TRUNCATED VERSION OF GABA-A b3 RECEPTOR MAY LEAD TO SUPERIOR HIGH-DEFINITION STRUCTURAL DATA. Tyrone Caruthers1, John Bracamontes2, Joe Henry Steinbach2. Department of Chemistry, Washington University, St. Louis, MO.1 Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO2.
Studying the biochemical structure of ligand-gated ion channels (LGICs) can lead to insights into topics such as the use of clinical drugs, including muscle relaxants, general anesthetics and sedatives. One such receptor, the GABA-A receptor, is an inhibitory chloride ion channel that is regulated by GABA binding. However, current research has not produced sufficient high-definition structural data for this receptor. The concern of my project is producing and isolating a truncated version of this receptor in hopes to solubilize and crystallize the external ligand-binding region.
The main problem with isolating the entire GABA-A receptor is that this receptor has hydrophobic membrane-spanning regions preventing it from being sufficiently soluble for creating crystals. However, recently a soluble Acetylcholine-binding protein (AChBP) has been crystallized and this protein is a structural homolog to the ligand-binding domain of LGICs. By comparing these two proteins, one can create a truncated hydrophilic version of the GABA-A receptor by imitating the structure of AChBP through excising the hydrophobic region of the GABA-A receptor. These truncated GABA-A subunits can possibly yield the necessary crystals for an analysis with crystallography. As opposed to using AChBP as a model for the ligand-binding site, using the truncated GABA-A subunits for crystal studies is a more direct way to understand the biochemical structure and, if successful, would lead to superior structural data.
This mutant was made by oligo mutagenesis. After amplification, this mutant genetic sequence was transfected into human embryonic kidney (HEK) cells. Affinity chromatography was used to purify the protein, and it can be determined if the protein is being expressed in the cell, on the membrane, or if it is being secreted. The recombinant protein was analyzed and quantified using SDS-PAGE followed by a western plot using the appropriate antibodies.
If my project produces a positive result, this may imply a more general approach to studying the exterior ligand-binding sites of different members of the LGIC family. Also high-definition structural data of the GABA-A receptor may lead to better understanding of drug interactions and potentially could improve clinical use of anesthetics.
Works Referenced:
Brejc K,. van Dijk WJ,. Klaassen RV, Schuurmans M, Van der Oost J, Smit AB, Sixma TK (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors Nature 411: 269-276