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Seizu

.eSeizures frequently result from an imbalance of excitation andinhibition due to a failure of inhibitory neurotransmission. Mostagents that enhance GABAA receptor (GABAAR) function haveantiseizure properties due to their ability to increase inhibitoryneurotransmitter tone. The evidence linking epilepsy withdysfunction of GABAergic inhibition is substantial, and has beenextensively reviewed14.

1. GABAARs and epilepsy

GABAARs are pharmacologically complex, with binding sites forbenzodiazepines (BZs), barbiturates, neurosteroids, general anes-thetics, loreclezole, and the convulsant toxins, picrotoxin andbicuculline. Protein subunits from seven different subunit fami-lies5 assemble to form pentameric6 transmembrane chloridechannels (Fig. 1). In mammals, 16 subunit subtypes have beencloned, including 6 a, 3 b and 3 g subtypes, as well as d, p7, e8 andu9 and alternatively spliced variants of the b2 and g2 subtypes.Subunit expression is regulated by region, cell type10 anddevelopmental stage11, reducing the number of isoformsexpressed in specic brain regions and individual neurons. Themost common composition includes two a1, two b2 and a singleg2 subunit; the d subunit is found instead of g in receptorsexpressed extrasynaptically. The subunits are arranged around a

central water-lled pore, which gates to conduct Cl ions whenGABA is bound (Fig. 1). Individual subunit subtypes conferdifferent sensitivities to GABAAR modulators including BZs

12,loreclezole13 and zinc ions14 (Table 1).

GABAARs are the target not only of the BZs, but other ASDsincluding barbiturates and agents like tiagabine and vigabatrin1

that increase GABA concentration at the synapse. Bicuculline andpicrotoxin, which are GABAAR antagonists, induce seizures inanimals and epileptiform activity in brain slice preparations.Several animal models of epilepsy have altered GABAAR number orfunction1,2,15. GABAAR subunit expression is altered in thehippocampi of experimental animals with recurrent seizures16

and in patients with temporal lobe epilepsy17,18. Angelmanssyndrome is a human neurodevelopmental disorder associatedwith severe mental retardation and epilepsy, which is linked to adeletion mutation on chromosome 15q11-1320 in a regionencoding the GABAAR b3 subunit

21. Two mutations in the g2subunit that impair GABAAR function

22, K289 M23 and R43Q24,have been linked to a human syndrome of childhood absenceepilepsy and febrile seizures, and a loss-of-function mutation inthe a1 subunit causes autosomal dominant Juvenile MyoclonicEpilepsy25. The R43Q mutation in the g2 subunit reduces BZsensitivity26 by altering GABAAR assembly

2729 and trapping thereceptor in the endoplasmic reticulum30. Other point mutations inGABAAR subunits have also been associated with generalizedepilepsies (see Fig. 2). Hence, GABAAR modulation by GABAergicASDs is likely critical to their antiseizure activity.

GABAAR-acting ASDs. Manipulation of subunit expression patterns or novel ASDs targeting the altered

receptors may provide a novel approach for seizure prevention.

2013 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

* Tel.: +1 501 686 7236; fax: +1 501 686 7850.

E-mail address: ljgreeneld@uams.edu

1059-1311/$ see front matter 2013 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.seizure.2013.04.015Review

Molecular mechanisms of antiseizure d

L. John Greeneld Jr.*

Dept. of Neurology, University of Arkansas for Medical Sciences, 4301W. Markham St.,

A R T I C L E I N F O

Article history:

Received 6 February 2013

Received in revised form 16 April 2013

Accepted 17 April 2013

Keywords:

Inhibition

Epilepsy

Antiepileptic drugs

GABA receptor

Seizures

Chloride channel

A B S T R A C T

The GABAA receptor (GABA

GABAARs s are used to ter

the subunit composition

actions at the GABAAR are

felbamate, retigabine, lo

antiseizure mechanisms. A

led to the development o

synthesis, transport or br

chloride gradient, which

additional target for GA

epilepticus and in chroni

jou r nal h o mep age: w wwg activity at GABAA receptors

500, Little Rock, AR 72205, United States

is a major target of antiseizure drugs (ASDs). A variety of agents that act at

ate or prevent seizures. Many act at distinct receptor sites determined by

he holoreceptor. For the benzodiazepines, barbiturates, and loreclezole,

primary or only known mechanism of antiseizure action. For topiramate,

mone and stiripentol, GABAAR modulation is one of several possible

pregnanolone, a progesterone metabolite that enhances GABAAR function,

anaxolone. Other agents modulate GABAergic tone by regulating the

down of GABA. GABAAR efcacy is also affected by the transmembrane

nges during development and in chronic epilepsy. This may provide an

rgic ASDs. GABAAR subunit changes occur both acutely during status

ilepsy, which alter both intrinsic GABAAR function and the response to

re

l s evier . co m/lo c ate /ys eiz

L.J. Greeneld Jr. / Seizure 22 (2013) 5896005902. Benzodiazepines

BZs were initially developed as anxiolytic agents in the 1950s.Chlordiazepoxide was introduced in 1960, followed by diazepam31

Fig. 1. Model of a GABAA receptor in the plasma membrane. A. A space-lling model of thwith the nicotinic acetylcholine receptor. There are two binding sites for GABA, betwee

subunits (arrow). C. A schematic view shows the topology of each subunit with a large ex

the second of which forms the chloride ion channel. Binding of GABA allows the channel

(IPSP). D. Putative arrangement of 5 subunits to form a pentamer with central chloride

Database PDB ID: 2BG9 from Unwin, N. (2005)198. Images modied from en.wikipedia

Table 1Antiseizure Drugs and their GABAAR Effects.

Anti-seizure drug Subunit specicity Site of action

Benzodiazepines a13, a5; g a/g interface,a1(H101)

Zolpidem a1 > a2,3; g a/g interface Barbiturates b subunits? M3 residues?

Loreclezole b2, b3 B2(N289) Ganaxolone a, b, d, # with e a1(Q241)

Topiramate a6 > a4 > a1-a2? unknown

Felbamate unknown unknown

Retigabine unknown unknown

Losigamone unknown unknown

Stiripentol a3, # with b1, e unknown Vigabatrin GABA transaminase

Gabapentin,

pregabalin

Glutamic acid decarboxylase

Valproic Acid Glutamic acid decarboxylase

Tiagabine GAT-1 (GABA transporter) and nitrazepam.32 In 1965, diazepam was rst used to treat statusepilepticus in humans.33 Clonazepam was introduced in the 1970sprimarily as an ASD,34 and clobazam, a 1,5 benzodiazepine, waslater developed as an ASD with less sedative effect.35 However, the

e pentomer in side view (A) and top view (B) based on the high sequence homology

n a and b subunits (bent arrows), and one for BZs between the alpha and gammatracellular loop containing a cysteine loop and 4 transmembrane domains (M1-M4),

to open and conduct Cl ions, resulting in the fast inhibitory post-synaptic potentialchannel lined by the M2 subunit. [Derived from the published structure: RCSB PDB

.org/wiki/GABA_A_receptor, used with permission (public domain)].

GABAAR action Other mechanisms

Left shift of GABA

C/R curve, "open frequency

Possible increased

GABAAR channel conductance

" channel open time &burst duration

Use-dependent Na+ channel block

Barbiturate-like? inhibits GABAAR at " concentrationBarbiturate-like, also "open frequency

Open channel block at high conc.

Enhanced GABA currents Na+/Ca2+ channel block, AMPA/kainate

receptor block

Barbiturate-like Blocks NMDA receptor currents

Increased GABA IPSCs Opens KCNQ2/3 potassium channels

Enhanced GABA

receptor current

Use-dependent Na+ channel block

Barbiturate-like CYP450 inhibition

increases [GABA]

" GABA synthesis? a2-d subunit of voltage-gated Ca2+ channel

" GABA synthesis? block of Na+, T-type Ca2+ channelsBlocks GABA reuptake

L.J. Greeneld Jr. / Seizure 22 (2013) 589600 591induction of tolerance limits their use as ASDs. The 1,5 BZs mayproduce less tolerance than the 1,4 BZs, but tolerance to 1,5 BZsdoes occur.36

BZ activity at GABAARs is a function of the drugs afnity for theBZ binding site and its intrinsic allosteric effect on the GABAAR. Theefcacy of individual compounds varies widely. Most BZs inclinical use are full agonists that maximally enhance GABAARactivity. Flumazenil, a competitive antagonist used to reverse BZ-induced sedation,37 binds to the BZ site without affecting GABAARfunction. Abecarnil,39 imidazenil,40 and bretazenil41 are partialagonists at the BZ site which have antiseizure efcacy in animalmodels and appear less prone to tolerance.42 Several b-carbolinesare inverse BZ agonists that inhibit GABA binding43 and caninduce seizures or anxiety.44

Antiseizure Activity. BZs are effective against most experimentalseizure types, but individual drugs vary in their potency/efcacy inspecic seizure models and their other clinical effects.45 BZs areparticularly effective against convulsive seizures induced bypentylenetetrazol46 and less effective against tonic seizuresinduced by maximal electroshock.47 BZs also slow the develop-ment of kindling.48

GABAAR Subunits and BZ Pharmacology. BZ augmentation ofGABAAR currents requires a g subunit, and the selectivity of BZresponsiveness is determined by which a subunits are present.5,49

The BZ binding site is located in a cleft between the extracellularamino termini of the a and g subunits.51 The a1 subunit results in a

Fig. 2. Model of a prototype GABAAR subunit (based on a1 subunit diagram from Olsen &generalized epilepsies (see also Macdonald et al., 2012200) in black, and locations of poreceptor with high afnity for the hypnotic, zolpidem, dening theBZ-1 (or V-1) receptor type.50 The a2 and a3 subunits result inreceptors with moderate zolpidem afnity, termed BZ-2 receptors.GABAARs containing the a5 subunit and/or the g3 subunit aresensitive to diazepam but not to zolpidem and are termed BZ-3receptors. GABAARs with the a4 or a6 subunits are insensitive tomost BZs.

The subunit composition not only determines the afnity forparticular BZs, but also the clinical/behavioral effect of the BZ atthat receptor. The role of the a subunits in BZ pharmacology wasrevealed by the discovery of a single histidine (H) residue found inall BZ-sensitive a subunits (H101 in the rat a1 subunit), but not inthe BZ-insensitive a4 or a6 subunits, which instead have acharged arginine (R) residue. This H residue was discovered in astrain of alcohol-non-tolerant rats, which were found to have aspontaneous point mutation in the a6 subunit (R100Q) that madetheir a6-containing GABAARs (found mostly in the cerebellum)diazepam-sensitive, accounting for their ethanol and BZ intoler-ance.53 Mutation of R100 to H in a6 dramatically increased BZbinding in this normally insensitive subunit, while mutation ofH101 to R in a1 reduced BZ sensitivity.52 BZ-insensitive a subunitmutations were subsequently knocked-in to identify BZactions at receptors containing that subunit. In homozygousa1(H101R) knock-in mice, the anxiolytic effect was intact, butBZs were not protective against pentylenetetrazol-inducedconvulsions and did not produce sedation or amnesia, suggesting

Tobin, 1990)199 showing approximate locations of point mutations associated with

int mutations associated with ASD sites of action. See text for details.

L.J. Greeneld Jr. / Seizure 22 (2013) 589600592that binding to the (wild type) a1 subunit is responsible forsedative, amnestic and antiseizure actions.54 Moreover, thesedative-hypnotic, zolpidem, showed no sedative effect ina1(H101R) mice.55 Unfortunately, these ndings underscorethe association between sedative and antiseizure efcacy at a1-containing GABAARs. Similarly, the anxiolytic

56 and myorelax-ant57 properties of BZs appear to derive from a2- and a3-containing GABAARs, while the a5 subunit was critical foramnestic effects.58 BZs may also have a true analgesic effectindependent of their sedative and anxiolytic actions, associatedwith the a2 and a3 more than a5 subunits.59 Since there is noevidence of biophysically distinct effects of BZs on receptorscomposed of different a subunits, the different behavioral effectsare likely due to the brain regions and neuronal populationsexpressing these specic GABAAR isoforms. New a2/a3 subunit-selective BZs appear to be anxiolytic but not sedating,60 and non-sedating antiseizure BZs that do not induce tolerance61 may alsobe possible.

BZ Actions at GABAARs. BZs increase the amplitude62 or decay

time63 of GABA-mediated inhibitory post-synaptic potentials(IPSPs). Current noise uctuation analysis64 and single channelstudies65 demonstrated that the BZs increased the openingfrequency of the GABAAR chloride channel. In patch-clamprecordings of CNS neurons, BZs produce a leftward shift of theconcentration-response curve for GABA,66 due to an increase in theafnity for GABA at its binding site, with no change in the kineticsof channel gating65 or single channel conductance. In contrast, aninverse agonist at the BZ site reduced the channel openingfrequency for a given GABA concentration. These ndings areconsistent with binding studies showing the allosteric interactionbetween the BZ and GABA binding sites. By increasing the afnityof the receptor for GABA through slowing the unbinding rate, theBZs increase the current produced by low GABA concentrations,but not by high GABA concentrations at which receptor binding issaturated, as observed in the synaptic cleft67. Thus, BZs generallydo not increase the amplitude of miniature inhibitory postsynapticcurrents (mIPSCs) from individual synapses, but instead prolongthe mIPSC decay phase68 by slowing the dissociation of GABA fromthe receptor.69 Prolongation of the mIPSC increases temporal andspatial summation of multiple synaptic inputs, which in turnincreases the amplitude of stimulus-evoked polysynaptic IPSCs.The BZs thus increase the inhibitory tone of GABAergic synapses,which reduces the hypersynchronous ring of neuron populationsthat underlies seizures.1

An alternative mechanism for BZ enhancement of GABAARcurrents has been proposed based on the controversial ndingthat BZs progressively increase GABAAR single channel conduc-tance.70 Unlike prior reports of a 27 pS main conductance leveland a 19 pS subconductance level,65 Eghbali et al.70 foundconductance levels ranging from 8 to 53 pS in response to GABAalone, and 7080 pS in the presence of diazepam, with up to 7-foldincrease in conductance observed when diazepam was added. Theincreases in conductance were seen predominantly in cell-attached patches onto neurons expressing native receptors, butwere also observed in outside-out patches. They found similarincreases in channel conductance induced by pentobarbital,71

neuroactive steroids,72 propofol,73 and GABA itself.74 The samegroup75 subsequently found that high conductance (>40 pS)GABAAR channels were not observed in recombinant a1-b1-g2GABAARs expressed in L929 cells, nor was conductance increasedby diazepam unless the GABAAR-associated protein (GABARAP)was also co-expressed, apparently facilitating clustering ofGABAAR proteins through its interaction with the cytoplasmicloop of the g2 subunit as occurs at synapses with native receptors.Since peptides mimicking the intracellular g2 loop (g2 381403)self-associate, and application of this peptide to the cytoplasmicsurface of inside-out patches prevented the diazepam-inducedincrease in conductance, they hypothesized that the apparentsingle channel conductance changes induced by BZs are due tosynchronized openings of multichannel clustered GABAARs viaconcerted action through the interacting cytoplasmic loops ofconjoined receptors. Such interactions might occur throughshared transmembrane domains between interacting receptors,as observed in G-protein-coupled receptors.76 This hypothesiscould be addressed using concatenated subunits, chimeras andother strategies. However, synchronization of multiple identicalchannels by such a mechanism should still require a discrete 9 pSunitary conductance of which the larger conductance states areinteger multiples, which has not been reported.

3. Barbiturates

Phenobarbital was synthesized by Emil Fischer at Bayer in 1911and introduced as an ASD by Alfred Hauptmann in 1912. Itstendency to produce sedation and cognitive slowing or confusion,as well as paradoxical hyperactivity in children, has curtailed itsuse in favor of more modern alternatives.77 In addition to theirenhancement of GABAAR currents, barbiturates also inhibitrepetitive action potential ring at neuronal sodium channels78

by reducing fractional open time and shifting the potential of half-maximal opening towards hyperpolarized potentials.79 Theseactions contribute to both their antiseizure action and theiradverse effect prole.

When co-applied with low concentrations of GABA, barbitu-rates increased the mean open time, but had no effect on thelengths of 3 distinct open state durations.80 The increase in meanopen time resulted from fewer openings in the shorter twodurations (O1 and O2) and more long duration (O3) openings.There was also an increase in long burst durations, but no changein the single channel opening frequency or in the closedfrequency duration histogram. The increase in channel opentime results in greater chloride current ux and increasedlikelihood that channel openings will summate, producing largerinhibitory currents. In the absence of GABA, high concentrationsof pentobarbital (EC50 0.33 mM) and phenobarbital (EC50 3 mM)directly activated GABAAR chloride currents with lower efcacy(smaller maximal currents) than GABA82. Barbiturate-activatedGABAAR currents were blocked by bicuculline and picrotoxin,and at high concentrations both phenobarbital and pentobarbi-tal produced open channel block that rapidly terminated theinduced currents. The concentrations involved in both directactivation and open channel block are far higher than usualtherapeutic levels, thus the main antiseizure mechanism is likelythe GABAAR allosteric effect in concert with synaptic andextrasynaptic GABA.

Unlike the BZs, barbiturate sensitivity does not require aspecic subunit composition. Homomeric b1 receptors expressedin Xenopus oocytes were directly activated by pentobarbital eventhough they were not responsive to GABA; these currents wereblocked by picrotoxin or penicillin but not bicuculline.83

Pentobarbital also induced current in both a1-b3 and b3homomeric GABAARs.

84 At saturating GABA concentrations,pentobarbital markedly potentiated a1-b3-d currents, increasingdesensitization and single channel open duration,85 suggestingthat barbiturates may be particularly effective at enhancing tonicGABAAR currents at extrasynaptic sites activated by low GABAconcentrations. The e subunit, which increases spontaneousGABAAR channel openings, signicantly reduced

86 but did notcompletely eliminate87 barbiturate responsiveness when co-expressed with a and b subunits. These ndings suggest thatbarbiturate sensitivity does not require either an a or a g subunitas it is present in b homomeric receptors; however, presence of

L.J. Greeneld Jr. / Seizure 22 (2013) 589600 593the d or e subunits that substitute for g can dramatically modifybarbiturate responsiveness by altering the efcacy of GABA foractivating the channel.

Chimera and mutagenesis studies have identied domains andresidues that contribute to barbiturate action, though in less detailthan for the BZs. Mutation of the proline (P228A) in the rsttransmembrane domain of the b1 subunit reduced barbiturateenhancement of GABA-evoked currents in a1-b1-g2L GABAARswhile increasing apparent GABA afnity, without otherwisealtering single channel kinetics.88 A chimera study usingconstructs created from the amino terminal end of the b3 subunitand the carboxyl terminal end of the r1 subunit, which formshomomeric GABAC receptors that are insensitive to barbiturates,found that residues of the b3 subunit involved in pentobarbitalbinding to GABAARs are located downstream from the middle ofthe M2 region.89 Similarly, mutation of a r1 tryptophan (W328) inthe third transmembrane domain to a hydrophobic residueproduced both allosteric and direct channel activation bypentobarbital.90

4. Loreclezole

Loreclezole has antiseizure activity in a variety of seizuremodels, acting more like a barbiturate than a BZ in that theincrease in seizure threshold produced by loreclezole waspotentiated rather than blocked by the BZ antagonist, umaze-nil.91 In the hippocampal slice, loreclezole, potentiated pairedpulse inhibition92 and inhibited epileptiform discharges inducedby low Ca2+ or low Mg2+. Loreclezole strongly potentiatedrecombinant GABAARs containing a b2 or b3 subunit but didnot enhance currents from b1-containing receptors.93 A singleasparagine residue (b2(N289) or b3(N290)) at the cytoplasmicend of the 2nd transmembrane domain confers sensitivity toloreclezole; this amino acid is a serine in the b1 subunit.94

Mutation of b1S290 to N conferred loreclezole sensitivity to b1-containing GABAARs, while mutation of b2N289 or b3N290 to Seliminated loreclezole enhancement. When both b1 and b3 wereco-expressed in the same receptor, loreclezole sensitivity wasabolished, suggesting a dominant effect of the b1 subunit.95

Coexpression with different alpha subunits altered the degree ofloreclezole potentiation (a1 = a2 = a3 > a5 > a4);96 expressionof a5 and b3 with the p subunit reduced loreclezole potentia-tion,97 while a1-b3-e receptors showed normal loreclezoleenhancement.87

At higher concentrations (> 6 mM) loreclezole causedconcentration-dependent inhibition of GABAAR currents byenhancing the rate of apparent desensitization.98 The effectwas inconsistent with open channel block as it was voltageindependent, non-competitive, and increased with increasingGABA concentration. The BZ site was not involved as theinhibition was not antagonized by umazenil and did not requirea g subunit. This nding has important clinical implications, sinceinadvertent high levels of drug could inhibit rather than enhanceGABAAR function and potentially trigger seizure activity.Loreclezole inhibited a1-b1-g2L GABAAR currents that werenot potentiated by low concentrations of loreclezole, suggestingseparate sites for enhancement and inhibition. At the singlechannel level, high loreclezole concentrations decreased a1-b1-g2L mean open time by decreasing the average durations of theopen states, and also increased the occurrence of a 20 ms closedstate. Loreclezole inhibition was equally effective when appliedto the intracellular side of the receptor, suggesting that itsinhibitory binding site was accessible from both sides of themembrane, and pre-application of loreclezole prior to GABAinhibited the subsequent GABAAR current, indicating thatbinding did not require an open channel. Loreclezole was alsofound to inhibit r1 homomeric GABAC receptors with an IC50 of0.5 mM, which was proposed as a rapid means of pharmacologicalidentication of these receptors.99

5. Ganaxolone

Ganaxolone (3a-OH-3b-methyl-5a-pregnan-20-one) is the3b-methylated synthetic analog of the neurosteroid, allopregna-nolone, a natural metabolite of progesterone that allostericallyenhances GABAAR current. It has a broad range of antiseizureactivity in animal epilepsy models100 including seizures inducedby bicuculline, t-butylbicyclophosphorothionate (TBPS, a highafnity ligand for the GABAAR picrotoxin site), aminophylline andcorneal kindling, and is well tolerated and effective againstseizures in humans. Ganaxolone is currently under evaluation forpartial onset seizures. It may have special utility in women withcatamenial epilepsy who have increased seizures during periods oflow progesterone in the menstrual cycle, as well as in children withinfantile spasms.101

The antiseizure effect of neurosteroids is not due to action at theprogesterone receptor (PR), as exogenous allopregnanolone pre-vented seizures in PR knockout mice.102 Moreover, progesteronesantiseizure effect requires metabolism to allopregnanolone, as itwas blocked by nasteride, a 5a-reductase inhibitor that blocksallopregnanolone synthesis from progesterone. Hence, the antisei-zure effect of allopregnanolone (and ganaxolone) is mediated byGABAARs.

Specic neurosteroids can allosterically modulate GABAARactivity, either positively or negatively, via distinct binding siteson GABAARs.

104 Since a variety of GABAAR-interacting steroidhormones are synthesized in the brain,105 these agents representendogenous modulators of GABAAR function. Allopregnanolone issynthesized in two steps from progesterone: 5a-reductaseconverts progesterone to 5a-dihydroprogesterone, then 3-ahydroxysteroid oxidoreductase reversibly converts 5a-dihydro-progesterone to 3a-OH-5a-pregnan-20-one (allopregnanolone).Addition of the 3b-methyl group on ganaxolone does not alterbinding to GABAARs, but prevents further metabolism of the 3a-OH group and thus prolongs its GABAAR-modulating actions.

100

Ganaxolone enhanced GABA and BZ binding via positive allostericmodulation of GABAAR activity, and at nanomolar concentrationspotentiated GABA-evoked currents at a1-b1-g2L, a2-b1-g2L ora3-b1-g2L GABAARs expressed in Xenopus oocytes, while directactivation of chloride ux occurred to a limited extent only atmicromolar concentrations.100

The effects of GABAAR-enhancing neurosteroids on singlechannel GABAAR currents were studied using androsterone(5a-androstan-3a-ol-17-one) and pregnanolone (5b-pregnan-3a-ol-20-one) but likely apply to other positive steroidGABAAR modulators including ganaxolone. Like the barbitu-rates and loreclezole, these agents increased the proportion ofopenings to the two longer open states (O2 and O3) withoutaltering the intrinsic durations of those states, and producedlonger burst durations.106 There was no change in singlechannel conductance. Unlike the barbiturates, however, theneurosteroids also increased single channel opening frequencyand reduced closed time durations in all but the shortest closedtime distributions (thought to represent intraburst closures).At high concentrations (10 mM), both agents reduced openchannel durations consistent with open channel (ickering)block.

The subunit selectivity of GABAAR-enhancing neurosteroidswas initially controversial. An early study107 of recombinantreceptors expressed from combinations of a1, a6, b3, g2 and dshowed loss of neurosteroid responsiveness with combinationscontaining the delta subunit. There was also reduced sensitivity of

L.J. Greeneld Jr. / Seizure 22 (2013) 589600594cerebellar granule neurons to neurosteroids later in in vitrodevelopment, when d subunit expression increases. These ndingswere interpreted as showing inhibition of neurosteroid respon-siveness by inclusion of the d subunit. However, mice in which thed subunit was knocked out showed decreased behavioralsensitivity to neurosteroids.108 Moreover, in receptors composedof a1 or a6, b3 and either g2L or d, the greatest potentiation bytetrahydrodeoxycorticosterone (THDOC) was seen in a1-b3-dGABAARs.

109 At high concentrations (1 mM), THDOC inhibited thisisoform. There is currently consensus that d subunit-containingreceptors are more sensitive to neurosteroid enhancement, thoughother subunits are also involved in mediating neurosteroid actions.Presence of the a6 subunit reduced neurosteroid sensitivity.110

THDOC (1 mM) enhanced a1-b3-d more than a6-b3-d currents,but increased the extent of desensitization and prolongeddeactivation for both receptor isoforms; a1-a6 and a6-a1chimeras (spliced in transmembrane domain M1) suggested thatdifferences in deactivation rate and its voltage-dependencecorrelated with N-terminal domains, while the extent of desensi-tization and its voltage-dependence correlated with C-terminaldomains.111 In dentate granule cells from epileptic animals, anincrease in a4 subunit expression was associated with decreasedneurosteroid enhancement.112 In receptors composed of one asubunit (from a2 through a5), b2 and g2S, neurosteroidpotentiation was dependent on the conserved glutamine residue(a1(Q241)) in the rst transmembrane domain of the respective asubunit.113 The d subunit did not affect neurosteroid binding butlikely inuenced the efcacy of neurosteroid potentiation. Muta-tion of a1Q241 to L abolished neurosteroid potentiation, whilemutation to W mimicked the effect of steroids and preventedfurther augmentation; the neighboring S240 residue also partici-pated in steroid binding.114 A subsequent study using concatenat-ed subunits demonstrated that a single functional binding site (notdisrupted by the Q241 mutation) was sufcient for neurosteroidmodulation.115

6. GABAAR effects of other ASDs

6.1. Topiramate

Topiramate is a heterotricyclic sulfamate with several mecha-nisms of action including inhibition of sodium and calciumchannels, inhibition of carbonic anhydrase, and augmentation ofGABA-evoked currents. Topiramates effects on GABAARs maycontribute both to its antiseizure efcacy and its side effect proleincluding memory problems, fatigue and psychomotor slowing. Itis approved for partial and generalized seizures, the LennoxGastaut syndrome, and migraine.

Topiramate inhibited voltage-gated sodium channels, with aleft shift of the steady state inactivation curve116 resulting inintermittent blockade of sustained repetitive action potentialring during prolonged depolarization.117 It also blocked repetitivering in hippocampal CA3 neurons from spontaneously epilepticrats, and reduced excitatory post-synaptic potentials andresponses to bath-applied glutamate, suggesting blockade ofpost-synaptic glutamate receptors,118 affecting kainate-evokedbut not NMDA-evoked currents.119 Topiramate also inhibited bothL-type and non L-type high voltage activated calcium currents120

and reduced bicarbonate production via inhibition of carbonicanhydrase,121 which may contribute to its antiseizure effect.

Topiramate (10 mM) enhanced chloride ux into cerebellargranule neurons stimulated by 10 mM GABA, but did notsignicantly increase chloride inux alone.122 It also enhancedGABA-evoked currents in cultured cortical neurons that wereinsensitive to the BZ, clonazepam, and clonazepam-potentiatedGABA currents in topiramate-insensitive neurons, conrming thattopiramates site of action on GABAARs is independent of the BZsite.123 Subunit selectivity studies have been inconsistent.Topiramate (1100 mM) reversibly inhibited Cl currents evokedby 110 mM GABA in Xenopus oocytes expressing a1-b2-g2S anda2-b2-g2S GABAARs, and reduced the apparent desensitization(current-fading rate) in a1-b2-g2S-expressing oocytes, butpotentiated GABA-evoked Cl currents and increased the desensi-tization rate in a6-b2-g2S GABAARs, with no effect on a4-b2-g2Sreceptors or mixed population GABAARs expressed from rat brainmRNA.124 In contrast, another study found that topiramate couldboth potentiate and directly activate b2 or b3-containingheteromeric GABAARs, with greatest effect on a4-b3-g2S;

125

positive or negative effects on b1-containing GABAARs dependedon the co-expressed alpha subunit. Additional studies are neededto clarify the site and mechanism of action.

6.2. Felbamate

Felbamate was launched as a promising novel ASD in 1993 withan uncertain mechanism of action, but its use was curtailed afterearly reports of aplastic anemia126 and hepatic failure.127

Felbamate inhibited binding of the GABA antagonist [3H]T-BOBwith a regional pattern different from that produced by GABAagonists, bicuculline, zinc or neurosteroids,128 and enhancedGABA-elicited Cl currents in cultured cortical neurons. Felbamateenhancement was not blocked by umazenil, and felbamate didnot affect pentobarbital potentiation or PTX inhibition of GABA-evoked currents, suggesting an independent site of action. Itprolonged the mean burst duration of GABA-activated singlechannel currents, suggesting a barbiturate-like effect.129 Felba-mate also blocked N-methyl-D-aspartate (NMDA) receptor cur-rents, an alternative possible antiseizure mechanism.130 Derivativecompounds including uorofelbamate and carisbamate that arenot associated with hematopoietic or hepatic toxicity may reviveinterest in felbamatelike agents.131

6.3. Ezogabine

Ezogabine (also known as retigabine) is a novel ASD effective ina variety of animal models. Its primary effect is enhancedactivation of heteromeric potassium channels composed of theKCNQ2 and KCNQ3 subunits132 which underlie the M currentthat is a major determinant of resting membrane potential andneuronal excitability. However, ezogabine also dose-dependentlyand reversibly potentiated GABAAR-dependent IPSC peak ampli-tude, decay times and total charge transfer.133 EPSCs wereunaffected, and paired pulse depression was unchanged, suggest-ing a post-synaptic effect at GABAARs. Ezogabine potentiatedGABA-induced currents in rat cortical neurons in a concentration-dependent fashion at 10 mM and above.132 This action was notantagonized by umazenil, indicating a non-BZ site of action.Subunit dependence and binding site are not known.

6.4. Losigamone

Losigamone is a novel ASD which inhibited the persistentcomponent of sodium currents in hippocampal neurons atdepolarized potentials, suppressed sustained repetitive ring134

and decreased the frequency of spontaneous action potentialswithout affecting miniature post-synaptic current amplitudes.135

Losigamone stimulated 36Cl inux into spinal cord neurons in theabsence of GABA, and potentiated 36Cl inux stimulated bysubmaximal GABA concentrations; both effects were blocked bybicuculline or picrotoxin.136 Losigamone did not affect the specicbinding of [3H]GABA, [3H]unitrazepam, or [35S]t-butyl-bicyclo-phosphorothionate (TBPS) to their receptors, and there was

L.J. Greeneld Jr. / Seizure 22 (2013) 589600 595no difference in the effect of the + or stereoisomers or the racemicmixture. The site of action at GABAARs is unknown.

6.5. Stiripentol

Stiripentol is as an adjunct ASD which was thought to act byinhibiting cytochrome P450 enzymes involved in metabolism ofconventional ASDs. Recently, stiripentol was found to enhancerecombinant GABAAR currents, with greater potentiation of a3-containing receptors and reduced potentiation with the b1 or esubunits.137 It caused a leftward shift in the GABA concentration-response relationship without increasing maximal GABA-evokedcurrents, and did not involve sites associated with neurosteroid orloreclezole potentiation.137 Saturating barbiturate sites withpentobarbital occluded stiripentol enhancement, and stiripentolincreased the duration but not the frequency of opening of GABAARchannels, suggesting a barbiturate-like mechanism.138

6.6. GABA-enhancing agents

An additional mechanism for enhancing GABAAR-mediatedinhibition involves increasing the concentration or duration ofGABA in the synaptic cleft and perisynaptic/extrasynaptic sites.Enhancing GABA synthesis or blocking its reuptake or catabolismcould prolong GABA IPSPs and increase perisynaptic spill-over,increasing tonic/extrasynaptic GABA currents which likely play amajor role in seizure prevention.

6.7. Gabapentin and pregabalin

Gabapentin was designed as a GABA analog, and some studieshave suggested that it modulates the action of the GABA syntheticenzyme, glutamic acid decarboxylase (GAD) and the glutamatesynthesizing enzyme, branched-chain amino acid transaminase,resulting in increased GABA synthesis.139 Gabapentin increasesnon-synaptic GABA responses from neuronal tissues in vitro andincreases GABA levels in brain.140 Its other (likely primary) mode ofaction involves binding to the a2-d binding site on L- or P/Q-typepresynaptic voltage-gated calcium channels, presumably inhibit-ing excessive neurotransmitter release141 by interfering withcalcium channel functional expression or trafcking142. Pregaba-lin, which binds to the a2-d calcium channel subunit with higherpotency, may have similar mechanisms of action. Most studiesemphasize the calcium channel as the primary site,143 though acontributory effect on GABA metabolism is possible.144

6.8. Valproic acid

Valproic acid may affect GABA production, among other ASDmechanisms. Valproate caused non-signicant increases in cere-bral GABA levels but elevated brain GAD activity signicantly.145

Valproic acid increased GABA synthesis and release in brainregions including substantia nigra,146 which is thought to beinvolved in the control of seizure generation and propagation.147 Italso reduced the release of gamma-hydroxybutyric acid andattenuated neuronal excitation induced by NMDA-type glutamatereceptors.147 Additional ASD mechanisms include enhancingsodium channel inactivation (like phenytoin) and reducing T-typeCa2+ channel currents (like ethosuximide),148 perhaps explainingits utility against both partial onset and absence seizures.

6.9. Tiagabine

Tiagabine is a derivative of nipecotic acid that binds to thepresynaptic GAT-1 GABA transporter and blocks GABA reuptake,without acting as a false neurotransmitter. In controlhippocampal slices, tiagabine alone induced a signicantchloride conductance, suggesting that GAT-1 activity controlsthe basal level of extracellular GABA.149 Tiagabine prolongedhippocampal IPSC duration in a lamina-specic fashion, withgreater effect in stratum radiatum (167%) than stratum oriens(115%).150 Tiagabine reduced the frequency of epileptiformdischarges in hippocampal slices exposed to low Mg2+ or 4-aminopyridine.151 The ability of tiagabine to prolong GABAcurrents was preserved in hippocampal slices from pilocarpine-treated epileptic rats, hence the GAT-1 transporter remains afunctional target for regulating GABA levels in this model oftemporal lobe epilepsy.152 Tiagabine was effective as an adjunctagent against partial onset seizures, though its prolongedtitration period, requirement for multiple daily doses and CNSside effect prole (dizziness, tremor, somnolence, mood dis-turbances and rare psychotic symptoms) have limited its use.153

6.10. Vigabatrin

Vigabatrin (g-vinyl-GABA) has a unique mechanism of action,functioning as a suicide inhibitor of GABA transaminase, theprimary enzyme for GABA catalysis. This leads to permanentinhibition of the affected enzyme, requiring synthesis of newGABA transaminase and hence prolonging the biological half-lifebeyond its pharmacokinetic half-life of 8 hours.154 It is effectiveagainst complex partial seizures,155 LennoxGastaut syndromeand West Syndrome, for which it has become an alternative toACTH156 particularly in children with tuberous sclerosis.157

Vigabatrin increases the extracellular concentrations of GABAboth in vitro158 and in vivo.159 The anti-seizure effects ofincreasing GABA concentration may be complex. Biphasicresponses to vigabatrin administration have been observed, withan early proconvulsant effect.160 A biphasic effect was also seen onafter-discharge duration with an early facilitation of the limbicpattern of epileptiform discharge and later suppression at higherdoses.161 Elevated GABA concentrations may increase neuronalsynchronization, particularly of thalamic neurons in absenceepilepsy, and there have been several reports of vigabatrinworsening absence seizures or precipitating absence statusepilepticus.162

A major limitation to the use of vigabatrin is the relatively highincidence of vision disturbance associated with chronic use. In 21of 30 children treated with vigabatrin who were screened for visiondisturbances despite no report of visual symptoms, 4 had visualeld constrictions, which did not improve after discontinuation ofthe drug.163 One study associated visual disturbances withinfantile spasms rather than the drug.164 The prevalence of visionproblems ranged from 10 to 40% of pediatric and adult patientsexposed to vigabatrin.165 This adverse effect may be directlyrelated to elevated GABA concentrations, as hippocampal neuronsgrown in depolarizing conditions (25 mM KCl) showed 20% loss ofMAP-2-positive neurons in the presence of vigabatrin, which wasmimicked by GABA (100 mM) and blocked by bicuculline orpicrotoxin.166 The cellular mechanism of this toxicity is notentirely clear, but in situations in which GABA is depolarizing,excessive GABA could promote calcium entry through L-type(slowly desensitizing) voltage-gated calcium channels,167 which inturn could result in activation of caspases and cell death.168

7. Excitatory GABAA currents

Early in CNS development, neurons express the Na+/K+/Cl

cotransporter, NKCC1, rather than the K+/Cl cotransporter, KCC2,which is expressed in adult neurons. NKCC1 increases intracellularCl resulting in a depolarizing Cl reversal potential, while KCC2exports Cl yielding the hyperpolarizing Cl reversal potential

L.J. Greeneld Jr. / Seizure 22 (2013) 589600596found in adult neurons.169 As a result, activation of GABAARs maybe excitatory during early development.170 Excitatory GABAARcurrents may play a trophic role in neuronal migration andconnectivity,171 but may also contribute to epileptogenesis.172

Endogenous GABA appears to be proconvulsant in early postnatalrat hippocampal slices, as GABAA antagonists blocked epileptiformactivity induced by depolarization with high external [K+]173.However, BZ and barbiturate antiseizure efcacy appear to beintact, likely because persistent opening of GABAAR channels (inthe presence of allosteric agents) may reduce the depolarizingchloride reversal potential, resulting in shunt inhibition.Alternatively, subthreshhold GABA-evoked depolarization mayinactivate sodium channels and prevent action potential ring.174

The current through GABAAR channels can also be altered bychanges in intracellular bicarbonate, [HCO3

],169 which can owthrough the channel.175 Changes in [HCO3

] may underlie reducedsynaptic GABA currents during development of BZ tolerance.176

Depolarizing GABAAR currents may also be a source of interictalspike activity, as observed in epileptic subiculum neurons inhippocampal brain slices from patients with temporal lobeepilepsy.177 Changes in the GABA current reversal potential mightalso explain why diazepam can be less effective in children withepileptic encephalopathies,178 and rarely can cause status epilep-ticus in patients with the Lennox Gastaut syndrome.179

Bumetanide, an inhibitor of NKCC-1 used clinically as a potentloop diuretic, has been proposed as an adjunctive agent to reduceintraneuronal chloride accumulation and reverse the depolarizingchloride gradient that makes GABAARs excitatory, particularly inthe neonatal setting.170 Bumetanide improved the responsivenessof hippocampal slices to phenobarbital in reducing epileptiformdischarges induced by low Mg2+,180 and also suppressed electro-graphic seizures in neonatal rats.172 Depolarizing GABA currentsmay occur in cortical more than subcortical brain regions, possiblyexplaining the electroclinical uncoupling that occurs with pheno-barbital in neonatal seizures.181

Bumetanide has been used successfully in a single reportedhuman neonate with seizures,182 and a clinical trial(NCT00830531) is in progress. However, elimination of thedepolarizing Cl gradient during development may have adverseconsequences; a permanent reduction in AMPAR-mediatedexcitatory neurotransmission and sensorimotor gating decitswere observed after bumetanide exposure in neonatal rats.183 Suchpotential adverse effects will have to be weighed against thebenets associated with seizure termination or prevention.

8. GABAAR subunit changes and ASD efcacy in epilepsy andstatus epilepticus

There is considerable evidence that GABAAR composition andfunction change with epilepsy, both acutely in the setting of statusepilepticus and in chronic epilepsy. During status epilepticus, thereis a rapid change in GABAAR function that reduces BZ sensitivity,

184

apparently due to activity-dependent internalization of GABAARsand replacement with BZ-insensitive receptors.185 Kainic acid-induced status epilepticus in young (postnatal day 9) rats alteredthe normal developmental pattern of GABAAR subunit matura-tion,186 preventing the normal switch from a2 to a1-containingGABAARs. These changes correlate with the nding in animalmodels15,184,187 and humans188,189 that BZs are effective early inthe course of status epilepticus but lose their potency and efcacyfor termination of status with longer seizure duration. Thesendings increase the urgency for treatment of status epilepticusearly in its course, and also suggest the need to nd alternativetreatments that retain efcacy in refractory status epilepticus, ormethods to prevent or reverse the GABAAR changes that occur withprolonged seizures.In the pilocarpine model of temporal lobe epilepsy, the a1subunit in dentate granule neurons was downregulated and a4subunit was upregulated,190 while in younger (postnatal day 20)animals, the a1 subunit was increased after status epilepticus.191

GABAAR subunit changes after pilocarpine-induced status areassociated with altered physiological properties including reducedneurosteroid sensitivity and an increase in a4-containing recep-tors colocalized with synapses rather than extracellular sites.112

Such changes might contribute to the propensity for spontaneousseizures or alternatively represent a functional adaptation toreduce seizure expression. Substitution of a1 with a4 mightreduce synaptic (phasic) but increase extrasynaptic (tonic)inhibition, and BZ sensitivity would be lost in a4-containingreceptors. Using an adenovirus vector to insert the a1 subunit genedriven by the a4 promoter resulted in a decrease in seizurefrequency, suggesting that the GABAAR subunit changes werecontributory toward epileptogenesis.192 Hence, altering GABAARsubunit expression in brain regions critical to seizure developmentmay provide a new therapeutic strategy for seizure prevention.Alternatively, novel ASDs targeting the specic epilepsy-associatedsubunit composition might provide a less invasive approachtoward seizure control.

Long term exposure to the GABAAR allosteric agentsthemselves can alter GABAAR composition and function. In ratpups exposed to therapeutic concentrations of diazepam orphenobarbital from postnatal day 10 through 40, then taperedfor 2 weeks and euthanized on postnatal day 90 showedincreased mRNA expression in dentate granule neurons forGAT-1, 3 and 4, GABAAR subunits a4, a6, b3, d and u and GABABreceptor subunit R1, and decreased mRNA expression for GAD65,GAD67 and GABAAR subunits a1 and a3.

193 Tolerance- anddependence-associated changes in GABAAR subunit compositionwith chronic use of BZs194,195 and barbiturates196,197 have beenreported, and may reect tolerance to either the antiseizure oradverse CNS effects. However, these ndings suggest that notonly seizures, but their prolonged treatment with GABAergicagents, can alter inhibitory neuronal function for extendedperiods or even permanently, with possible cognitive andbehavioral consequences that must be considered whencontemplating chronic GABAergic ASD use.

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L.J. Greeneld Jr. / Seizure 22 (2013) 589600600

Molecular mechanisms of antiseizure drug activity at GABAA receptorsGABAARs and epilepsyBenzodiazepinesBarbituratesLoreclezoleGanaxoloneGABAAR effects of other ASDsTopiramateFelbamateEzogabineLosigamoneStiripentolGABA-enhancing agentsGabapentin and pregabalinValproic acidTiagabineVigabatrin

Excitatory GABAA currentsGABAAR subunit changes and ASD efficacy in epilepsy and status epilepticusReferences