GABA receptors

Question 1

What is GABA?

Answer 1

GABA (γ-Aminobutyric acid) is a 4-carbon carboxylic acid and is the main inhibitory neurotransmitter in the mammalian brain.

Question 2

How do we know GABAA receptors are permeable to Cl- ions?

Answer 2

The association with Cl- ions is known due to experiments where the I-V relationship was plotted and the reversal potential was close to the predicted Nernst potential for Cl-.

Question 3

What is phasic activation of GABAA receptors?

Answer 3

Phasic activation of GABAA receptors results in a transient postsynaptic response due to GABA binding to the receptors on the postsynaptic membrane and then being taken up into glial cells and neurons via transporters (e.g. GAT1 or GAT3).

Question 4

How many genes code for GABAA receptor subunits?

Answer 4

There are 19 genes that code for different GABAA receptor subunits which assemble to create different receptor subtypes.

Question 5

What is the speed of response for GABAA vs GABAB receptors?

Answer 5

Fast synaptic response mediated by ionotropic GABAA receptors and a slower response mediated by metabotropic GABAB receptors.

Question 6

What are GABAA receptors?

Answer 6

GABAA receptors are pentameric ligand-gated ion channels (LGICs) which mediate the fast, inhibitory GABA response.

Question 7

How do GABAA receptors cause inhibition?

Answer 7

This is due to chloride ions (Cl-) moving into the cell, hyperpolarizing it and moving the membrane potential away from threshold thereby reducing the likelihood of an action potential.

Question 8

What is tonic activation of GABAA receptors?

Answer 8

Tonic activation of GABAA receptors occurs because receptors have a higher affinity for GABA so low, “ambient” levels of GABA can cause spiking.

Question 9

Which neurotransmitters are involved in the inhibitory response and how do we know this?

Answer 9

Spinal inhibitory currents were blocked by bicuculline, a GABA receptor antagonist, and also partially by the glycine antagonist strychnine. The dual component to the response suggests that GABA and Glycine were localised to the same presynaptic vesicle and co-released.

Question 10

Explain shunting inhibition.

Answer 10

Membrane potential = reversal potential. Channels are open and therefore membrane conductance increases, however, there is no net flow of ions. Since V=IR, and R is reduced (conductance increased) then it takes more current to elicit a change in voltage. This acts divisively on the amplitude of the postsynaptic potential as opposed to the subtractive nature of a hyperpolarizing IPSP.

Question 11

Explain hyperpolarising inhibition.

Answer 11

GABA binds to the GABAA receptors on the postsynaptic side which opens the Cl- channel. An influx of Cl- hyperpolarizes the membrane moving it away from the threshold required to trigger an action potential.

Question 12

Length of shunting vs hyperpolarising inhibition.

Answer 12

Shunting inhibition only lasts as long as the channels are open, but hyperpolarizing inhibition lasts longer.

Question 13

Describe the experiment which shows bicarbonate ions are permeable to the GABAA channel.

Answer 13

Voltage clamp recordings from a Xenopus oocyte which expressed “GABAC” (homomeric ρ1) receptors. Chloride ions were substituted for anions and cations and the reversal potential of each was measured. It had shifted for the anions, showing that the channel was permeable to them, and had a more positive value for bicarbonate ions (HCO3-) which means it is less permeable than Cl-.

Question 14

What is the relative permeability for HCO3- ions and how is this calculated?

Answer 14

The relative permeability to Cl- (1) was calculated using a variation on the Goldman-Hodgkin-Katz equation and showed GABAC receptors conduct bicarbonate (HCO3-) with a relative permeability of around 0.27 ± 0.03.

Question 15

How can the pH of a cell alter the inhibitory response?

Answer 15

Driving force for HCO3- is always outward due to the passive distribution of H+ ions which creates a higher internal pH. The loss of negative charge increases the membrane potential so is depolarising.

Question 16

Where do HCO3- ions come from?

Answer 16

CO2 enters the cell and is converted into HCO3- which leaves the cell when the GABAA receptor ion channels open. CO2 is always replacing the loss of HCO3-.

Question 17

What experimental method was used to show that GABA can be excitatory in immature neurons?

Answer 17

Gramicidin forms pores in the membrane not permeable to Cl- which prevents the intracellular Cl- concentration from changing. Using gramicidin-perforated-patch experiments on immature rat cerebellar granule cells (P7), and using glutamate blocks, they recorded some spontaneous GABAergic depolarising potentials that sometimes triggered action potentials. This disappeared when bicuculline, a GABAA antagonist, was applied, showing that the depolarization was due to GABA. For more mature cells at stage P21, the depolarising action of GABA was lost.

Question 18

Is GABA always inhibitory?

Answer 18

No, GABA release doesn’t always result in inhibition and is dependent on the driving force of Cl- ions.

Question 19

Why is GABA excitatory in immature neurons?

Answer 19

Cl- gradients are set up by cation-chloride co-transporters. In immature neurons there is more of Na–K–2Cl co-transporter isoform 1 (NKCC1) which is Cl- accumulating, meaning that when GABAA receptor channels open the Cl- ions move out of the cell and it depolarises.

Question 20

Why is GABA inhibitory in mature neurons?

Answer 20

After a developmental change, there is more K-Cl co-transporter isoform 2 (KCC2) present which is Cl- extruding, and so the driving force for Cl- ions moves them into the cell and it hyperpolarizes.

Question 21

How else can phasic activation of GABAA receptors occur?

Answer 21

GABA can also diffuse out of the synaptic cleft and activate postsynaptic receptors on adjacent synapses as well as on presynaptic terminals. The effect this has is still considered phasic because the release event and postsynaptic response are temporally related.

Question 22

Which experiment detected tonic activation of GABAA receptors?

Answer 22

Tonic activation can occur independent of a release event. Using voltage-clamp recordings in rat cerebellar granule cells in the presence of glutamate blockers, spontaneous IPSPs were measured, these were then blocked with the addition of bicuculline. The resting membrane conductance was also decreased with the addition of GABA antagonists which indicates that the GABAA channels are blocked and tonic inhibition can no longer occur.

Question 23

Does tonic activation occur in immature cells?

Answer 23

From voltage recordings at different ages of rat cerebellar granule cell it was shown that at P7 there were spontaneous postsynaptic currents, however these were discrete and became briefer and smaller in more mature cells.

Question 24

Which two ways can GABA be synthesised?

Answer 24

GABA is an offshoot of the TCA cycle and can be made from glutamate by glutamic acid decarboxylase (GAD). The “GABA-shunt” is the name for the alternate route that α-ketoglutarate from the TCA cycle is converted into succinate via the production of glutamate and then GABA.

Question 25

Describe vesicle uptake and release of GABA.

Answer 25

Once GABA has been synthesised in the cytosol, it is taken up into vesicles by vesicular GABA transporter (VGAT). Single vesicular release will activate the GABAA receptors on the postsynaptic terminal and result in phasic inhibition that is temporally related to the release. Multiple vesicles can also be released caused by stronger inputs and the higher concentration of GABA allows it to diffuse out of the cleft to activate neighbouring GABAA receptors creating a longer and slower IPSP.

Question 26

What mechanism allows tonic activation of GABAA receptors?

Answer 26

For tonic inhibition to occur, it is the low ‘ambient’ levels of GABA that remain in the cleft which activate higher affinity receptors. It is the release of GABA from vesicles after an action potential that maintain this low level, even with the action of the GABA uptake transporters (GAT1 and GAT3). For example, in the cerebellar glomerulus, there is a large number of GABA releasing Golgi cell synapses on dendrites from granule cells, which could contribute to the persistent low levels of GABA.

Question 27

What is the structure of the GABAA receptor?

Answer 27

GABAA receptors are part of the cys-loop superfamily of LGICs made up from five subunits each with four transmembrane spanning domains and form a pentameric structure with a central ion pore permeable to Cl-/HCO3- created by the second transmembrane domain.

Question 28

What is the common assembly of the GABAA receptor subunits?

Answer 28

There are 19 GABAA receptor subunit genes grouped by sequence similarity and this subunit diversity predicts large receptor heterogeneity. The most common assembly of subunits is two α, two β, and one γ, structured so that the subunits alternate e.g. βαβαγ.

Question 29

What experiments were done to elucidate the structure of the GABAA receptor?

Answer 29

The structure of the GABAA receptor is homologous with that of other pentameric LGICs such as nicotinic acetylcholine receptors (nAChR). Using electron cryomicroscopy of nAChR from Torpedo to find the structure of the receptor, information about the ligand binding domain was revealed and implicated the two α subunits. In GABAA receptor homology modelling, GABA binds at the interface between α and β, so has two potential binding sites.

Question 30

What role does the γ2 subunit play in GABAA receptor pharmacology?

Answer 30

For normal phasic activation of GABAA receptors, the receptors must be present on the postsynaptic density and the γ2 subunit has been shown to have a role in clustering GABAA receptors. γ2 -/- mice show a normal number of receptors however they have reduced channel conductance and there is a loss of channel clustering, showing that γ2 is not essential in forming the receptors but does have an impact on function.

Question 31

What role does the δ subunit play in GABAA receptor pharmacology?

Answer 31

Using immunogold electron microscopy in the rat cerebellar granule cell layer, the δ subunit was found to be present in the extrasynaptic dendritic and somatic membrane but not in the synapse itself. Tonic activation is thought to occur by activation of receptors outside the synapse and so the presence of the δ subunit in those regions could indicate its necessity in producing tonic inhibition.

Question 32

Which subunits have a higher affinity for GABA, implicating them in tonic activation?

Answer 32

Experiments that knocked-out the α6 and δ subunits from rat cerebellar granule cells found that the tonic IPSPs were absent, suggesting the need for these subunits in receptors to produce tonic inhibition. These subunits possibly have a higher affinity for GABA and so will activate when there is a low concentration of ambient GABA.

Question 33

What purpose does inhibition play in the brain?

Answer 33

The purpose of phasic inhibition in the brain is to prevent the over-excitation of neurons which could lead to pathological conditions like epilepsy. However, GABAA receptors can have different functional roles, such as synchronising neuronal activity as a form of information coding.

Question 34

Explain how GABA interneurons can synchronise neuronal activity.

Answer 34

In hippocampal interneurons, activating presynaptic basket cells phase-locked the firing of postsynaptic pyramidal cells. These GABAergic interneurons set firing at theta (θ) frequencies (4-7 Hz) and can synchronise the firing of many pyramidal cells in a network due to their divergence.

Question 35

Explain how GABA interneurons can act in feedforward mechanisms to create coincidence detection.

Answer 35

GABAergic interneurons can act in feedforward mechanisms, where they are activated simultaneously with the target neuron and then also feedforward onto the target neuron, inhibiting its response. In CA1 hippocampal pyramidal cells, di-synaptic feedforward inhibition is shown to reduce the window for which temporal summation occurs. Using GABAA receptor antagonists increased this window and abolished the coincidence detection that is due to feedforward inhibition.

Question 36

Explain how feedforward inhibition can shape the excitatory response of neurons.

Answer 36

In experiments done on the cerebellar cortex of the rat, Purkinje cells were synapsed by parallel fibres. The parallel fibres were stimulated, which in turn activated the Purkinje cell and interneurons which rapidly shortened the EPSP. With the addition of a GABAA antagonist, the EPSP became much larger and more sustained, showing that the GABAergic interneurons were shaping the original EPSP to be smaller and briefer.

Question 37

Explain how tonic inhibition can shape incoming EPSPs.

Answer 37

Tonic activation of GABAA receptors increases the cells input conductance which means that the amplitude of subsequent EPSPs is reduced (V=IR). This is a type of shunting inhibition and will make it harder for the cell to integrate EPSPs within the spatial and temporal window to trigger an action potential. Tonic inhibition differs from high-frequency phasic inhibition in that the integration of phasic inhibition occurs in the soma whereas for tonic inhibition any integration that occurs is with the GABA concentration in the synaptic cleft.