ANESTHETIC MECHANISMS AND GABA-A RECEPTOR STRUCTURE

Project: Research project

Project Details

Description

The GABA-A receptors are the major inhibitory post-synaptic
receptors in the mammalian central nervous system. Many drugs used in the
induction of general anesthesia, including benzodiazepines, volatile and
intravenous anesthetics and barbiturates potentiate GABA-induced currents. The
extent of potentiation depends on the specific subunit-subtype composition of
the GABA-A receptors. Progress has been made towards identifying the binding
sites for these drugs. Mutagenesis and photoaffinity labeling studies
identified some amino acids that may form the binding sites for these drugs.
Understanding the three dimensional structure of the drug binding sites and the
conformational changes induced by drug binding, however, will require a high
-resolution, x-ray crystal structure. The crystallization of integral membrane
proteins has been a difficult problem and it is unlikely that a crystal
structure of the entire GABA receptor will be obtained in the near future. A
partial solution to obtaining high-resolution, three-dimensional structural
information about integral membrane proteins has been to produce and
crystallize the extra-membrane domains of the proteins as separate,
water-soluble proteins. The extracellular domain of the GABA receptors contains
residues that form the GABA and benzodiazepine binding sites. The major goals
of this application are 1) to identify a suitable expression system that will
overproduce the extracellular domain of the GABA receptor as a water-soluble
protein, 2) to characterize the physical and functional properties of the
extracellular domain protein and 3) to initiate crystallization trials with the
protein. We have truncated the GABA rho-1 subunit just prior to the start of
the first membrane-spanning domain. These studies have been initiated with the
rho-l subunit because it forms homomeric channels. The resultant 259 amino acid
protein is secreted from Drosophila S2 cells. Its mobility on a sucrose-density
gradient is consistent with its forming a pentamer. One hundred milligram
quantities can be produced and purified from E. coli inclusion bodies.
Conditions to refold the guanidine-solubilized protein will be investigated. If
successful the refolded protein will be characterized and used in
crystallization trials. If refolding is unsuccessful over-expression in
eukaryotic expression systems including yeast and mammalian and insect cells in
culture will be tried. While the risk of this project is high the impact would
also be high if it results in a high-resolution structure of the extracellular
domain of an ionotropic GABA receptor subunit. If this approach is successful
it will provide new understanding of the structure of the ligand binding sites
within the extracellular domain. It will also provide insights into the
conformational changes induced by agonist and drug binding and thereby provide
an understanding of anesthetic action at a molecular level.
StatusFinished
Effective start/end date9/1/008/31/03

Funding

  • National Institute of General Medical Sciences: $125,625.00
  • National Institute of General Medical Sciences: $125,625.00

ASJC

  • Structural Biology

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