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dc.creatorSanghvi, Mitesh
dc.date.available2011-02-18T23:10:32Z
dc.date.issued2008-08
dc.identifier.urihttp://hdl.handle.net/2346/19538en_US
dc.description.abstractThe 5-HT3 receptor is a member of the Cys-loop family of ligand-gated ion channels (LGICs) and mediates excitatory fast synaptic transmission in the central and peripheral nervous system. Despite the clear physiological importance of the 5-HT3 receptor, only a small number of published studies have directly examined the structure of the receptor. The principal objective of this work is to express and affinity-purify the mouse 5-HT3A receptor and then begin detailed structural characterization of the receptor. A mouse 5-HT3A receptor containing a C-terminal ƒÑ-bungarotoxin (ƒÑBgTx) pharmatope tag was constructed and stably transfected into HEK293 cells. To obtain sufficient quantities of receptor protein for affinity-purification, ƒÑBgTx-5-HT3A receptor-HEK cells were cultured in 140 mm tissue culture dishes (~1000 dishes). Typically, cells were treated with 100 ƒÝM serotonin 24 h prior to harvesting resulting in a ~ 2.5 fold increase in receptor expression. ƒÑBgTx-5-HT3A receptors were affinity-purified using an ƒÑBgTx-derivatized affinity column. The lipid-protein interface and the agonist-binding site of purified 5-HT3ARs were directly examined using photoaffinity labeling with the hydrophobic probe ([125I]TID) and [3H]5-HT, respectively. The preliminary results of these studies include: 1) [125I]TID photoincorporates into the 5-HT3A receptor and the labeling maps to two proteolytic fragments, designated V8-17K and V8-8K; 2) N-terminal sequencing of each rpHPLC-purified fragment revealed that V8-17 starts at Val195 and based on its apparent molecular weight extends through the M1, M2, and M3 transmembrane segments. V8-8K starts at Val424 and contains the M4 transmembrane segment; 3) Within the M4 transmembrane segment, [125I]TID photoincorporated into Ser451, which corresponds (in the aligned sequence) to Thr422 in the lipid-exposed face of the Torpedo muscle-type nACh receptor ƒÑ1M4 segment; 4) [3H]5-HT photoincorporates into the 5-HT3A receptor in a specific manner (MDL72222 significantly inhibits the labeling). Collectively, the results obtained so far are consistent with each of these two important LGICs displaying a high-degree of structural homology. Additional studies are in progress to further identify lipid exposed segments/residues in the M1 and M3 transmembrane segments as well as to probe the structure of the agonist binding site in the 5-HT3A receptor. The results will be compared with those previously determined for the Torpedo nAChR in order to provide a more detailed structural comparison of these two important LGICs.
dc.format.mimetypeapplication/pdf
dc.language.isoeng
dc.publisherTexas Tech Universityen_US
dc.subjectAffinity purificationen_US
dc.subjectNicotine acetylcholine (nACh) receptoren_US
dc.subject5-HT3A receptoren_US
dc.titleStructural studies with affinity-purified 5-Hydroxytryptamine-3A (5-HT3A) receptors
dc.typeDissertation
thesis.degree.namePh.D.
thesis.degree.levelDoctoral
thesis.degree.disciplineTTUHSC -- Pharmacology and Neuroscience
thesis.degree.grantorTexas Tech University
thesis.degree.departmentTTUHSC -- Pharmacology and Neuroscience
dc.contributor.committeeMemberLombardini, J. Barry
dc.contributor.committeeMemberMachu, Tina K.
dc.contributor.committeeMemberNorman, Reid L.
dc.contributor.committeeMemberUrbatsch, Ina L.
dc.contributor.committeeMemberFreeman, Are
dc.contributor.committeeChairBlanton, Michael P.
dc.degree.departmentTTUHSC -- Pharmacology and Neuroscienceen_US
dc.rights.availabilityUnrestricted.


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