Study On The Molecular Mechanism Of P2X7 Receptor Mediates Activation And Apoptosis In Human HEK293 Cells

P2X purinoceptors are cell membrane ion channels, gated by adenosine 5'-triphosphate (ATP) and other nucleotides;they have been found to be widely expressed on mammalian cells, and, by means of their functional properties, can be differentiated into three sub-groups. The first group is almost equally well activated by ATP and its analogue alphabetamethyleneATP, whereas the second group is not activated by the latter compound. A third type of receptor (also called P2Z) is distinguished by the fact that repeated or prolonged agonist application leads to the opening of much larger pores, allowing large molecules to traverse the cell membrane. This increased permeability rapidly leads to cell death, and lysis.Molecular cloning studies have identified seven P2X receptor subtypes, designated P2X1-P2X7. These receptors are proteins that share 35-48% amino acid identity, and possess two putative transmembrane (TM) domains, separated by a long (-270 residues) intervening sequence, which is thought to form an extracellular loop. Around 1/4 of the residues within the loop are invariant between the cloned subtypes, including 10 characteristic cysteines.Studies of the functional properties of heterologously expressed P2X receptors, together with the examination of their distribution in native tissues, suggests they likely occur as both homo- and heteromultimers in vivo.When the P2X7 receptor is expressed, it is found to have different functional properties from those of P2X1-P2X6. Key properties of the current produced are little rectification or desensitisation, and strong potentiation of responses when the concentration of extracellular Ca²⁺ and/or Mg²⁺ are reduced. It is also found to be relatively insensitive to ATP. In certain studies, prolonged activation of expressed P2X7 receptors causes cell permeabilisation, and lysis, suggesting that the P2X7 receptor may correspond to the native P2Z receptor, which responds similarly.The clone of rat P2X7 receptor was inserted into pcDNA3-hi-EGFP vector with T4 ligase and transformed into E.coli DH5a. The positive recombinant plasmid wasselected and identified via sequence assay and restrictive enzyme digestion. The recombinant plasmid was then transfected into HEK293 cell by Lipofect 2000. The cells containing stable transformants were selected by the ability of resistance to G418. The stably transfected cell line was identified by ISH and functional examination. The eukaryotic expression vector named pcDNA3-hi-EGFP-rP2X7 was successfully constructed and the stably transfected HEK293 cell line which expressed p2X7 was obtained.We applied cDNA micraarray named human 14k gene expression array (SHC-R-HC-100-20) to achieve the expression profiles of the stably transfected HEK293 cell line treated with extrocellar ATP under different conditions. The results show that 46 genes are significant upregulated and 2 genes downregulated in the group of 0.05 mmol/L ATP;35 genes are significant upregulated and 1 genes downregulated in the group of O.lmmol/L ATP;51 genes are significant upregulated and 13 genes downregulated in the group of 0.4mmol/L ATP;98 genes are significant upregulated and 8 genes downregulated in the group of 0.8 mmol/L ATP. The data also means that with the higher level of ATP, more genes are in the trend of expression up-regulation by induced. As a result, the gene expression amount and level depend largerly on the concentration and time length of treated extrocelluar ATP.Examined by RT-PCR, we found that ATF3, BTG2, V-FOS, TGF1, HRK, BCL2, DUSPK GADD45A, FBJ, STC2, SGK genes show same expression profiles as in cDNA microarray. These genes functions cover a large range from apoptosis, inflammation, celluar signal transduction and cell cycle. Some of gene were firstly found have a closely relationship with P2X7 receptor. The transfected HEK293 cell samples treated with different concentration of extracelluar ATP was combined with the surface of the CM 10 proteinchip. Then reading data of SELDI-TOF-MS was analyzed by Blomarker Wizard Software to get a classification rule of tree, which is standard configuration that can distinguish the different protein peak. As a result, at the key M/Z values of 3551.9, 3939.1, 4127.3, 4282.1, 5353.9, 6272.3, 6541.5 show significant increasing accompanied with the high level of extracelluar ATP. On the contract, the M/Z values of 8175.2, 8286.7, 8659.3, 8692.1 show significantedcreasing at the same time.The present study use HEK293 model stabily transfected with rat P2X7 receptor to analysis the cDNA and protein expression change after treatment with extracelluar ATP as a ligand under different conditions. A great quantity of related results will increase the understanding of the activation and apoptosis mechanism regulated by P2X7 receptor.