Transcript Towards a Molecular Description of the GABA

Towards a Molecular
Description of the GABA-A
and Glycine Neuroreceptors.
Jeanette Hobbs
Overview
• Introduction
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Justification
What are GABA & Glycine?
Neurotransmitters
Neuroreceptors
Ligand-gated Ion Channels
The GABA-A & glycine receptor
Benzodiazepine binding site
• Experimental - Crystallization
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Objective
Previous attempts
Protein Purity
Crystallization Screens
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 structurebased drug design.
Introduction-neurotransmitters
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Chemically four classes of neurotransmitters:
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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
The brain stem
and spinal cord
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.
Direction of Impulse
Neuroreceptors
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Transmitter is the key – GABA or glycine
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Receptor is the lock – GABA-A or glycine
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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
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!
Ligand-gated ion channel neuroreceptors
Channel closed
ClClCl-
Cl-
ClNT neurotransmitter
ClCell membrane
Cl-
Ligand-gated ion channel neuroreceptors
Cl-
ClCl-
pore
Cl-
Cl-
Cl-
Cl-
ClCl-
ClNT
ClCl-
Cl-
ClCl-
Cl-
Cl-
Cl-
Cl-
Ligand-gated ion channel neuroreceptors
NT
Ligand-gated ion channel neuroreceptors
The GABA-A Receptor ?
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Major mammalian inhibitory neurotransmitter receptor
Pentameric integral membrane protein containing an ion channel
selective for chloride ions.
Extracellular part
Transmembrane part
• Target of numerous clinically used drugs, e.g. the
benzodiazepines.
• Sedative, anxiolytic, anticonvulsive and myorelaxant effects
Benzodiazepine Binding Site
• Allosterically stimulate the function of the GABA-A
receptor.
Allosteric stimulation
• Binds at its own separate site away from the active site.
Allosteric stimulation
NT
Cl-
ClClCl-
ClValium
Cl-
ClCl-
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ClClCl-
Cl-
Cl-
Benzodiazepine receptor-ligand interactions
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
Nicotinic acetylcholine receptor at 4.6Å resolution: transverse
tunnels in the channel wall.
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Miyazawa, A., Fujiyoshi, Y., Stowell, M. & Unwin, N. J. Mol. Biol. 288, 765786. (1999).
Three-dimensional view obtained from the fully
averaged structure
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
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.
How?
Dr. M. Goeldner
Synthetic Organic
Chemistry
Dr. J. Hobbs
Crystallization of
GABA-A
receptor residue.
Prof. P. Kuhn
Crystallography
Dr. E. Sigel
Mutagenesis,
expression,
ligand binding
studies
Dr. H. Xue
Domain
recognition,
expression and
purification
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 131residue is identical in sequence to the corresponding
region of the human homolog.
Heteromers of the GABA-A receptor
Larger
pore
pentamer
hexamer
Smaller
pore
tetramer
SDS PAGE of GABA-A and Glycine Sample
• Sodium Dodecyl Sulphate PolyAcrylamide Gel
Electrophoresis
Laser Scattering for GABA-A protein residue
For Glycine Protein residue
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.
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.
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.
Questions ?
• Electrophoresis kit to test for purity of protein once it has
been delivered here?
• Is the protein we have still OK to use > 2 mths old @ 4°C?
• Do we have an endless supply of the protein?