Title: Towards%20a%20Molecular%20Description%20of%20the%20GABA-A%20and%20Glycine%20Neuroreceptors.%20%20Jeanette%20Hobbs
1Towards a Molecular Description of the GABA-A
and Glycine Neuroreceptors.Jeanette Hobbs
2Overview
- Introduction
- Justification
- What are GABA Glycine?
- Neurotransmitters
- Neuroreceptors
- Ligand-gated Ion Channels
- The GABA-A glycine receptor
- Benzodiazepine binding site
- Experimental - Crystallization
- Objective
- Previous attempts
- Protein Purity
- Crystallization Screens
3WHY?
- No structural data
- YET, targets of pharmacologically and clinically
important drugs, e.g. benzodiazepines (BZ).
- Bottleneck in identification of protein elements
that constitute pharmocophore sites, hampering
structure-based drug design.
4Introduction-neurotransmitters
- Chemically four classes of neurotransmitters
- Acetylcholine
- Excitatory amino acids glutamate and Inhibitory
GABA glycine - Amines seratonin, dopamine, histamine
- Neuropeptides - endorphins
- GABA is gamma-amino-butyric acid
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6 7What do Neurotransmitters do?
- Neurotransmitters ferry information from the end
of one nerve to the "beginning" of another by
activating a large molecule at the far end of the
synapse called a neuroreceptor.
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9Neuroreceptors
- Transmitter is the key GABA or glycine
- A transmitter that binds briefly to a receptor,
causing channels to open and ions to move in or
out of that neuron - ligand-gated ion channel neuroreceptors
10Neuroreceptors contd.
- Activation causes a net change in the electrical
properties (membrane potential) of that neuron
and determines its activity. - Increase in Chloride ions HYPERPOLARIZATION
- Neurons less likely to fire.
- Calming, tranquilizing, prevents us being
overwhelmed by stressful situations!
11Ligand-gated ion channel neuroreceptors
Channel closed
Cell membrane
12Ligand-gated ion channel neuroreceptors
Cl-
NT
13Ligand-gated ion channel neuroreceptors
14Ligand-gated ion channel neuroreceptors
15The GABA-A Receptor ?
- Major mammalian inhibitory neurotransmitter
receptor - Pentameric integral membrane protein containing
an ion channel selective for chloride ions.
Extracellular part Transmembrane part
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17- Target of numerous clinically used drugs, e.g.
the benzodiazepines. - Sedative, anxiolytic, anticonvulsive and
myorelaxant effects
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19Benzodiazepine Binding Site
- Allosterically stimulate the function of the
GABA-A receptor.
20Allosteric stimulation
- Binds at its own separate site away from the
active site.
21Allosteric stimulation
Cl-
Valium
Cl-
Cl-
Cl-
Cl-
22Benzodiazepine receptor-ligand interactions
23GABA-A and Benzodiazepine
- Structure of drug binding sites and differences
in different receptor isoforms are NOT KNOWN, as
is the overall receptor structure. - Do know about a homologous protein complex, the
nicotinic acetylcholine receptor (does not
recognize benzodiazepine). - However, has been studied extensively. Structure
has been determined based on - Negatively stained 2-dimensional crystals
- Electron microscope image analysis
- To 4.6 Å
24Nicotinic acetylcholine receptor at 4.6Å
resolution transverse tunnels in the channel
wall.
- Miyazawa, A., Fujiyoshi, Y., Stowell, M. Unwin,
N. J. Mol. Biol. 288, 765-786. (1999).
25Three-dimensional view obtained from the fully
averaged structure
26Cross-sections through the fully averaged
structure running along the central axis of the
receptor, and rotated about this axis to bring in
successive 5-fold related views
27Objectives for overall project
- Find structure of different conformational states
of the binding pocket for the drugs of the
benzodiazepine types. - To determine the differences of this pocket in
different receptor subtypes. - Express the receptor subunit fragments in E.coli
followed by crystallization and 3-dimensional
crystallographic analysis.
28How?
29Experimental Details
- Structural elucidation impeded by large size of
receptor. - Need a minimal domain as the first step in
dissecting the receptor structure. - 131-residue fragment in the extra cellular region
- Cys166 to Leu296 of ?1 subunit of bovine GABA-A
receptor. - Contains five residues shown to be associated
with BZ modulation of receptor function. - Full-length polypeptide of 456 amino acid
residues, the 131-residue is identical in
sequence to the corresponding region of the human
homolog.
30Heteromers of the GABA-A receptor
31Larger pore
pentamer
hexamer
Smaller pore
tetramer
32SDS PAGE of GABA-A and Glycine Sample
- Sodium Dodecyl Sulphate PolyAcrylamide Gel
Electrophoresis
33Laser Scattering for GABA-A protein residue
34For Glycine Protein residue
35Crystallization Screens
- Crystallization screens from Emerald
Biostructures-Wizard I II, and Cryo I II. - Crystal Growth Matrices are sets of 48 unique
solutions for macromolecular crystal growth.
36Wizard I II
- Highly effective random sparse matrices
-
- Sixteen different crystallants.
- Eleven different buffers, ranging from pH 4.2
to10.5, ensure a broad sampling of
crystallization space.
37Cryo I II
- Every formulation will flash-freeze to a clear
amorphous glass in liquid nitrogen or in the
cryo-stream at 100K. - Eleven different cryocrystallants and sparing use
of glycerol ensures a broad sampling of possible
cryo conditions. - Crystals can be frozen directly from their growth
chambers, avoiding the additional step of
pre-equilibration with an artificial cryo-solvent
that can damage the crystal.
38Questions ?
- Electrophoresis kit to test for purity of protein
once it has been delivered here? - Is the protein we have still OK to use gt 2 mths
old _at_ 4C? - Do we have an endless supply of the protein?