PHAR 402, Dr. Lu

 

THE GABAA-RECEPTOR-BENZODIAZEPINE RECEPTOR-CHLORIDE ION CHANNEL COMPLEX

 

g-Aminobutyric acid (GABA) is perhaps the most comprehensively studied inhibitory neurotransmitter in the mammalian central nervous system (CNS).  It has been estimated that about 40% of synapses in the brain are GABAergic.  It is now well recognized that cellular excitability leading to convulsive seizures can be attenuated by GABAergic stimulation in the CNS.  Current evidence also indicates that most anxiolytics and hypnotic-sedative drugs such as benzodiazepines and barbiturates exert their pharmacological actions via interactions with a discrete neuronal site on the GABAA Receptor-Benzodiazepine Receptor-Chloride Ion Channel Complex.

Unlike other neurotransmitter receptors such as DA and 5-HT discussed earlier, the model of the GABA receptor consists of a multiple receptor complex which provides binding sites for a variety of drugs and/or endogenous biologically active compounds.  Stimulation of these binding sites contributes to the responses related to the GABAergic system itself.  For this reason it is referred to as a GABAA Receptor-Benzodiazepine Receptor-Chloride Ion Channel Complex.  You should refer to pages 220 to 226 of the text for a discussion of the GABAergic synapse as the primary site of action of benzodiazepines and also refer to pages 194 to 197 for a brief discussion of how some anticonvulsants work via activation of GABAergic receptors.

 

Objectives

A student should be able to:

1.             Reproduce the biosynthetic and metabolic pathways of GABA.

2.             Identify the step at which sodium valproate exerts its anticonvulsant response in the above pathways.

3.             Reproduce the model of the GABAA Receptor-Benzodiazepine Receptor-Chloride Channel Complex and explain the effect on the receptor and the biological response of each type of drug or endog­enous compound, which interacts with the receptor complex.

4.             Explain the location and function of GABA’s presynaptic receptor.

5.             Account for the mechanism of action of baclofen and its structural relationship to GABA and its structural differences from the more classical muscle relaxants.

6.             Account for the mechanism of action of irreversible GABA transaminase (GABA-T) inhibitors.

7.             Identify the receptor bound conformation of GABA.

8.             Explain why GABA must be present in order for barbiturates or benzodiazepines to exert their pharmacological activity.

  

Biosynthesis and Metabolism of GABA:

GABA metabolism is shown in Figure 1.

                 

                                          Figure 1.  Biosynthetic Pathway and Metabolism of GABA

 

GABA is formed in neurons by the decarboxylation of the amino acid L-glutamic acid.  The rate-determining enzyme which catalyzes this step is L-glutamic acid decarboxylase (GAD).  The essential cofactor of the enzyme is pyridoxal phosphate (Vitamin B6).  The enzyme responsible for eliminating GABA is GABA transaminase (GABA-T).  This enzyme also depends on pyridoxal phosphate as its cofactor.  The resulting product, succinic semialdehyde (SSAD) is converted to succinic acid, which has negative-feedback inhibition on the enzyme GAD, thereby decreasing the conversion of L-glutamic acid to GABA.  Therefore, if insufficient quantities of GABA are present, GAD is activated due to the lack of end-product inhibition resulting from a lower concentration of the end-product, succinic acid.  An example of an anticonvulsant agent working by preventing this feedback inhibition is sodium valproate (Divalproex SodiumÒ).  Its structure is shown in Figure 2.         

 

                          

                        

                      Figure 2.  Structure of Sodium Valproate

 

Sodium Valproate has been used successfully to control epilepsy by blocking succinic semialdehyde dehydrogenase (SSAD dehydrogenase).  As succinic acid production decreases, the activity of GAD increases, which results in a higher concentration of the inhibitory neurotransmitter, thus preventing convulsive seizure formation.  (Please note that sodium valproate may potentiate the action of other CNS depressants such as benzodiazepines and barbiturates.)

On the other hand, compounds possessing the ability to inhibit GAD, the key enzyme in GABA biosynthesis, can cause seizures and convulsions!  The enhanced excitability of neurons results from a decrease in the concentration of GABA.  A specific inhibitor of this enzyme is allylglycine (Figure 3).

 

                        

                          Figure 3.  Allylglycine

 

GABA-T (GABA transaminase) Inhibitors:

Another target for anticonvulsant drug action is GABA transaminase (GABA-T), the key metabolic enzyme for GABA degradation.  There exist both reversible and irreversible GABA-T inhibitors.  Reversible GABA-T inhibitors competitively inhibit the enzyme, therefore if not given in sufficiently large enough quantities the inhibition could easily overcome, making them less desirable as anticonvulsants.   On the other hand, the inhibitory activity resulting from irreversible GABA-T inhibitor is not as easily overcome which creates a more efficacious outcome.  There are two ways to irreversibly inhibit GABA-T enzymes.  Gabaculine exerts its activity by covalently binding to Vitamin B6, the essential cofactor for GABA-T reaction (Figure 4).

                                   Figure 4.  Gabaculine and how it binds Vitamin B6

But as a result of only binding to the cofactor, the inhibitory action exhibited by Gabaculine can be overcome by increasing vitamin B6 intake. Thus, for Gabaculine to be effective as an anticonvulsant we need to restrict dietary intake of vitamin B6.  (Q.1. Explain why it is not advisable to restrict dietary intake of vitamin B6 to insure the GABA-T inhibitory action of Gabaculine?).  Therefore, only g-Vinyl-GABA (Vigabatrin®), a different type of irreversible GABA-T inhibitor, has been approved for use as an anticonvulsant because it covalently binds GABA-T as well as vitamin B6 (Figure 5).

 

 

 

Figure 5.        g-Vinyl-GABA and how it irreversibly inhibits GABA-T

 

 

The GABAA Receptor-Benzodiazepine Receptor-Chloride Ion Channel Complex

 

Figure 6 depicts a model of GABAA receptor-benzodiazepine receptor-chloride ion channel complex.  The inhibitory nerve transmission related to the complex appears to be controlled by the action of several compounds which affect nerve transmission by interacting with individual receptors located in such close proximity that each exerts an effect on the chloride ion channel of a nerve.

 

 

 

Figure 6.  The GABAA Receptor-Benzodiazepine Receptor-Chloride Ion Channel Complex

 

It has been shown that this receptor complex contains a receptor for the inhibitory neurotransmitter GABA, an additional binding site for the benzodiazepines (BZDs) and a third binding site for some barbiturates and convulsants such as picrotoxin (see page 271 of your text for structures and a brief discussion of how picrotoxinin works as an analeptic agent).

It is apparent that if the convulsants and anticonvulsants act on the same site, they must also exert opposite effects.  Agonist and antagonist activity at the GABA receptor is related to the ability of these compounds to open or close the chloride channel.  It has been found that when GABA and benzodiazepines act on their receptors, they enhance the binding of each.  It is hypothesized that the binding of a BZD agonist to the receptor induces a conformational change which allosterically modulates the binding of GABA (i.e., BZD binding induces the GABA receptor to shift from a low affinity state to a high affinity state and also stabilizes the receptor in a conformation that permits the ion channel to remain open).  Similarly GABA binding also enhances BZD binding to its receptor via the same mechanism.  For this reason, GABA receptor agonists and benzodiazepine receptor agonists are positive allosteric effectors to each other.

The anticonvulsant activity of some barbiturates such as pentobarbital is also mediated through their action at the picrotoxin/barbiturate binding site which increase the influx of chloride ions causing hyperpolarization and thereby preventing the cell from firing.   Again, barbiturates facilitate GABA binding via a positive allosteric effect on GABA receptors similar to BZDs. This conclusion is based on the observation that pentobarbital blocks convulsions induced by GABA antagonists or by maximal electroshock seizure (MES) and also on the fact that pentobarbital’s anticonvulsant effect, but not its hypnotic effect, could be blocked by bicuculline.

On the other hand, convulsants such as picrotoxinin, bind to this same site thereby prevents GABA binding by inducing an opposite conformational change that cause a closing of the chloride channels, giving rise to their convulsant action.  (Q.2.  It has been suggested that in the absence of GABA, benzodiazepines would be ineffective, Explain this using the GABA receptor complex model).

 

GABA Agonists

Certain compounds have been shown to act directly with the GABA receptor.  The receptor-bound conformation of GABA has been suggested through structure-activity relationship studies and is shown in Figure 7.

 

Figure 7.  Receptor-bound Conformation of GABA

 

Structural evidence substantiating this conclusion comes from the potent GABA agonist, THIP and  (Figure 7).  THIP binds to the GABA receptor with high affinity and lacks the flexibility possessed by GABA, thereby giving insight to the receptor-active conformation.

Progabide (GabreneÒ), a prodrug of GABA, is a broad-spectrum anticonvulsant which bears some structural resemblance to that of benzodiazepines.  However, its anticonvulsant action is due to in vivo conversion to GABA upon entering the CNS and thus exhibits a direct action on the GABA receptor.  (Q.3. Show by chemical equation how Progabide is converted to GABA in vivo?).

The only compound shown to be a GABA antagonist is (+)-bicuculline (Figure 8), a known CNS convulsant from natural sources.

 

Figure 8.  Progabide (prodrug of GABA) and (+)-Bicuculline (GABA antagonist)

While agonists and antagonists for the GABA binding site have been discovered, none are used clinically at the present time.

 

Benzodiazepine Receptor Agonists

Benzodiazepines have a potent interaction with the GABA inhibitory neurotransmitter system.  It has been shown that benzodiazepines act allosterically to increase the affinity of the receptor for GABA, thus allowing the chloride channel to open.  Structures of two benzodiazepine agonists are given in Figure 9.

 

             

                       Figure 9.  Benzodiazepine Agonists

 

 

 

Benzodiazepine Receptor Antagonists

Flumazenil (Figure 10) is a pure benzodiazepine receptor antagonist which only prevents benzodiazepines from binding to their receptor but does not affect the GABAA receptor.  For this reason, it can be used in the treatment of a benzodiazepine overdose.

                      

 Figure 10.  Flumazenil (AnexateÒ)- Benzodiazepine Antagonist

 

 

Inverse Agonists of the Benzodiazepine Receptor

Recently, a new type of drug has been discovered - referred to as inverse agonists.  These compounds bind to benzodiazepine receptors and exert the opposite effect to that of conventional benzodiazepines, producing signs of increased anxiety and convulsions.  ß-CCE (ß-carboline carboxylate ethyl ester) is an example (Figure 11).  Flumazemil also prevents b-CCE from binding to its receptor.

 

             

Figure 11.  ß-CCE - Inverse Agonist

Compounds Affecting GABA Release and Reuptake

 

There are also presynaptic autoregulatory receptors for GABA.  These receptors, when stimulated by GABA, prevent further release of GABA from the GABAergic neurons.

A number of compounds have been found which increase or inhibit GABA release.  Baclofen, a selective GABAB agonist, (Figure 12) enhances the release of GABA and is used clinically as a muscle relaxant.  Note its similarity in structure to GABA, and its structural difference from most of the muscle relaxants.  (Q.4.  Redraw the structure of baclofen and encircle the portions resembling GABA).  It should be pointed out that the exact location and the functional role of the GABAB receptor are still unknown.  However, it appears to be located on the presynaptic glutaminergic neurons which regulate GABA release. Please note that the presynaptic autoreceptors in the GABAergic neurons are not the same as GABAB receptors since theirs activation will inhibit further release of GABA from the GABA neurons.

 

                  

              Figure 12.  Baclofen (LioresalÒ)

 

Most muscle relaxants are glycols or glycol derivatives (esters or ethers).  Some of these are depicted in Figure 13. These do not act as GABAergic compounds.

              

                       Figure 13.  Two glycol derivative muscle relaxants

 

Several compounds have been shown to inhibit GABA uptake.  Nipecotic acid and R(+)-4-methylaminocrotonic acid (Figure 14), increase GABA levels at postsynaptic receptors as a result of inhibiting the uptake of the neurotransmitter, GABA.  Tiagabine, a derivative of nipecotic acid, is a potent GABA uptake inhibitor currently in clinical trials as an anticonvulsant.  Thus, it is possible to design potent uptake inhibitors with very different structure features.

 

 

               

                             Figure 14.  GABA uptake inhibitors

Gabapentin (NeurontinÒ) is an anticonvulsant with no direct action on the GABA receptor even though it is structurally related to GABA. It is not an inhibitor of GABA reuptake or degradation by GABA-T.  For this reason, its anticonvulsant action is said to be mediate via a GABA independent mechanism.   However, recent observation that (R)-3-methyl-GABA (Figure 15) enhances GABA biosynthesis by activating the enzyme GAD.  Thus, it is possible that gabapentin may also act by this mechanism.

 

  

Figure 15. Gabapentin and (R)-3-methyl-GABA

 

Future Directions

 

Several subtypes of benzodiazepine receptors have been identified.  It is possible that each of these receptor subclasses may independently mediate the anxiolytic, sedative, muscle relaxant, anticonvulsant, and other similar properties, respectively, associated with benzodiazepines.  The BZ1 (w1) receptor subtype has been shown to be associated with the hypnotic and sedative activity of benzodiazepines, while the BZ2 (w2) receptor subtype has been associated with the anxiolytic, anticonvulsant, and muscle relaxant effects of benzodiazepines.  It should be noted that at least one other benzodiazepine receptor subtype, BZ3 (w3), has been identified.

The discovery of the benzodiazepine receptor and its subtypes has lead to the identification of several non-benzodiazepine agents.  Some of these agents have been shown to be more selective than benzodiazepines.  The structure of one such agent is given in Figure 16.

 

                      

                     Figure 16.  TZP

 

The compound TZP has an increased affinity for the w1 receptor subtype and lower affinity for the w2 receptor.  Recently, the location and a possible functional role for the diazepam-binding inhibitor (DBI), a neuropeptide that binds with high affinity to mitochondrial DBI receptors in the extraneuronal glial cells (supporting cells of the nervous system), has been suggested.  DBI receptors regulate transfer of cholesterol into glial cells where neurosteroids such as pregnenolone sulfate and 3a-,5a-tetrahydro-deoxycorticosterone are synthesized.  These neurosteroids, in turn, bind and modulate the behavior of neuronal GABA receptors.  The discovery of DBI receptors in the glial cell membranes have provided an additional new target for drug design and at the same time allows us to understand how atypical anxiolytic-hypnotics such as suriclone, zopiclone (ImovaneÒ), and related analogues [Alpidem (AnanxylÒ), Zolpidem (StilnoxÒ)] work to induce a conformational change at the BZD site. (Figure 17)

 

Figure 17.  Drugs acting on the DBI binding site

 

In addition to the discovery of non-benzodiazepine agents, non-barbiturate agents have also been found (Figure 18).

 

                  

                       Figure 18.  Etazolate

Etazolate binds to the same site as barbiturates even though it does not possess the traditional barbiturate structure.  This non-barbiturate anxiolytic agent has the advantage of not inducing liver enzymes and has considerably less addicting liability.