The control of redox state of free thiols on ryanodine receptor (RyR) has been implicated as an important mechanism to regulate RyR channel activity and tune its responses to the physiological modulators. Both the skeletal and cardiac RyRs have been shown to be activated by S-nitrosylation of free thiols on them by a chemical process analogous to the oxidation of ¡°critical¡± or ¡°hyperreactive¡± thiols on RyR proteins. Inositol 1,4,5-triphophate receptors (IP3Rs) that control Ca2+ release from internal stores in non-excitable cells were found to be activated by oxidation, which emphasizes the redox reaction as a common mechanism to regulate intracellular Ca2+ channels. Therefore, the study on nitric oxide-mediated regulation of these ion channels will be important to understand the regulation of Ca2+ homeostasis in all cells including excitable and non-excitable cells since it regulates internal Ca2+ stores via RyR and /or IP3 receptors.
The aims were to investigate the chemical reaction underlying the thiol-oxidation and activation of ryanodine receptors (RyRs) by various types of NO donors namely authentic NO¡¤, S-nitrosothiols and other NO¡¤ species such as nitroxyl anions (i.e. HNO). The different actions of these various NO¡¤ species were used to better evaluate the physiological significance of RyR activation by biologically relevant forms of NO, to investigate the role of oxygen on these chemical reactions and to identify the critical cysteine residues involved in redox mediated regulation of RyRs.
The main findings are that RyRs are direct targets of S-nitrosothiols which trans-nitrosate hyper-reactive thiols and activate RyRs at biologically relevant concentration. In contrast, NO¡¤ gas cannot modify RyRs at biological circumstances found in cells. HNO is considerably more potent activator of RyR1 than NO¡¤, activates RyRs at nM concentrations. The study with truncated RyR1 indicated that all of transmembrane domains are located close to the C-terminus of the protein and the ¡®critical¡¯ regulatory thiols are part of conserved cysteines residing in it.
Further studies will be required to elucidate the interplay of oxidants and reductants found in the cytosolic milieu of all cells and how these activators and inhibitors act to regulate the opening and closure of Ca2+ release channels.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-11122003-081026 |
| Date | 02 February 2004 |
| Creators | Cheong, Eunji |
| Contributors | Guy Salama, William Walker, Daniel Farkas, Harvey Borovetz |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-11122003-081026/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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