truncated forms. Therefore, although the full-length receptor is expressed in the corneal epithelium, the presence of truncated subunits in the trimeric assembly of the receptor could inhibit the receptor's ability to form a large pore and induce a cytotoxic response. This is further supported by the work of Becker et al. who showed that lack of a single carboxyl tail in the trimeric P2X7
assembly is dominant-negative for receptor activity 
. Together these data suggest a heterogeneous population of homo- and heterotrimeric receptors displaying a phenotype that resembles that of the heterotrimer.
The observation that corneal epithelial cells express a P2X7 variant may ensure that cell death does not regularly occur and disrupt corneal transparency and vision. While the epithelium maintains the ability to express the full-length receptor we hypothesize that in cell remodeling and/or pathology such as diabetes, that the balance is altered and the variant becomes upregulated. Further investigation into the expression and regulation of the P2X7 receptor should reveal an elegant mechanism for P2X7 to function as a multifaceted receptor that can mediate cell proliferation and migration or cell death.