The Role of Negatively Charged Amino Acids in Channel Function, pH Potentiation, and Zinc Modulation of the ATP-gated Rat P2X2 Receptor.
dc.contributor.author | Friday, Sean Celester | en_US |
dc.date.accessioned | 2008-08-25T20:58:05Z | |
dc.date.available | NO_RESTRICTION | en_US |
dc.date.available | 2008-08-25T20:58:05Z | |
dc.date.issued | 2008 | en_US |
dc.date.submitted | 2008 | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/60879 | |
dc.description.abstract | P2X receptors are a family of genes encoding seven homologous subunits which form ion channels that are permeable to Na+, K+, and Ca2+ ions upon activation by ATP. Expressed in excitable and non-excitable cells, the P2X2 receptor has been shown to have broad physiological importance in neuromuscular junction development, gustatory sensation, and the regulation of urinary bladder function. The ATP-evoked responses of P2X2 receptors are allosterically modulated by divalent zinc and protons. We have used site-directed mutagenesis and two-electrode voltage clamp electrophysiology to test whether any of the 34 extracellular aspartate and glutamate residues of the ATP-gated rat P2X2 receptor are important for general channel function, pH potentiation, or zinc modulation. All of the extracellular acidic residues tested produced functional channels with only small changes in ATP potency or maximal response. Seven candidates (D82, E85, E91, E115, D136, D209, and D281) showed significantly reduced zinc potentiation, and one candidate (E84) showed significantly reduced zinc inhibition. Further tests with cysteine versions of the eight identified candidates and thiol-reactive MTSET suggested that only D136 remains a good candidate to directly bind zinc at the potentiation site, and there were no good candidates for zinc inhibition. When the 34 alanine substituted mutants were screened for ability to potentiate to acidic pH, E63 showed significantly reduced pH potentiation as compared with wild type P2X2. Molecular level understanding of the structure-function relationship of allosteric sites of these receptors might allow for development of selective drugs that target allosteric sites, which could lead to safer therapies with fewer side effects than current approaches with neurotransmitter agonists and antagonists. | en_US |
dc.format.extent | 1436143 bytes | |
dc.format.extent | 1373 bytes | |
dc.format.mimetype | application/pdf | |
dc.format.mimetype | text/plain | |
dc.language.iso | en_US | en_US |
dc.subject | P2X Receptor | en_US |
dc.subject | MTSET | en_US |
dc.subject | Zinc and PH Modulation | en_US |
dc.subject | Extracellular Acidic Residues | en_US |
dc.title | The Role of Negatively Charged Amino Acids in Channel Function, pH Potentiation, and Zinc Modulation of the ATP-gated Rat P2X2 Receptor. | en_US |
dc.type | Thesis | en_US |
dc.description.thesisdegreename | PhD | en_US |
dc.description.thesisdegreediscipline | Molecular, Cellular, and Developmental Biology | en_US |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | en_US |
dc.contributor.committeemember | Hume, Richard I. | en_US |
dc.contributor.committeemember | Akaaboune, Mohammed | en_US |
dc.contributor.committeemember | Duan, Cunming | en_US |
dc.contributor.committeemember | Neubig, Richard Robert | en_US |
dc.contributor.committeemember | Uhler, Michael D. | en_US |
dc.subject.hlbsecondlevel | Molecular, Cellular and Developmental Biology | en_US |
dc.subject.hlbtoplevel | Science | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/60879/1/fridays_1.pdf | |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
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