Towards%20a%20Molecular%20Description%20of%20the%20GABA-A%20and%20Glycine%20Neuroreceptors.%20%20Jeanette%20Hobbs

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Towards a Molecular Description of the GABA-A
and Glycine Neuroreceptors.Jeanette Hobbs
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Overview

• 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

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WHY?

• 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.

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Introduction-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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• Glycine

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What 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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Neuroreceptors

• 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

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Neuroreceptors 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!

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Ligand-gated ion channel neuroreceptors
Channel closed
Cell membrane
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Ligand-gated ion channel neuroreceptors
Cl-
NT
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Ligand-gated ion channel neuroreceptors
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Ligand-gated ion channel neuroreceptors
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The 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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• Target of numerous clinically used drugs, e.g.
  the benzodiazepines.
• Sedative, anxiolytic, anticonvulsive and
  myorelaxant effects

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Benzodiazepine Binding Site

• Allosterically stimulate the function of the
  GABA-A receptor.

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Allosteric stimulation

• Binds at its own separate site away from the
  active site.

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Allosteric stimulation
Cl-
Valium
Cl-
Cl-
Cl-
Cl-
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Benzodiazepine receptor-ligand interactions
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GABA-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

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Nicotinic 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).

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Three-dimensional view obtained from the fully
averaged structure
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Cross-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
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Objectives 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.

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How?
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Experimental 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.

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Heteromers of the GABA-A receptor
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Larger pore
pentamer
hexamer
Smaller pore
tetramer
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SDS PAGE of GABA-A and Glycine Sample

• Sodium Dodecyl Sulphate PolyAcrylamide Gel
  Electrophoresis

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Laser Scattering for GABA-A protein residue
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For Glycine Protein residue
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Crystallization 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.

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Wizard 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.

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Cryo 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.

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Questions ?

• 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?