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TRPM2 and P2X7: Structure-Function and Regulation - Words | Free Paper Example
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.
As the importance of oxidative stress dissertation on permeation 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. Cellular functions can be better understood that rely on the entry of divalent cations mediated by TRPM2 channels.
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, dissertation on permeation.
P2X7 functional activity is prominent dissertation on permeation inflammation, elimination of intracellular pathogens, the release of neurotransmitters by astrocytes, and chronic inflammatory and neuropathic pain Xing Liu et al. Hence, understanding its regulation by Zn and Cu dissertation on permeation help in the better management of the diseases, dissertation on permeation.
TRPM channels are considered to be integral membrane proteins consisting of eight subunits. TRPM 2 is reported to dissertation on permeation 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, Dissertation on permeation was revealed when the coiled-coil domain was deleted or its hydrophobic residues in positions a and d of the heptad repeats Jiang, 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, 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.
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.
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, dissertation on permeation. TPM2 facilitates dissertation on permeation 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, Gln, and Asp Rong Xia et al.
However, their exact role in TRPM2 channels still needs to be confirmed Rong Xia et al. 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 dissertation on permeation ligand-gated ion channels because of its affinity for the ligand ATP acting from the extracellular milieu Lin-Hua Jiang, 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 dissertation on permeation. It is not known whether ATP activation also leads to increase impermeability to the larger cations dissertation on permeation as N -methyl-D-glucamine and the cationic propidium dye quinolinium, dissertation on permeation, 4-[ 3-methyl-2 3 H -benzoxazolylidene methyl][3- triethylammonium propyl] diiodide YO-PRO-1 Lin-Hua Jiang et al.
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. 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 3 H -benzoxazolylidene methyl][3- triethylammonium propyl]diiodide YO-PRO-1there is a need dissertation on permeation 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, But it is not known about the involvement of residues primarily involved in dissertation on permeation 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, 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, dissertation on permeation.
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.
The cell lines will be subjected to transfection using lipofectamin reagents. cDNA encoding the hTRPM2 protein with a C-terminal Glu-Glu or EE epitope tag EYMPME will be subcloned in pcDNA3 vector. Overlapping extension and site-directed mutagenesis will be performed to enable the deletion of the coiled-coil domain residues from dissertation on permeation Whole-cell recordings will be carried out using an Axopatch B amplifier at mV.
Immunoprecipitation will be carried out to obtain the targeted protein. Cells will also be subjected to biotin-labelling and immunoprecipitation with anti-FLAG M2 antibody Zhu-Zhong Mei et al. Finally, the immunoprecipitated proteins obtained from the cell lysates will be detected by Western blotting. In another experiment, constructs encoding human TRPM2 will be used. Mutations will be introduced and confirmed by sequencing. The products will be then inserted into pCR 2.
The vector sequence will be then excised from the anticipated PCR product, TRPM2N-pCR2. Biotin labelling, western blotting and whole-cell recording will be performed. The relative permeability to calcium and magnesium will be derived using the Goldman Hodgkin-Katz equation Rong Xia et al, dissertation on permeation.
Next, for determining PX27 receptor-mediated pore formation, human embryonic kidney HEK cells expressing P2X7 receptors will be obtained, as mentioned in a previous description. However, lipofectamine will be used for transfecting wild-type and mutant rat P2X7 receptors.
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. Immunohistochemistry will be performed. The uptake of YO- PRO will be measured with a Zeiss Axiovertdissertation on permeation, and finally, concentration responses were measured. To find the regulation by zinc and copper, human embryonic kidney HEK 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. Alanine substitution introduced by site-directed mutagenesis dissertation on permeation be confirmed by sequencing. Whole-cell recordings will be carried out by maintaining cells at mV. Here, copper chloride and zinc sulphate salts will be dissolved in a standard external solution. By using a rapid solution changer RSC 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 M confocal microscope Xing Liu et al. TRPM2 and P2X7: Structure-Function and Regulation.
Table of Contents. Significance of research. Review of background of the study. Research approaches. Learn More. Our academic experts can deliver a custom essay specifically dissertation on permeation you. Use discount. Learn more. Jiang, LH. Lin-Hua Jiang, Francois Rassendren, Annmarie Surprenant, Alan North, R.
Lin-Hua Jiang, Francois Rassendren, dissertation on permeation, Amanda Mackenzie, Yi-Hong Zhang, Annmarie Surprenant, dissertation on permeation, Alan North, R. Rong Xia, dissertation on permeation, Zhu-Zhong Mei, Hong-Ju Mao, Wei Yang, Li Dong, dissertation on permeation, Helen Bradley, David J.
Beech, Lin-Hua Jiang. Xing Liu, dissertation on permeation, Annmarie Surprenant, Hong-Ju Mao, Sebastien Roger, Rong Xia, Helen Bradley, Lin-Hua Jiang. Zhu-Zhong Mei, Rong Xia, David J Beech, Lin-Hua Jiang. Zhu-Zhong Mei, Hong-Ju Mao, Lin-Hua Jiang. The following paper on TRPM2 and P2X7: Structure-Function and Regulation was written by a student and can be used for your research or references. Make sure to cite it accordingly if you wish to use it. Human Development Theories Analysis.
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