dc.creator | Lo, Wen-yi | |
dc.date.accessioned | 2020-08-23T15:52:00Z | |
dc.date.available | 2010-12-01 | |
dc.date.issued | 2008-12-01 | |
dc.identifier.uri | https://etd.library.vanderbilt.edu/etd-11242008-214043 | |
dc.identifier.uri | http://hdl.handle.net/1803/14766 | |
dc.description.abstract | This project is concerned with identifying and characterizing structural determinants of GABAA receptor biogenesis. I used flow cytometry to measure surface levels of GABAA receptor subunits on HEK293T cells coexpressing wild-type alpha1 subunits and mutant beta2 subunits containing segmental deletions or point mutations to identify beta2 subunit structural determinants that are important for biogenesis of alpha1beta2 GABAA receptors. I located novel sites in the beta2 subunit N-terminal domain and major M3-M4 cytoplasmic loop that are necessary to attain maximal surface GABAA receptor levels. I used a combination of multiple sequence alignment, glycosidase digestion, brefeldin A treatment and analytic centrifugation and demonstrated that a structural determinant (D450) at the boundary of the beta2 subunit major cytoplasmic loop and M4 transmembrane domain is conserved among all subunits of the Cys-loop superfamily and is required for receptor assembly. Furthermore, using homology modeling and glycosidase digestion, I found that beta2 subunit N-terminal residue, N104, is a glycosylation site that is located on the minus side of the subunit-subunit interaction region and that its N-glycan processing in the Golgi apparatus is affected by the incorporation of gamma2 subunits into alpha1beta2gamma2 pentamers. Together, my studies demonstrated that both beta2 subunit N-termini and M3-M4 cytoplasmic loops contain structural determinants for GABAA receptor biogenesis. | |
dc.format.mimetype | application/pdf | |
dc.subject | PNGaseF | |
dc.subject | blocking forward trafficking | |
dc.subject | sucrose density gradient | |
dc.subject | endo H | |
dc.title | Structural determinants of GABAA receptor biogenesis | |
dc.type | dissertation | |
dc.contributor.committeeMember | Robert L. Macdonald | |
dc.contributor.committeeMember | Roger J. Colbran | |
dc.contributor.committeeMember | Jerod Scott Denton | |
dc.type.material | text | |
thesis.degree.name | PHD | |
thesis.degree.level | dissertation | |
thesis.degree.discipline | Neuroscience | |
thesis.degree.grantor | Vanderbilt University | |
local.embargo.terms | 2010-12-01 | |
local.embargo.lift | 2010-12-01 | |
dc.contributor.committeeChair | Danny G. Winder | |