The Protein Production Core's mission is to produce heterologously expressed, purified and functionally reconstituted human neuronal GABAA and glycine (GlyR) receptors in the large quantities required for photolabeling and sequencing. This is a challenging resource-intensive objective best handled by a central facility. During the current period the Core has thoroughly developed several inducible mammalian cell lines capable of expressing large quantities of GABAA receptors bearing accessible purification epitopes. With such cell lines the successive steps of solubilization, purification and reconstitution have been optimized. GABAA receptors with ?1?3 and ?1?3?2L subunit compositions are now in production and have enabled new general anesthetic sites to be found. The Core can now routinely fulfill its overall specific aim of supplying Projects 1 &2 with nanomole quantities of well characterized, purified receptors functionally reconstituted that are required for photolabeling studies. The Core has had initial success with a new cell line expressing ?4?3? GABAA receptors. The specific receptors to be produced in this way are all human neuronal members of the Cys-loop superfamily and will be produced in HEK293 TetR cells, ensuring correct processing. The focus is on the inhibitory receptors, specifically the GABAA and glycine receptors: synaptic receptors composed of GABAA subunits ?1?3?2L, and extrasynaptic receptors composed of GABAA subunits ?4?3?. In addition ?1?3 and ?4?3 receptors will act as controls. GlyR cell lines will express ?1? subunits. Selected subunits will be mutated in order to: (1) determine subunit stoichiometry using site directed spin labeling, and (2) to aid the projects distinguish between the five pseudo symmetric transmembrane intersubunit general anesthetic binding sites. A subsidiary goal is to support the needs of investigators (primarily Project 3) using electrophysiological techniques. They will be able to achieve efficiencies by using the Core's resources to design wild type and mutant constructs for their studies This latter service will not detract from the Core's main labor-intensive goal of developing stable, inducible cell lines and over-expressing human neuronal receptors for photolabeling and biochemical studies. The Core will be situated in the Mallinckrodt Pharmacology Laboratory at MGH. This Core supports the PPG's overall aim of defining the structure activity relationships of drugs that target the various different anesthetic-binding sites on GABAA and glycine (GlyR) receptors in order to improve the safety of these dangerous drugs.
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