GABA and Glycine Receptors Flashcards
What does GABA stand for?
Gamma aminobutyric acid
What type of neurotransmitters are gaba and glycine?
Inhibitory
What is the precursor aminoacid for glycine?
Serine
Converted to glycine by serine hydroxymethyl transferase
What enzymes converts glutamate in GABA?
Glutamic acid decarboxylase
What are the types of GABA receptors?
GABAa (ionotropic anions)
GABAb (metabotropic Gi linked)
GABAc (ionotropic found in retina)
What family of receptors are glycine and GABAa in?
Cys-loop receptors
As well as nAchR
What does a subunit of a GABAa look like?
Extracellular N (ligand blinding) TM1 TM2 TM3 (Large intracellular loop site of phosphorylation to regulate channel properties) TM4 Extracellular c terminus
How many subunits come together for from a glycine and GABAa receptor?
5 (pentamers)
Glycine subunit pattern?
Alpha - beta - alpha - alpha - beta
GABAa receptors are typical formed from what subunits?
2 x alpha (same subtype)
2 x beta (same subtype)
1 x gamma
How many GABAa alpha subtypes exist?
6
How many GABAa beta subtypes exist?
4
How many GABAa gamma subtypes exist?
3
Where does GABA bind to in GABAa receptors?
The interface between the alpha and beta subunit
Therefore 2 GABA molecules are needed
Benzodiazepines are a positive allosteric modulator for the GABAa receptors…what subunits are required for BDZ to bind?
Alpha (1,2,3,5)
Gamma (2)
Bind between the alpha and gamma subunit
What percentage homologous exists between subunits in same group?
Approx 70%
What percentage homology exists between different gabaa subtype groups?
30%-40%
Alpha and gamma more closely related than alpha and beta
Subtypes of GABAa receptors can be found through many areas of the brain.
Give 2 examples
Hippocampus - alpha 1 and 5
Cerebellum (granule cells) - alpha 6
Most common beta subunits expressed in brain?
(Beta 2 and 3) > beta 1
Most common gamma subunits?
2>1>3
Why is subunit diversity important?
Different channel kinectics
Differently regulated and vary modulators effects
Different affinity got agonist
Different channel properties
Different locations - targeted to different regions of the cell
Different pharmacology
What amino acid at what position is essential for bdz binding?
What amino acid do alpha 4 and 6 have in the same position?
How can this knowledge be used to study BDZs?
Histidine at position 101 on alpha subunits 1,2,3 and 5
Alpha 4 and 6 have an arginine
Knock in mutations replacing the histidine for a different amino acid (arginine) allow for the function of bdz working on that particular subunit to be studied
What is the function of BDZs at the alpha 1 subunit responsible for?
Sedation
Anticonvulsant
What is the function of BDZs at the alpha 2 subunit responsible for?
Anxiolysis
40% of GABAa receptors have these subunits
2xalpha1
2xbeta2
1xGamma 2
Name some GABAa agonists
Gaba
Muscimol
B-alanine
Name some glycine receptor agonists
Glycine
Taurine
B-alanine
Name some GABAa antagonists
Bicuculline (competitive)
Picrotoxin (non competitive)
H+
Name a glycine receptor antagonist
Picrotoxin (non competitive)
Why is the beta subunit important for glycine receptors?
Intracellular binding to scaffolds and receptor targeting
What protein binds to one of the beta subunits of two glycine receptors?
Gephyrin
What is gephyrin and how is it important?
93kDa protein
P1 isoform interacts with beta subunit of glycine receptors
May be present with other without glycine receptors
Believed to have many functions but distribution of glycine receptors is one of them
In terms of activation, desensitisation and deactivation describe which is faster
Activation = GABAa (0.7ms)>glycine(1.3ms) Deactivation = GABAa>glycine Desensitisation = GABAa>glycine
What is the EC50 and hill slope of glycine receptor dose responce curve for glycine?
EC50=54 (micro)M
Hillslope = 1.6
What is the EC50 and hillslope for GABAa receptors for GABA?
EC50 = 45(micro)M Hillslope = 1.1
Measured in cerebellar purkinje cell
What does an upwards and downwards deflection on a current graph thing mean for GABAa receptors?
Upward current means chloride ions moving in
Downwards current means chloride ions moving out
If extracellular concentration of Cl- = 140mM
And intracellular concentration is 6mM
What is the estimated equilibrium potential?
-84.2mV meaning at RMP chloride ions will move in
What is the actual equilibrium potential for a GABAa receptor?
About -70mV
Why is there a difference in equilibrium potentials between chloride and GABAa?
GABAa receptors also permeable to bicarbonate ions
What is the extra and intracellular concentration of bicarbonate and what is the equilibrium potential?
Out = 20-30mM In= 10-20 mM E = -10mV
Meaning at RMP ions will move out
If RMP is -70mV and equilibrium potential for GABAa is -70mV, what will happen on application of GABA.
Membrane potential will remain the same
Chloride ions will move in (towards is EP), bicarbonate ions moving out (towards it EP) will balance the movement of charge (electroneural)
However, since bicarbonate will leave its associative H+ will remain in cell therefore ph will drop on GABA application
What is the difference in chloride concentrations between mature and developing neurons?
Mature = low intracellular concs
Immature = high
mature neurons have KCC2 channels which export 2 Chloride ions
What scientist discovered that kcc2 expression changes with age?
Rivera et al 1999
Used northern blotting of rat hippocampi of different ages
Shows KCC2 expression increases over time
What technique allows for intracellular chloride to be measured?
Gramicidin perforated patch
Inserts monovalent action channels into membrane which allows measurement of current
What cannot traditional whole cell patch clamp be used to measure intracellular chloride?
Pipette content will enter the cell and disrupt true intracellular chloride concentration (prevents accurate recording)
Gramicidin allows for true intracellular chloride to be measured…how?
By not dializing the cell the true chloride conc remains within the cell. Create the voltage current relationship to find equilibrium potential. If we then know the extracellular concentration of chloride (which is a variable we have set) we can use the nernst equation to work out intracellular conc
What are the implications of varying intracellular chloride concentration?
Will effect direction current will flow
E.g. With gramicidin current at -50mV is upward deflection
Without gramicidin intracellular concentration of chloride increases and for the same voltage step it is a downward deflection
What will be the equilibrium potential for GABAa in a developing neuron?
What are the implications if this?
Higher than that of a mature neuron (-34mV)
Therefore, GABA application in a developing neuron may be excitatory
At voltages between the equilibrium potentials of a mature and immature neuron
Mature neurons will hyperpolarise
Immature will depolarise
What term describes how both stimulatory and inhibitory inouts come together at the axon hillock to dictates if an action potential fires?
Neuronal intergration
What is special about the axon hillock?
High density of VG sodium channels lowers the threshold for action potential firing
What factors influence the extent of neuronal integration?
Amplitude
Proximity
Location size/shape of synapse
What is the effect of an inhibitory synapse more proximal to the cell body than an excitatory synapse?
GABA will open chloride channels
Will causes greater electrical shunting
Will decrease the EPSP (Lowe the chance of action potential firing)
Postive charge will be cancelled out by negative chloride ions
What will the effect of GABA be in developing neurons?
It may be excitatory and may induce and action potential alone for potentate excitatory glutamate EPSPs
In developing neurons what concentration of intracellular Cl-?
Up to 40mM
In mature neurons what is the intracellular concentration of Cl-?
4-10mM
