The N-methyl-D-aspartate (NMDA) receptor, a subtype of glutamate-gated ion channel, has been shown to be a major target of ethanol in the central nervous system (CNS). Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDAR GluN1 and GluN2A subunits that influence ethanol sensitivity. Among the alcohol sensitive sites, a methionine residue is highly conserved in all GluN1 and GluN2 subunits. We proposed the methionine position (Met-821) in the M4 domain of the GluN2C subunit can regulate ethanol sensitivity and ion channel gating. 14 mutations were made at the methionine position, 7 substitutions yielded functional receptors, which can influence ethanol sensitivity, glutamate potency and desensitization compared to wild type NMDAR containing GluN2C subunit. The other 7 mutations showed small spontaneous currents with apparent ethanol inhibition. This is the first time we observed that mutations in the M3, M4 domains of the NMDAR exhibited spontaneous activities. The predicted structure of the NMDAR indicates that alcohol sensitive positions in the M3-M4 intersubunit interfaces between the two subunit types interactively regulate ethanol sensitivity and ion channel gating. We proposed that the Met-821 position interact with the Gly-638 or Phe-639 position in the GluN1 M3 domain to regulate ethanol sensitivity and ion channel gating. Dual tryptophan mutants G638W/M821W and F639W/M821W showed small spontaneous currents with apparent ethanol inhibition. Tryptophan is the largest and most hydrophobic amino acid, which is predicted to disrupt the channel function, so it is not surprising that dual tryptophan mutants yielded abnormal functional receptors. To test the interaction between these two pairs of positions, cysteine mutations were made at Gly-638, Phe-639, and Met-821. Dual cysteine mutants G638C/M821C and F639C/M821C yielded functional receptors. G638C/M821C showed significant interaction with respect to ethanol inhibition, suggesting these pair of positions interactively regulate ethanol sensitivity and ion channel gating. DTT reducing experiments showed DTT-potentiated currents and increased deactivation time constant Tau in the dual cysteine mutant G638C/M821C. In the present studies, we showed that the Met-821 position involved in regulating ethanol sensitivity and ion channel gating. We also showed Gly-638 and Met-821 positions in the M3-M4 intersubunit interfaces between GluN1 and GluN2C subunits interactively regulate ethanol sensitivity. The results we observed from GluN2C-containing NMDAR are different from the previous discoveries in the NMDAR containing GluN2A subunit. The difference may mainly lies in the sequence difference between GluN2A and GluN2C M4 domains and small hydrophobic environment formed near the methionine position.
|Advisor:||Peoples, Robert W.|
|Commitee:||Buchanan, James T., Lobner, Douglas C., Peoples, Robert W.|
|School Location:||United States -- Wisconsin|
|Source:||MAI 52/06M(E), Masters Abstracts International|
|Keywords:||Alcohol action modulation, Cysteine cross-link, Glun2c, Ion channel gating, Nmda, Spontaneous gating|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be