Browsing by Subject "Affinity purification"
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Item Structural Studies with Affinity-Purified 5-Hydroxytryptamine-3A (5-HT3A) Receptors(Texas Tech University, 2008-08) Sanghvi, Mitesh; Blanton, Michael P.; Lombardini, J. Barry; Machu, Tina K.; Norman, Reid L.; Urbatsch, Ina L.; Freeman, AreThe 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.Item Structural studies with affinity-purified muscle and neuronal nicotinic acetylcholine receptors(Texas Tech University, 2007-05) Hamouda, Ayman K.; Blanton, Michael P.; Lombardini, J. Barry; Popp, R. Lisa; Pressley, Thomas A.; Machu, Tina K.Nicotinic acetylcholine receptors (nAChRs) are a heterogeneous group of ligand gated ion channels that mediate the function of the endogenous neurotransmitter acetylcholine. A more refined understanding of the molecular structure of individual neuronal nAChRs is a prerequisite for the development of subtype-selective ligands. Such ligands will be valuable in the understanding and treatment of certain CNS diseases as well as nicotine addiction. Using an acetylcholine derivatized affinity column originally used for purification of Torpedo nAChR, we have purified a4b2 and a4b4 neuronal nAChRs from HEK293 cell lines that stably express a4b2 or a4b4 nAChRs respectively. The lipid-protein interface and the agonist-binding site of purified neuronal nAChRs were directly examined using photoaffinity labeling with [125I]TID and [125I]Epibatidine respectively. The preliminary results of these studies include: 1) [125I]TID photoincorporates into the M4 and M1 transmembrane segments of both a4 and b2 subunits; 2) Within a4M4/b2M4 segments, [125I]TID labeled amino acid residues a4Cys582/b2Cys445 which are identical (in the aligned sequence) to the [125I]TID-labeled residues Cys418 in the lipid exposed face of Torpedo a1M4; 3) [125I]TID also labeled a4Arg572 within a4M4 which is situated in the same position as His408 in the lipid exposed face of Torpedo a1M4; 4) Within a4M1 segment, [125I]TID labeled Cys226, Cys231 and Leu232 which correspond to [125I]TID labeled residues Cys222, Phe227 and Leu228 in the lipid exposed face of Torpedo a1M1. In similar fashion, [125I]TID labeled b2Cys220 and b2Val221 which correspond to [125I]TID labeled Cys222 and Leu223 in the lipid exposed face of Torpedo a1M1. 5) Further studies are needed to determine if [125I]TID photoincorporates into the M2 and M3 transmembrane segments; 6) Photoaffinity labeling of a4b2 and a4b4 nAChRs with [125I]Epibatidine reveals that [125I]Epibatidine photoincorporates specifically into the b4 subunit with no (or insignificant photoincorporation) within a4 and b2 subunits; 7) [125I]Epibatidine photoincorporation ion the a4 subunit was limited to the agonist binding pocket as the majority of labeling was displaceable by addition of nicotine; 8) The site of specific [125I]Epibatidine photincorporation within the b4 subunit was mapped to loop E (b4Val102-Glu131) within the complementary components of the agonist binding site.