Showing posts with label Positive allosteric modulators. Show all posts
Showing posts with label Positive allosteric modulators. Show all posts

Friday, 6 February 2015

Tuning GABAa receptors, plus Oxytocin

Today’s post will hopefully not get too complicated.

As has been mentioned in this blog, and also at leading institutions like MIT, it does seem possible to fine-tune certain receptors in the brain that have become dysfunctional in autism.  In the case of MIT they were “tuning” a receptor called mGluR5, which they suggested was either hypo or hyper, in other words too much or too little, depending on what the underlying disease variant was.

This was done with something called an allosteric modulator, either a positive one called PAM, or a negative one called NAM.

They found that a particular glumate receptor, called mGluR5, was dysfunction in many autism-like conditions.  But the nature of the dysfunction varied, so different people would require different treatments to return the receptor performance back to normal (top dead center).   So it really becomes like tuning your car engine. 
As I have progressed in my review of the literature it becomes clear that numerous receptors are “out of tune”; so a better analogy is tuning something like a piano.


"Tuning" the shape (but not number) of dendritic spines also appears not to be as fanciful as it sounds.

Back to GABAA

Regular readers will know that one of the key dysfunctional receptors in autism is called GABAA.

This subject is very complicated.  In effect what appears to have happened in autism is that the neurons have not matured as they should, and so GABAA receptors continue to function in their “normal” immature state.  The concentration of chloride remains high since the NKCC1 transporter continues to exist, whereas KCC2/3 should have developed.  The result is that when the receptor is stimulated, instead of causing an inhibitory/calming effect it causes an excitatory effect.

This is fortunately treatable by inhibiting the flow of chloride into the cells, through NKCC1, using a drug called Bumetanide.

However this is not the end of the story.

At least 11 binding sites on GABAA receptors

As you can learn from Wikipedia:-

The active site of the GABAA receptor is the binding site for GABA and several drugs such as muscimol, gaboxadol, and bicuculline. The protein also contains a number of different allosteric binding sites which modulate the activity of the receptor indirectly. These allosteric sites are the targets of various other drugs, including the benzodiazepines, nonbenzodiazepines, barbiturates, ethanol, neuroactive steroids, inhaled anaesthetics, and picrotoxin, among others.

We are particularly interested in the allosteric binding sites.
The only one that is usually referred to, in any depth, is the site for benzodiazepines, but there are at least 11 different binding sites.

gamma-Aminobutyric acid (GABA)a receptors for the inhibitory neurotransmitter GABA are likely to be found on most, if not all, neurons in the brain and spinal cord. They appear to be the most complicated of the superfamily of ligand-gated ion channels in terms of the large number of receptor subtypes and also the variety of ligands that interact with specific sites on the receptors. There appear to be at least 11 distinct sites on GABAA receptors for these ligands.

These sites include:-

·        GABA Binding Site
·        Benzodiazepine Binding Site
·        Neurosteroid Binding Site
·        Convulsant Binding Site
·        Barbiturate Binding Site
·        b Subunit Binding Site(s)

In an earlier post I highlighted the discovery by Professor Catterall, that tiny doses of a particular Benzodiazepine drug called Clonazepam had a strange effect on the GABAA receptor.

Clonazepam is a known Positive Allosteric Modulator (PAM) of the GABAA site.  In mature neurons it amplifies the calming effect when the GABA binding site is stimulated.  In mouse models of autism (we assume therefore immature neurons)   where GABA is still excitatory, the tiny dose seemed to switch it to inhibitory.

This suggests a new function, rather than a PAM, the effect was to invert the function entirely.

Now it appears that similar things may indeed also be possible at some of the other 9+ binding sites (I exclude GABA Binding Site itself)

As complicated as this subject may sound, it actually gets even more complicated since the GABA receptors are made up of sub-units.  It appears that mutations in these subunits may be a cause of some epilepsies and, I propose, some “oddities” in autism.

Recent studies have again shown that many genetic dysfunctions found in autism relate to GABA, this short article is not so recent, but gives a nice summary:-

GABA is the major inhibitory neurotransmitter in the brain. It essentially acts as a brake for brain activation. Several aspects of GABA regulation have been linked to ASD, from early brain development to adult brain function.
Variations in GABA receptor subunits have been strongly associated with ASD. GABA receptors come in two major forms: fast, “ionotropic” GABAA receptors let negatively charged chloride ions flow into the neuron, and slow, “metabotropic” GABAB receptors produce chemical messages inside the neuron. GABAA receptors, the most common form in the brain, contain five subunits that shape their properties. Genome-wide association studies have linked the GABAA receptor subunit genes GABRA4 (α4 subunit), GABRB1 (β1 subunit), and GABRB3 (β3 subunit) to autism.[1][2] In addition, deletion of a chromosomal region that contains a cluster of a variety of GABA receptor genes (region 15q11-13) causes Angelman Syndrome.[3][4]
Genes controlling the development of GABA-releasing neurons have also been associated with ASD. Autism-linked variations in the ARX and DLX family of transcription factors interfere with proper expression of GABA.[5][6][7] Absence of such GABA-releasing neurons would negatively affect early brain development as well as adult brain stability.

Notably, variations in other ASD-linked genes affect GABA signaling. New evidence shows that the gene MECP2, the mutation of which causes Rett Syndrome, is critical for normal function of GABA-releasing neurons.[8] When MECP2 expression was blocked in GABAergic neurons of mice, GABA expression and release were reduced and the mice exhibited autistic behaviors.

ASD is a complex disorder that is likely to be caused by a combination of mutations in a variety of genes. GABA receptors are a promising therapeutic target because of their important role in monitoring brain excitation. Identification and exploration of autism-linked mutations in other GABA-related genes could shed light on the pathogenesis of autism.

Over to Switzerland

At the University of Bern a small research group is looking  at the world of  GABAA receptors, here is what they say:-

“Many scientists and companies are put off by the complexity of the field of GABAA receptors, but it is exactly this complexity that offers numerous possibilities of fine-tuned pharmacological interventions.” 

Here is one of their recent papers, that shows both what is known and how very much remains unknown.

Ion Conductance
The GABAA receptors are generally GABA-gated anion channels selective for Cl ions, with some permeability for bicarbonate anions (49). Exceptionally, in C. elegans, a cation-selective GABA-gated channel has been discovered (50). Excitatory neurotransmitters increase the cation conductance to depolarize the membrane, whereas inhibitory neurotransmitters increase the anion conductance to tendentially hyperpolarize the membrane. However, if the gradient for Cl ions decreases due to down-regulation of KCC2 chloride ion transporters, opening of GABAA receptors may cause an outward flux of these anions, leading to depolarization of the membrane and thereby to excitation. This phenomenon has been implicated in neuropathic pain (51). During early development (52) and in neuronal subcompartments (53), GABA similarly confers excitation. 
Although it is relatively simple to address questions at the level of individual receptor subunit isoforms, we can only speculate how many GABAA receptors are expressed in our brain and what their subunit composition is, not to mention subunit arrangement.

Many scientists and companies are put off by the complexity of the field of GABAA receptors, but it is exactly this complexity that offers numerous possibilities of fine-tuned pharmacological interventions.

It may be anticipated that genetic alterations of subunits of the GABAA receptor affect any of the above mentioned processes and thereby contribute to inherited human diseases. A start has been made with the analysis of point mutations that cause epilepsy

Why is all this relevant ?

We have in recent posts discovered that at least two anti-convulsants (carbamazepine and phenytoin) appear to modulate GABAA receptors in unexpected ways when given in tiny doses.

We also found out that valproate also seems to possess such qualities.  The exact mode of action of valproate is not known and perhaps it also acts a modulator of one of the many binding sites on the GABAA receptors.

We do think that valproate is working somehow via GABA.

It turns out that Carbamazepine has also been shown to potentiate GABA receptors made up of alpha1, beta2, and gamma2 subunits.

I have already established that the effect of tiny doses of Valproate is not the same as tiny doses of Clonazepam.

The next step would be to look at the effect of tiny doses of carbamazepine, phenytoin and potentially anything else that modulates those mysterious  GABAAsites.  They are clearly all there for a reason.  It seems that their role goes beyond just the allosteric modulation (amplification/reduction) of GABA’s effect.  It is likely much more subtle and they affect emotional behaviour.

Given the difficulty/impossibility of research on human brains, in the end we may need to revert to the medical world’s often used “scientific” discovery methods known as trial and error, and stumbled upon.

For the moment that will be left to Professors Sigel and Catterall and their mice, and Dr Bird, in Australia, with his human subjects.

Oxytocin and Bumetanide share the same mode of action in autism

Whilst on the subject of GABAA, I should come back to Oxytocin.

The conclusion of this Ben-Ari paper from last year is that Oxytocin and Bumetanide share the same effect in autism; they lower the level of chloride within the neurons and help switch GABA back to inhibitory.

It seems that oxytocin from the mother may be the signal to the developing brain to lower Cl levels.  Oxytocin has many other functions in the body.

Small doses of oxytocin/Syntocinon, have been shown to be effective in some people with autism.  One reader from Portugal has written on this blog how effective it has been in his young son.

Oxytocin/Syntocinon is not available everywhere, but is being reintroduced to the US.

I am wondering if in some people, who are not responders, bumetanide/oxytocin lowers the level of chloride, but not enough to show any benefit.  People using Bumetanide, which has a short half-life, comment that the effect fades through the day and that splitting the same daily dose 3 times a day is beneficial over 2 times a day.  This might suggest that combining Oxytocin with Bumetanide might give better results, by maintaining the downward pressure on chloride levels and keeping GABA more inhibitory and for longer.

In the longer term, an analog of Bumetanide is needed without the diuretic effect and with a delayed release, to maintain a constant effective level.  This is known to the researchers, but would require a big financial investment.

Larger doses of oxytocin are likely to produce effects elsewhere in the body.

If anyone tries the combination of Bumetanide + oxytocin, let me know.

Thursday, 18 September 2014

GABA A Receptors in Autism – How and Why to Modulate Them

This post will get complicated, since it will look at many aspects of the GABA A receptor, rather than just a small fraction that usually appear in the individual pieces of the scientific literature. 

It was prompted by comments I have received from regular readers, regarding Bumetanide, Clonazepam, epilepsy and whether there might be alternatives with the same effect.  So it is really intended to answer some complex issues. 

There are some new interesting facts/observations that may be of wider interest, just skip the parts that too involved.

Regarding today’s picture, most readers of this blog are female and by the way, while the US is the most common location by far,  a surprisingly high number of page views come from France, Hong Kong, South Africa and Poland.


We have seen that GABA is one of the brain's most important neurotransmitters and we know that various forms of GABA dysfunction are associated with autism, epilepsy and indeed schizophrenia.

One recurring aspect in the research is the so-called excitatory-inhibitory balance of GABA.

The way the brain is understood to function assumes that GABA should be inhibitory and NMDA should be excitatory.

What makes GABA inhibitory is the level of the electrolyte chloride within the cells.  If the level is “wrong”, then GABA may be excitatory and the fine balance required with the NMDA receptor is lost.  The brain then cannot function as intended.

Source: Sage Therapeutics, a company that is developing new drugs that target GABAA and NMDA receptors

To understand what is going wrong in autism and how to treat it we need to take a detailed look at the GABAA receptor and all the anion transport mechanism associated with it.  Most research looks at either the receptor OR the transporters and exchangers.

Anion Transport Mechanisms of the GABAA receptor

You will either need to be a doctor, scientist or very committed to keep reading here.

We know that level of chloride within the cells is critical to whether GABA behaves as excitatory or inhibitory.  This has all been established in the laboratory.

The usual target in autism is the NKCC1 transporter that lets chloride INTO cells, but as you can see in the two figures below, there are other ways to affect the concentration of chloride.  

·        The KCC2 transporter lets chloride out of the cells

·        The sodium dependent anion exchanger (NDAE) lets chloride out of the cells

·        The sodium independent anion exchanger 3 (AE3), lets chloride in.  It extrudes intracellular HCO3- in exchange for extracellular Cl-.

All this does actually matter since we will be able to link it back to a known genetic dysfunction and it would suggest alternative therapeutic avenues.  We can also see how epilepsy fits into the picture.

NKCC1 in Autism

Without doubt, the transporter that controls the flow of chloride into the brain is the expert field of Ben Ari.

His recent summary paper is below:-

He showed that by reducing the level of chloride in the autistic brain using the common diuretic Bumetanide, a marked improvement in many peoples’ autism could be achieved

This post is really about expanding more on what he does not tell us.

KCC2 in Autism

In typical people, very early in life the KCC2 transporter develops and as a result level of chloride falls inside the cells, since the purpose of the transporter is to extrude chloride.

It appears that in autism this mechanism has been disrupted.  The existing science can show us what has gone wrong.

The following study shows that KCC2 is itself regulated by neuroligin-2 (NL2), a cell adhesion molecule specifically localized at GABAergic synapses.

It gets more interesting because the scientists looking for genetic causes of autism have already identified the gene that encodes NL2, which they call NLGN2 (neuroligin 2) as being associated with autism and schizophrenia (adult onset autism).




GABAA receptors are ligand-gated Cl- channels, and the intracellular Cl- concentration governs whether GABA function is excitatory or inhibitory. During early brain development, GABA undergoes functional switch from excitation to inhibition: GABA depolarizes immature neurons but hyperpolarizes mature neurons due to a developmental decrease of intracellular Cl- concentration. This GABA functional switch is mainly mediated by the up-regulation of KCC2, a potassium-chloride cotransporter that pumps Cl- outside neurons. However, the upstream factor that regulates KCC2 expression is unclear.


We report here that KCC2 is unexpectedly regulated by neuroligin-2 (NL2), a cell adhesion molecule specifically localized at GABAergic synapses. The expression of NL2 precedes that of KCC2 in early postnatal development. Upon knockdown of NL2, the expression level of KCC2 is significantly decreased, and GABA functional switch is significantly delayed during early development. Overexpression of shRNA-proof NL2 rescues both KCC2 reduction and delayed GABA functional switch induced by NL2 shRNAs. Moreover, NL2 appears to be required to maintain GABA inhibitory function even in mature neurons, because knockdown NL2 reverses GABA action to excitatory. Gramicidin-perforated patch clamp recordings confirm that NL2 directly regulates the GABA equilibrium potential. We further demonstrate that knockdown of NL2 decreases dendritic spines through down-regulating KCC2.


Our data suggest that in addition to its conventional role as a cell adhesion molecule to regulate GABAergic synaptogenesis, NL2 also regulates KCC2 to modulate GABA functional switch and even glutamatergic synapses. Therefore, NL2 may serve as a master regulator in balancing excitation and inhibition in the brain.

KCC2 in Peripheral nerve injury (PNI)

Autism is not the only diagnosis associated with reduced function of the KCC2 transporter; Peripheral nerve injury (PNI) is another.

In this condition researchers sought to counter the failure of KCC2 to remove chloride from within the cell by increasing the flow chloride through the Cl-/HCO3- anion exchanger known as AE3.


Peripheral nerve injury (PNI) negatively influences spinal gamma-aminobutyric acid (GABA)ergic networks via a reduction in the neuron-specific potassium-chloride (K(+)-Cl(-)) cotransporter (KCC2). This process has been linked to the emergence of neuropathic allodynia. In vivo pharmacologic and modeling studies show that a loss of KCC2 function results in a decrease in the efficacy of GABAA-mediated spinal inhibition. One potential strategy to mitigate this effect entails inhibition of carbonic anhydrase activity to reduce HCO3(-)-dependent depolarization via GABAA receptors when KCC2 function is compromised. We have tested this hypothesis here. Our results show that, similarly to when KCC2 is pharmacologically blocked, PNI causes a loss of analgesic effect for neurosteroid GABAA allosteric modulators at maximally effective doses in naïve mice in the tail-flick test. Remarkably, inhibition of carbonic anhydrase activity with intrathecal acetazolamide rapidly restores an analgesic effect for these compounds, suggesting an important role of carbonic anhydrase activity in regulating GABAA-mediated analgesia after PNI. Moreover, spinal acetazolamide administration leads to a profound reduction in the mouse formalin pain test, indicating that spinal carbonic anhydrase inhibition produces analgesia when primary afferent activity is driven by chemical mediators. Finally, we demonstrate that systemic administration of acetazolamide to rats with PNI produces an antiallodynic effect by itself and an enhancement of the peak analgesic effect with a change in the shape of the dose-response curve of the α1-sparing benzodiazepine L-838,417. Thus, carbonic anhydrase inhibition mitigates the negative effects of loss of KCC2 function after nerve injury in multiple species and through multiple administration routes resulting in an enhancement of analgesic effects for several GABAA allosteric modulators. We suggest that carbonic anhydrase inhibitors, many of which are clinically available, might be advantageously employed for the treatment of pathologic pain states.


Using behavioral pharmacology techniques, we show that spinal GABAA-mediated analgesia can be augmented, especially following nerve injury, via inhibition of carbonic anhydrases. Carbonic anhydrase inhibition alone also produces analgesia, suggesting these enzymes might be targeted for the treatment of pain

Treatment of neuropathic pain is a major clinical challenge that has been met with minimal success. After peripheral nerve injury, a decrease in the expression of the K–Cl cotransporter KCC2, a major neuronal Cl extruder, leads to pathologic alterations in GABAA and glycine receptor function in the spinal cord. The down-regulation of KCC2 is expected to cause a reduction in Cl extrusion capacity in dorsal horn neurons, which, together with the depolarizing efflux of HCO−3 anions via GABAA channels, would result in a decrease in the efficacy of GABAA-mediated inhibition. Carbonic anhydrases (CA) facilitate intracellular HCO−3 generation and hence, we hypothesized that inhibition of CAs would enhance the efficacy of GABAergic inhibition in the context of neuropathic pain. Despite the decrease in KCC2 expression, spinal administration of benzodiazepines has been shown to be anti-allodynic in neuropathic conditions. Thus, we also hypothesized that spinal inhibition of CAs might enhance the anti-allodynic effects of spinally administered benzodiazepines. Here, we show that inhibition of spinal CA activity with acetazolamide (ACT) reduces neuropathic allodynia. Moreover, we demonstrate that spinal co-administration of ACT and midazolam (MZL) act synergistically to reduce neuropathic allodynia after peripheral nerve injury. These findings indicate that the combined use of CA inhibitors and benzodiazepines may be effective in the clinical management of neuropathic pain in humans.

In conclusion, the major finding of the present work is that ACT and MZL act synergistically to inhibit neuropathic allodynia. In light of the available in vitro data reviewed above, a parsimonious way to explain this synergism is that CA inhibition blocks an HCO−3 -dependent positive shift in the Er of GABA and/or glycine-mediated currents and the consequent tonic excitatory drive mediated by extrasynaptic GABAA receptors, while preserving shunting inhibition that is augmented by benzodiazepine actions at postsynaptic GABAA receptors. Obviously, further work is needed at the in vitro level in order to directly examine the cellular and synaptic basis of the ACT-MZL synergism and clinical studies are required to determine the safety of intrathecally applied CA inhibitors in humans. Since MZL and ACT, as well as several other inhibitors of CA [37], are clinically approved, we propose that their use in combination opens up a novel approach for the treatment of chronic neuropathic pain

Midazolam and Acetazolamide

The therapeutic as well as adverse effects of midazolam are due to its effects on the GABAA receptors; midazolam does not activate GABAA receptors directly but, as with other benzodiazepines, it enhances the effect of the neurotransmitter GABA on the GABAA receptors (↑ frequency of Cl− channel opening) resulting in neural inhibition. Almost all of the properties can be explained by the actions of benzodiazepines on GABAA receptors. This results in the following pharmacological properties being produced: sedation, hypnotic, anxiolytic, anterograde amnesia, muscle relaxation and anti-convulsant.

Acetazolamide, usually sold under the trade name Diamox in some countries.  Acetazolamide is a diuretic, and it is available as a (cheap) generic drug.

In epilepsy, the main use of acetazolamide is in menstrual-related epilepsy and as an adjunct in refractory epilepsy.

Acetazolamide is not an immediate cure for acute mountain sickness; rather, it speeds up part of the acclimatization process which in turn helps to relieve symptoms.  I am pretty sure, many years ago, it was Diamox that I took with me when crossing the Himalayas from Nepal into Tibet.  We did not have any problems with mountain sickness.

If periodic paralysis above rings some bells it should.  Two forms already mentioned in this blog are Hypokalemic periodic paralysis and Andersen Tawil syndrome.  We even referred to a paper suggesting the use of Bumetanide.

Acetazolamide is a carbonic anhydrase inhibitor, hence causing the accumulation of carbonic acid Carbonic anhydrase is an enzyme found in red blood cells that catalyses the following reaction:

hence lowering blood pH, by means of the following reaction that carbonic acid undergoes

Anion exchanger 3 (AE3) in autism

Anion exchange protein 3 is a membrane transport protein that in humans is encoded by the SLC4A3 gene. It exchanges chloride for bicarbonate ions.  It increases chloride concentration within the cell.  AE3 is an anion exchanger that is primarily expressed in the brain and heart

Its activity is sensitive to pH. AE3 mutations have been linked to seizures

Bicarbonate (HCO3-) transport mechanisms are the principal regulators of pH in animal cells. Such transport also plays a vital role in acid-base movements in the stomach, pancreas, intestine, kidney, reproductive organs and the central nervous system.


Chloride influx through GABA-gated Cl channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl gradient established in part by K+–Cl cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na+-driven Cl–HCO3 exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K+–Cl cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl gradient such that Cl flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl gradient. Our results show that Na+-driven Cl–HCO3 exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.


During early development, γ-aminobutyric acid (GABA) depolarizes and excites neurons, contrary to its typical function in the mature nervous system. As a result, developing networks are hyperexcitable and experience a spontaneous network activity that is important for several aspects of development. GABA is depolarizing because chloride is accumulated beyond its passive distribution in these developing cells. Identifying all of the transporters that accumulate chloride in immature neurons has been elusive and it is unknown whether chloride levels are different at synaptic and extrasynaptic locations. We have therefore assessed intracellular chloride levels specifically at synaptic locations in embryonic motoneurons by measuring the GABAergic reversal potential (EGABA) for GABAA miniature postsynaptic currents. When whole cell patch solutions contained 17–52 mM chloride, we found that synaptic EGABA was around −30 mV. Because of the low HCO3 permeability of the GABAA receptor, this value of EGABA corresponds to approximately 50 mM intracellular chloride. It is likely that synaptic chloride is maintained at levels higher than the patch solution by chloride accumulators. We show that the Na+-K+-2Cl cotransporter, NKCC1, is clearly involved in the accumulation of chloride in motoneurons because blocking this transporter hyperpolarized EGABA and reduced nerve potentials evoked by local application of a GABAA agonist. However, chloride accumulation following NKCC1 block was still clearly present. We find physiological evidence of chloride accumulation that is dependent on HCO3 and sensitive to an anion exchanger blocker. These results suggest that the anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons.

Sodium dependent anion exchanger (NDAE)

Not much has been written about these exchangers, outside of very technical literature.

Sodium-coupled anion exchange is activated by intracellular acidification (Schwiening and Boron, 1994), suggesting that regulation of the chloride gradient by NDAEs may be closely linked to the regulation of cellular pH. As prolonged neuronal activity can cause neuronal acidification by efflux of bicarbonate through GABAA receptors (Kaila and Voipio, 1987), sodium-coupled anion exchange may help to maintain a hyperpolarizing chloride reversal potential and thus promote the inhibitory action of GABA. Thus activation of sodium-coupled anion exchange by acidosis may also contribute to seizure termination by promoting a more negative chloride reversal potential and thus promoting the inhibitory effects of GABA.

The GABAA receptor  (background is cut and paste from Wikipedia)

In order for GABAA receptors to be sensitive to the action of benzodiazepines they need to contain an α and a γ subunit, between which the benzodiazepine binds. Once bound, the benzodiazepine locks the GABAA receptor into a conformation where the neurotransmitter GABA has much higher affinity for the GABAA receptor, increasing the frequency of opening of the associated chloride ion channel and hyperpolarizing the membrane. This potentiates the inhibitory effect of the available GABA leading to sedative and anxiolytic effects.

Structure and function

Schematic diagram of a GABAA receptor protein ((α1)2(β2)2(γ2)) which illustrates the five combined subunits that form the protein, the chloride (Cl-) ion channel pore, the two GABA active binding sites at the α1 and β2 interfaces, and the benzodiazepine (BDZ) allosteric binding site

The receptor is a pentameric transmembrane receptor that consists of five subunits arranged around a central pore. Each subunit comprises four transmembrane domains with both the N- and C-terminus located extracellularly. The receptor sits in the membrane of its neuron, usually localized at a synapse, postsynaptically. However, some isoforms may be found extrasynaptically. The ligand GABA is the endogenous compound that causes this receptor to open; once bound to GABA, the protein receptor changes conformation within the membrane, opening the pore in order to allow chloride anions (Cl) to pass down an electrochemical gradient. Because the reversal potential for chloride in most neurons is close to or more negative than the resting membrane potential, activation of GABAA receptors tends to stabilize or hyperpolarise the resting potential, and can make it more difficult for excitatory neurotransmitters to depolarize the neuron and generate an action potential. The net effect is typically inhibitory, reducing the activity of the neuron. The GABAA channel opens quickly and thus contributes to the early part of the inhibitory post-synaptic potential (IPSP).


GABAA receptors are members of the large "Cys-loop" super-family of evolutionarily related and structurally similar ligand-gated ion channels that also includes nicotinic acetylcholine receptors, glycine receptors, and the 5HT3 receptor. There are numerous subunit isoforms for the GABAA receptor, which determine the receptor's agonist affinity, chance of opening, conductance, and other properties.
In humans, the units are as follows:
There are three ρ units (GABRR1, GABRR2, GABRR3), however these do not coassemble with the classical GABAA units listed above,[18] but rather homooligomerize to form GABAA-ρ receptors (formerly classified as GABAC receptors but now this nomenclature has been deprecated[19] ).
Five subunits can combine in different ways to form GABAA channels. The minimal requirement to produce a GABA-gated ion channel is the inclusion of both α and β subunits, but the most common type in the brain is a pentamer comprising two α's, two β's, and a γ (α2β2γ)
The receptor binds two GABA molecules, at the interface between an α and a β subunit

The important subunits for this post are:-
Very little is written about this subunit.

While the effect of editing on protein function is unknown, the developmental increase in editing does correspond to changes in function of the GABAA receptor. GABA binding leads to chloride channel activation, resulting in rapid increase in concentration of the ion. Initially, the receptor is an excitatory receptor, mediating depolarisation (efflux of Cl- ions) in immature neurons before changing to an inhibitory receptor, mediating hyperpolarization(influx of Cl- ions) later on. GABAA converts to an inhibitory receptor from an excitatory receptor by the upregulation of KCC2 cotransporter. This decreases the concentration of Cl- ion within cells. Therefore, the GABAA subunits are involved in determining the nature of the receptor in response to GABA ligand. These changes suggest that editing of the subunit is important in the developing brain by regulating the Cl- permeability of the channel during development. The unedited receptor is activated faster and deactivates slower than the edited receptor.

Editing of the I/M site is developmentally regulated

A switch in the GABA response from excitatory to inhibitory post-synaptic potentials occurs during early development where an efflux of chloride ions takes place in immature neurons, while there is an influx of chloride ions in mature neurons (Ben-Ari 2002). GABA switches from being excitatory to inhibitory by an up-regulation of the cotransporter KCC2 that decreases the chloride concentration in the cell. However, if GABA itself promotes the expression of KCC2 is still under debate (Ganguly et al. 2001; Ludwig et al. 2003; Titz et al. 2003). Further, the α subunits are critical elements in determining the nature of the GABAA receptor response to GABA (Böhme et al. 2004). The α3 mRNA (Gabra-3) is present at high levels in several forebrain regions at birth with a major decline after post-natal day 12 (P12), when the expression of α1 is going up (Laurie et al. 1992). The change from α3 to α1 may cause the switch in GABA behavior from excitatory to inhibitory post-synaptic potentials during development.
GABAA receptors respond to anxiolytic drugs such as benzodiazepines and are thus important drug targets. The benzodiazepine binding site is located at the interface of the α and γ2 subunits (Cromer et al. 2002). Antagonists that bind to this site enhance the effect of GABA by increasing the frequency of GABA-induced channel opening events. Post-transcriptional modifications of the α3 subunit, such as the I/M editing described here, could be important in determining the mechanistic features that are responsible for the diversity of GABAA receptors and the variability in sensitivity to drugs


A number of ligands have been found to bind to various sites on the GABAA receptor complex and modulate it besides GABA itself.


  • Agonists: bind to the main receptor site (the site where GABA normally binds, also referred to as the "active" or "orthosteric" site) and activate it, resulting in increased Cl conductance.
  • Antagonists: bind to the main receptor site but do not activate it. Though they have no effect on their own, antagonists compete with GABA for binding and thereby inhibit its action, resulting in decreased Cl conductance.
  • Positive allosteric modulators: bind to allosteric sites on the receptor complex and affect it in a positive manner, causing increased efficiency of the main site and therefore an indirect increase in Cl conductance.
  • Negative allosteric modulators: bind to an allosteric site on the receptor complex and affect it in a negative manner, causing decreased efficiency of the main site and therefore an indirect decrease in Cl conductance.
  • Open channel blockers: prolong ligand-receptor occupancy, activation kinetics and Cl ion flux in a subunit configuration-dependent and sensitization-state dependent manner.
  • Non-competitive channel blockers: bind to or near the central pore of the receptor complex and directly block Cl- conductance through the ion channel.

The GABAA receptor include a site where benzodiazepine can bind.  These are drugs that include like valium. Binding at this site increase the effect of GABA.  Since this receptor is meant to be inhibitory, giving valium should make it strong inhibitory, ie calming. 

It was noted that in autism the effect of valium was often the reversed, instead of calming it further increased anxiety.

The Valium is working just fine, it is magnifying the effect the effect of GABA, the problem is that the receptor is functioning as excitatory, the Valium is making it over-excitatory.  Now we come to the reason why.

We know that the excitatory-inhibitory balance is set by the chloride concentration within the cells.  We also know that exact mechanism that determines this level.


BTBR mice have reduced spontaneous GABAergic inhibitory transmission
Nonsedating doses of benzodiazepines improved autism-related deficits in BTBR mice
Impairment of GABAergic transmission reduced social interaction in wild-type mice
Behavioral rescue by low-dose benzodiazepine is GABAA receptor α2,3-subunit specific


Autism spectrum disorder (ASD) may arise from increased ratio of excitatory to inhibitory neurotransmission in the brain. Many pharmacological treatments have been tested in ASD, but only limited success has been achieved. Here we report that BTBR T+ Itpr3tf/J (BTBR) mice, a model of idiopathic autism, have reduced spontaneous GABAergic neurotransmission. Treatment with low nonsedating/nonanxiolytic doses of benzodiazepines, which increase inhibitory neurotransmission through positive allosteric modulation of postsynaptic GABAA receptors, improved deficits in social interaction, repetitive behavior, and spatial learning. Moreover, negative allosteric modulation of GABAA receptors impaired social behavior in C57BL/6J and 129SvJ wild-type mice, suggesting that reduced inhibitory neurotransmission may contribute to social and cognitive deficits. The dramatic behavioral improvement after low-dose benzodiazepine treatment was subunit specific—the α2,3-subunit-selective positive allosteric modulator L-838,417 was effective, but the α1-subunit-selective drug zolpidem exacerbated social deficits. Impaired GABAergic neurotransmission may contribute to ASD, and α2,3-subunit-selective positive GABAA receptor modulation may be an effective treatment.
These results indicate that different subtypes of GABAA receptors may have opposite roles in social behavior, with activation of GABAA receptors containing α2,3 subunits favoring and of GABAA receptors with α1  subunits reducing social interaction, respectively.

Because of their broad availability and safety, benzodiazepines and other positive allosteric modulators of GABAA receptors administered at low nonsedating, nonanxiolytic doses that do not induce tolerance deserve consideration as a near-term strategy to improve the core social interaction deficits and repetitive behaviors in ASD.

These results are most consistent with the hypotheses that reduced inhibitory neurotransmission is sufficient to induce autistic-like behaviors in mice and that enhanced inhibitory neurotransmission can reverse autistic-like behaviors.


I have received various comments about epilepsy.  Epilepsy has many variants, just like autism.  Epilepsy is often comorbid with autism.  GABA dysfunction is known to be closely involved in some types of autism and some types of epilepsy.

It is known that Bumetanide has very different effects in different types of epilepsy.
The question that naturally arises is whether you can give Bumetanide to someone who has autism and epilepsy and if you cannot, is there an alternative with the same desired effect?

Well it appears that any method that changes chloride levels is likely to affect epilepsy.  It appears that all three methods (NKCC1, KCC2 and AE3) would likely have the same impact on epilepsy.

But would it be a good effect or a bad effect?

Would it interact with any existing anti-epilepsy drugs?

I suspect that Bumetanide might be an effective anti-epileptic in people with autism and that other GABA related drugs might no longer be needed.  Quite likely the effect of Bumetanide and the anti-epileptic targeting GABA might be too much.  So the blog reader that pointed out that the bumetanide clinical trial excluded children with epilepsy has highlighted an important point.

While epilepsy is not fully understood and there are various variants, it would seem plausible that the epilepsy common in core classic autism and early regressive autism is the same type and that it is linked to the same excitatory/inhibitory dysfunction.

You may be wonder if other diuretics have anti-epileptic properties. Here is a paper by a Neurologist from Denver on the subject:-

Why is there an excitatory/inhibitory dysfunction in Autism?

People are writing entire books on the GABA excitatory/inhibitory balance.  I was curious as to why this dysfunction exists at all in autism. 

We learnt from Ben-Ari in earlier posts all about this switch from excitatory to inhibitory that is supposed to occur very early on in life, we now have two reasons why this may fail to happen in autism:-

1.     Editing modifies the GABAA receptor subunit α3.  The change from α3 to α1 may cause the switch in GABA behavior from excitatory to inhibitory post-synaptic potentials during development.  This change appears not to occur in some types of autism.  We see from the Clonazepam research that α3 and  α1 have opposite effects in autism.   In autism, activation of GABAA receptors containing α2,3 subunits favours social interaction  and activation of α1  subunits reduces social interaction.


2.     The GABA functional switch is mainly mediated by the up-regulation of KCC2, a potassium-chloride cotransporter that pumps Cl- outside neurons.  NL2 also regulates KCC2 to modulate GABA functional switch. Therefore, NL2 may serve as a master regulator in balancing excitation and inhibition in the brain.  The gene that encodes NL2 is called NLGN2 (neuroligin 2).  Dysfunction in gene NLGN2 is known to occur in both autism and schizophrenia (adult onset autism).


We came full circle back to Bumetanide and Clonazepam as most likely the safest and most effective therapy to adjust the E/I (excitatory/inhibitory) balance in autism. KCCI agonists do not seem to exist.  The bicarbonate exchanger agonist Acetazolamide/Diamox is another common diuretic and I see no reason why it would not also be effective, but we would then affect bicarbonate levels.  Since these ions play a role in controlling pH levels, I think we might risk seeing some unintended effects.  We know that Bumetanide is safe in long term use.  We know that all diuretics that change chloride level within the cell and will affect epilepsy; so it is a case of “better the devil you know”. 

I finally understood exactly why tiny dose of Clonazepam are effective and how this fits in with the changes the Bumetanide has produced.  Thankfully, such tiny doses are free of the typical side effects expected from benzodiazepines.  One tablet lasts 10 days.

It also answers somebody else’s question about starting with Clonazepam before the Bumetanide.  If you did that you might well make things much worse, you would magnify the unwanted excess brain cell firing.  Once you added bumetanide things would then reverse and brain cell firing would be inhibited.

I rather like the parallel with neuropathic pain, the other condition we looked at with reduced KCC2 transporter function, the researchers there proposed the combination of a diuretic (Acetazolamide) to lower cellular chloride (via exchanger AE3) and a benzodiazepine (Midazolam) as a positive allosteric modulator.  This is extremely similar to Ben Ari’s bumetanide (diuretic affecting transporter NKCC1) plus Catterall’s tiny doses of clonazepam (benzodiazepine) as a positive allosteric modulator.

As for epilepsy and bumetanide, we know that bumetanide has different effects on different types of autism. It seems plausible that people with autism might tend to have the same type of epilepsy.  In any case Monty, aged 11 with ASD, does not have epilepsy/seizures and I suspect taking bumetanide has decreased the chance he ever will.  Of course I cannot prove this, it is just conjecture.