Significance of research
This investigation could enable us to understand diverse physiological and pathophysiological processes that are linked with the channel assembly and expression of TRPM 2 channels in neuronal, cardiovascular, immune, and endothelial cells (Rong Xia et al., 2008). As the importance of oxidative stress is gaining importance in health studies, this study could assist the medical professionals to probe the oxidative stress-induced activation of TRPM2 that might lead to insulin secretion, endothelial permeability, and cell death (Rong Xia et al., 2008). Cellular functions can be better understood that rely on the entry of divalent cations mediated by TRPM2 channels. As TRPM 2 channels show a wide range of expression in the brain, heart, placenta, lung, kidney, spleen, and ovary, this study could become a significant landmark in the development of pharmacological agents (Zhu-Zhong Mei, Hong-Ju Mao & Lin-Hua Jiang, 2007). This research could facilitate the delivery of therapeutic agents of the ion dimension. This could be because P2X7 receptor activation mediated by ATP is central in determining the permeation pathway to a good number of small and large cations (Lin-Hua Jiang et a., 2005). P2X7 functional activity is prominent in inflammation, elimination of intracellular pathogens, the release of neurotransmitters by astrocytes, and chronic inflammatory and neuropathic pain (Xing Liu et al., 2007). Hence, understanding its regulation by Zn and Cu could help in the better management of the diseases.
Review of background of the study
TRPM channels are considered to be integral membrane proteins consisting of eight subunits. TRPM 2 is reported to be of mammalian origin and resembles voltage-gated potassium channel subunits and TRPV (TRP vanilloid) functionally. It was reported that subunit interaction mediated by the coiled-coil domain is essential for the assembly of TRPM 2 channels (Jiang, 2007). This was revealed when the coiled-coil domain was deleted or its hydrophobic residues in positions a and d of the heptad repeats (Jiang, 2007). Hence, the highly conserved C-terminal coiled-coil domain was regarded as the key molecular determinant in mediating subunit interaction that is necessary for tetrameric assembly of the TRPM channels (Jiang, 2007).
This has strengthened an earlier report that emphasized the association between the highly conserved coiled-coil domain and TRPM2 subunit interaction (Zhu-Zhong Mei et al., 2006).
Therefore, the expression patterns of proteins representing the coiled-coil domain may serve to furnish details on the importance of subunit interaction in the maintenance of channel assembly and its integrity (Zhu-Zhong Mei et al., 2006). Although these reports have focused on the functional role of coiled-coil domains as a general structural element, large studies may be required for further confirmation. Further, as TRPM2 proteins comprise an ion channel core domain and cytoplasmic NH2 and COOH termini, sequence alignment of the pore regions revealed two conserved cysteine residues at positions 996 and 1008, which are reported to be involved in the function of TRPM2 channel function (Zhu-Zhong Mei, Hong-Ju Mao & Lin-Hua Jiang, 2006). This is because SH_ groups of cysteine were thought to form disulfide bonds to maintain the tertiary structure and conformation of the channel complex during channel gating (Zhu-Zhong Mei, Hong-Ju Mao & Lin-Hua Jiang, 2006).TPM2 facilitates the permeability of Ca and other divalent cations, and this function was reported to be determined by conserved acidic or negatively charged residues in the selective filter such as Glu-960, Gln-981, and Asp-987(Rong Xia et al., 2008). However, their exact role in TRPM2 channels still needs to be confirmed (Rong Xia et al., 2008). The activation of TRPM 2 depends on adenosine diphosphoribose (ADPR) and related molecules, including NAD, H2O2 in addition to intracellular Ca2_(Zhu-Zhong Mei, 2006). However, ADPR mediated activation was reported to be crucial. Hence, its involvement needs to be investigated.
Next, the other important receptor channel to be given considerable research interest is P2X7receptors. Being formed from the assembly of subunits P2X1-P2X7, it is considered as ligand-gated ion channels because of its affinity for the ligand ATP acting from the extracellular milieu (Lin-Hua Jiang, 2000). But, the information on residues contributing to the ATP binding site was not clear.
It was reported that activation of ATP leads to the opening of an integral ion channel that is permeable to small cations (Lin-Hua Jiang et al., 2005). It is not known whether ATP activation also leads to increase impermeability to the larger cations such as N-methyl-D-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene) methyl]-1-[3-(triethylammonium) propyl] diiodide (YO-PRO-1) (Lin-Hua Jiang et al., 2005). A study on permeation is important because this would enable us to understand the opening of a permeation pathway through which nucleotides themselves could leave cells or through which large molecules could enter cells. The other significance of ATP enabled P2X7 receptor activation is the release of interleukins, cytoskeletal rearrangements, L-selectin shedding, and activation of phospholipase D and p38 MAP kinase (Lin-Hua Jiang et al., 2005). Since ATP was reported to stimulate the activation of the action-permeable channel and within several seconds the uptake of fluorescent dyes such as ethidium and quinolinium,4-[(3-methyl-2-(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonium) propyl]diiodide (YO-PRO-1), there is a need to investigate this particular functional activity of PX27 receptor. Further, divalent cations such as zinc and copper were reported to interact with the ectodomain receptor and inhibit its function (Xing Liu, 2007). But it is not known about the involvement of residues primarily involved in the inhibition process. So there is a need to conduct a study to gain insights into the vital components that influence the divalent cation to mediate PX27 inhibition. This could be because a functional alteration may affect the vital biological processes characteristic of the PX27 receptor. These are inflammation, elimination of intracellular pathogens, the release of pro-inflammatory cytokines by macrophages and release of neurotransmitters by astrocytes, and chronic inflammatory and neuropathic pain (Xing Liu, 2007).
In view of the above information, we would like to propose a project whose aims are a). To study the channel assembly and ion permeation that could help us to understand protein expression and functional activity of TRPM2 channels, and
b). To study the pore formation mechanism and the inhibitory actions of divalent cations zinc and copper concerned with the functional activity of P2X7 receptor. It is anticipated that this proposal would meet some of the earlier unaddressed issues.
Human embryonic kidney (HEK) 293 cells obtained from American Type Culture Collection (ATCC) will be maintained in Dulbecco’s modified Eagle medium (DMEM). The cell lines will be subjected to transfection using lipofectamin2000 reagents. cDNA encoding the hTRPM2 protein with a C-terminal Glu-Glu (or EE) epitope tag (EYMPME) will be subcloned in pcDNA3 vector. TRPM2 subunit will be amplified using the primers 5’-ATG GAG CCC TCA GCC CTG AGG AAA GC and 5’-GCG CTC GTT GTG GAT GAG G, for the forward and backward reactions, respectively. Overlapping extension and site-directed mutagenesis will be performed to enable the deletion of the coiled-coil domain (residues from 1167 to 1201).
Whole-cell recordings will be carried out using an Axopatch 200B amplifier at -40 mV. Immunoprecipitation will be carried out to obtain the targeted protein. The samples will be then loaded and separated in a 10 % SDS- PAGE. Cells will also be subjected to biotin-labelling and immunoprecipitation with anti-FLAG M2 antibody (Zhu-Zhong Mei et al., 2006). Finally, the immunoprecipitated proteins obtained from the cell lysates will be detected by Western blotting. By using Zamboni’s fixative cells will be fixed, and double staining will be performed (Zhu-Zhong Mei et al., 2006). In another experiment, constructs encoding human TRPM2 will be used. Mutations will be introduced and confirmed by sequencing. For this purpose, the sequence encoding part of the TRPM2 will be amplified by 5_TCTCTAGAATGGAGCCCTCAGCCCTGAGG-3_ and 5_TCAGTACAGGTAGAGCAAGGTGTCC-3_ for the forward and backward reactions, respectively. The products will be then inserted into pCR 2.1. The vector sequence will be then excised from the anticipated PCR product, TRPM2N-pCR2.1 (Rong Xia et al., 2006).
Biotin labelling, western blotting and whole-cell recording will be performed. Cells will be subjected to _40 mV, and voltage ramps with a 1-s duration from_120mVto 80mVwere applied every 5 s. The relative permeability to calcium and magnesium will be derived using the Goldman Hodgkin-Katz equation (Rong Xia et al., 2006). Next, for determining PX27 receptor-mediated pore formation, human embryonic kidney (HEK)-293 cells expressing P2X7 receptors will be obtained, as mentioned in a previous description. However, lipofectamine 2000 will be used for transfecting wild-type and mutant rat P2X7 receptors. Overlapping PCR will be used for deleting the 18-amino acid cysteine-rich (CCRSRVYPSCKCCEPCAV) region of the receptor (Lin-Hua Jiang et al., 2005). PCR amplification of overlapping fragments of P2X7 cDNA will be carried out with the help of primers that facilitate the deletion of the 54 nucleotides. Subcloning of the fragment that lacked the 54 nucleotides will be carried out and will be confirmed by sequencing. Whole-cell recordings will be carried out using the membrane potential at _60 mV. Immunohistochemistry will be performed. The uptake of YO- PRO will be measured with a Zeiss Axiovert 100, and finally, concentration responses were measured.
To find the regulation by zinc and copper, human embryonic kidney (HEK293) cells will be transfected with plasmids. Wild type and mutant rat P2X7 receptors will be engineered to comprise a C-terminal EYMPME epitope (EE tag) (Xing Liu et al., 2007). Alanine substitution introduced by site-directed mutagenesis will be confirmed by sequencing. Whole-cell recordings will be carried out by maintaining cells at -60 mV. Here, copper chloride and zinc sulphate salts will be dissolved in a standard external solution. By using a rapid solution changer RSC-160 system, the application of the agonists, zinc, and copper will be carried out. Immunocytochemistry will be performed by staining the transfected cells and using mouse primary anti-EE antibody and secondary fluorescein isothiocyanate-conjugated anti-mouse IgG antibody. Finally, the images will be captured using a Zeiss AxioVert 200M confocal microscope (Xing Liu et al., 2007).
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- Lin-Hua Jiang, Francois Rassendren, Annmarie Surprenant, Alan North, R. “Identification of Amino Acid Residues Contributing to the ATP-binding Site of a Purinergic P2X Receptor. “J Biol Chem 275.44(2000): 34190–34196.
- Lin-Hua Jiang, Francois Rassendren, Amanda Mackenzie, Yi-Hong Zhang, Annmarie Surprenant, Alan North, R.”N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X7 receptors.” Am J Physiol Cell Physiol 289(2005): C1295–C1302, 2005.
- Rong Xia, Zhu-Zhong Mei, Hong-Ju Mao, Wei Yang, Li Dong, Helen Bradley, David J. Beech, Lin-Hua Jiang. ”Identification of Pore Residues Engaged in Determining Divalent Cationic Permeation in Transient Receptor Potential Melastatin Subtype Channel.” J Biol Chem 283.41, 27426–27432.
- Xing Liu, Annmarie Surprenant, Hong-Ju Mao, Sebastien Roger, Rong Xia, Helen Bradley, Lin-Hua Jiang. “Identification of key residues coordinating functional inhibition of P2X7receptors by zinc and copper.” Molecular Pharmacology Fast Forward (2007): doi:10.1124/mol.107.039651.
- Zhu-Zhong Mei, Rong Xia, David J Beech, Lin-Hua Jiang. “Intracellular coiled-coil domain engaged in subunit interaction and assembly of melastatin-related transient receptor potential channel 2.”J Bio Chem (2006): Manuscript M607591200.
- Zhu-Zhong Mei, Hong-Ju Mao, Lin-Hua Jiang. “Conserved cysteine residues in the pore region are obligatory for human TRPM2 channel function.” Am J Physiol Cell Physiol 291(2006): C1022–C1028.