Cys-loop receptors: Investigating the nicotinic acetylcholine receptor transmembrane domain and overexpression of a prokaryotic homolog (Gloeobacter violaceus Ion Channel)

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Cys-loop receptors: Investigating the nicotinic acetylcholine receptor transmembrane domain and overexpression of a prokaryotic homolog (Gloeobacter violaceus Ion Channel)

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Title: Cys-loop receptors: Investigating the nicotinic acetylcholine receptor transmembrane domain and overexpression of a prokaryotic homolog (Gloeobacter violaceus Ion Channel)
Author: Pauwels, Jonathan
Abstract: We investigated the thermal motion of the transmembrane domain in murine muscle type nicotinic acetylcholine receptors (nAChR) and developed a bacterial expression system for the overexpression and purification of a prokaryotic ligand-gated ion channel from Gloeobacter violaceus (GLIC). nAChR, along with receptors for γ-aminobutyric acid, serotonin, glycine and glutamate, is a member of the superfamily of pentameric ligand-gated ion channels known as cys-loop receptors and GLIC is a closely related bacterial homolog. In the first set of experiments, we investigated differences measured by disulfide trapping at the 20’ position in the M2 transmembrane helices of nAChR and GABAA receptors. Due to their homology, it is commonly assumed these receptors have a great deal of structural and functional similarity. At the 20’ position in GABAA receptors disulfide trapping has shown inter-subunit crosslinking between both adjacent and non-adjacent (across the channel) residues. Whereas, similar experiments at the 20’ position in nAChR only showed inter-subunit crosslinking between adjacent subunits. In this study, we attempted to elucidate the fundamental difference between nAChR and GABAA receptors that led to these divergent findings. Specifically, we engineered neutralizing residues at the 20’ position in nAChR in an attempt to promote a biochemical environment more favorable to the formation of disulfides. Next, we developed a procedure for the overexpression and purification of GLIC. As a bacterial homolog, GLIC is an invaluable asset in the study of the basic structure and function of cys-loop receptors. After comparison of BL21(DE3), C41(DE3) and C43(DE3) chemically competent cells in the transformation and expression of GLIC, we determined C41(DE3) provided the best expression system for our construct. We used centrifugation and chromatographic techniques to purify GLIC. Our procedure provides large amounts of pure protein and can easily be adapted to chimeric GLIC proteins for downstream functional and structural studies.
URI: http://hdl.handle.net/2346/45375
Date: 2012-05

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