Janet L. Fisher

The anticonvulsant stiripentol acts directly on the GABAA receptor as a positive allosteric modulator

Stiripentol (STP) has been used as co-therapy for treatment of epilepsy for many years. Its mechanism of action has long been considered to be indirect, as it inhibits the enzymes responsible for metabolism of other anti-convulsant agents. However, a recent report suggested that STP might also act at the neuronal level, increasing inhibitory GABAergic neurotransmission. We examined the effect of STP on the functional properties of recombinant GABA(A) receptors (GABARs) and found that it was a positive allosteric modulator of these ion channels. Its activity showed some dependence on subunit composition, with greater potentiation of alpha3-containing receptors and reduced potentiation when the beta1 or epsilon subunits were present. STP caused a leftward shift in the GABA concentration-response relationship, but did not increase the peak response of the receptors to a maximal GABA concentration. Although STP shares some functional characteristics with the neurosteroids, its activity was not inhibited by a neurosteroid site antagonist and was unaffected by a mutation in the alpha3 subunit that reduced positive modulation by neurosteroids. The differential effect of STP on beta1- and beta2/beta3-containing receptors was not altered by mutations within the second transmembrane domain that affect modulation by loreclezole. These findings suggest that STP acts as a direct allosteric modulator of the GABAR at a site distinct from many commonly used anti-convulsant, sedative and anxiolytic drugs. Its higher activity at alpha3-containing receptors as well as its activity at delta-containing receptors may provide a unique opportunity to target selected populations of GABARs.

Effect of the α subunit subtype on the macroscopic kinetic properties of recombinant GABAA receptors

Abstract The GABAA receptors (GABARs) are chloride-permeable ligand-gated ion channels responsible for fast inhibitory neurotransmission. These receptors are structurally heterogeneous, and in mammals can be formed from a combination of sixteen different subunit subtypes. Much of this variety comes from the six different α subunit subtypes. All neuronal GABARs contain an α subunit, and the identity of the α subtype affects the pharmacological properties of the receptors. The expression of each of the different α subtypes is regulated developmentally and regionally and changes with both normal physiological processes such development and synaptic plasticity, and pathological conditions such as epilepsy. In order to understand the functional significance of this structural heterogeneity, we examined the effect of the α subtype on the receptors response to GABA. Each of the six α subtypes was transiently co-expressed with the β3 and γ2L subunits in mammalian cells. The sensitivity to GABA was measured with whole-cell recordings. We also determined the activation, deactivation, desensitization, and recovery kinetics for the six isoforms using rapid application recordings from excised macropatches. We found unique characteristics associated with each α subunit subtype. These properties would be expected to influence the post-synaptic response to GABA, creating functional diversity among neurons expressing different α subunits.

The natural products magnolol and honokiol are positive allosteric modulators of both synaptic and extra-synaptic GABAA receptors

The National Center for Complementary and Alternative Medicine (NCCAM) estimates that nearly 40% of adults in the United States use alternative medicines, often in the form of an herbal supplement. Extracts from the tree bark of magnolia species have been used for centuries in traditional Chinese and Japanese medicines to treat a variety of neurological diseases, including anxiety, depression, and seizures. The active ingredients in the extracts have been identified as the bi-phenolic isomers magnolol and honokiol. These compounds were shown to enhance the activity of GABA(A) receptors, consistent with their biological effects. The GABA(A) receptors exhibit substantial subunit heterogeneity, which influences both their functional and pharmacological properties. We examined the activity of magnolol and honokiol at different populations of both neuronal and recombinant GABA(A) receptors to characterize their mechanism of action and to determine whether sensitivity to modulation was dependent upon the receptors subunit composition. We found that magnolol and honokiol enhanced both phasic and tonic GABAergic neurotransmission in hippocampal dentate granule neurons. In addition, all recombinant receptors examined were sensitive to modulation, regardless of the identity of the α, β, or γ subunit subtype, although the compounds showed particularly high efficacy at δ-containing receptors. This direct positive modulation of both synaptic and extra-synaptic populations of GABA(A) receptors suggests that supplements containing magnolol and/or honokiol would be effective anxiolytics, sedatives, and anti-convulsants. However, significant side-effects and risk of drug interactions would also be expected.

Dynamic temporal and spatial regulation of mu opioid receptor expression in primary afferent neurons following spinal nerve injury

Despite using prescribed pain medications, patients with neuropathic pain continue to experience moderate to severe pain. There is a growing recognition of a potent peripheral opioid analgesia in models of inflammatory and neuropathic pain. The goal of this study was to characterize the temporal and spatial expression of mu opioid receptor (mOR) mRNA and protein in primary afferent neurons in a rat L5 spinal nerve ligation model of persistent neuropathic pain. Bilateral L4 and L5 dorsal root ganglia (DRGs), L4 and L5 spinal cord segments, and hind paw plantar skins were collected on days 0 (naïve), 3, 7, 14, and 35 post-spinal nerve ligation or post-sham surgery. We found that expression of mOR mRNA and protein in primary afferent neurons changed dynamically and site-specifically following L5 spinal nerve ligation. Real-time RT-PCR, immunohistochemistry, and Western blot analysis demonstrated a down-regulation of mOR mRNA and protein in the injured L5 DRG. In contrast, in the uninjured L4 DRG, mOR mRNA transiently decreased on day 7 and then increased significantly on day 14. Western blot analysis revealed a persistent increase in mOR protein expression, although immunohistochemistry showed no change in number of mOR-positive neurons in the uninjured L4 DRG. Interestingly, mOR protein expression was reduced in the skin on days 14 and 35 post-nerve injury and in the L4 and L5 spinal cord on day 35 post-nerve injury. These temporal and anatomically specific changes in mOR expression following nerve injury are likely to have functional consequences on pain-associated behaviors and opioid analgesia.

A mutation in the GABAA receptor α1 subunit linked to human epilepsy affects channel gating properties

A genetic component is associated with the development of many forms of epilepsy. Recently, mutations in the GABAA receptor have been linked to several inherited epilepsies. One of these mutations is a non-conservative change of alanine to aspartate in the third transmembrane domain of the alpha1 subunit. To determine the functional consequences of this alteration, mutated alpha subunits were transiently transfected along with wild-type beta3 and gamma2L subunits into HEK-293T cells. The mutated alpha1(A294D) subunit reduced GABA sensitivity of the receptor, increased the deactivation rate and slowed desensitization. The mutation caused a reduction in channel open time but no change in single channel conductance. Studies with additional mutants, altering the charge and/or size of the side-chain, indicated that both size and hydrophobicity of the residue at this location influence channel gating. The effects on GABA sensitivity, deactivation rate and channel open time are consistent with a reduced efficacy of channel gating, and would be expected to decrease GABAergic neurotransmission. The alpha1 subtype is the most widely expressed of the alpha subunits, with expression increasing throughout development. Therefore, production of the mutated subunit could cause global hyperexcitability throughout the brain, leading to generalized seizures with juvenile onset.

Subunit‐specific desensitization of heteromeric kainate receptors

Kainate receptor subunits can form functional channels as homomers of GluK1, GluK2 or GluK3, or as heteromeric combinations with each other or incorporating GluK4 or GluK5 subunits. However, GluK4 and GluK5 cannot form functional channels by themselves. Incorporation of GluK4 or GluK5 into a heteromeric complex increases glutamate apparent affinity and also enables receptor activation by the agonist AMPA. Utilizing two‐electrode voltage clamp of Xenopus oocytes injected with cRNA encoding kainate receptor subunits, we have observed that heteromeric channels composed of GluK2/GluK4 and GluK2/GluK5 have steady state concentration–response curves that were bell‐shaped in response to either glutamate or AMPA. By contrast, homomeric GluK2 channels exhibited a monophasic steady state concentration–response curve that simply plateaued at high glutamate concentrations. By fitting several specific Markov models to GluK2/GluK4 heteromeric and GluK2 homomeric concentration–response data, we have determined that: (a) two strikingly different agonist binding affinities exist; (b) the high‐affinity binding site leads to channel opening; and (c) the low‐affinity agonist binding site leads to strong desensitization after agonist binding. Model parameters also approximate the onset and recovery kinetics of desensitization observed for macroscopic currents measured from HEK‐293 cells expressing GluK2 and GluK4 subunits. The GluK2(E738D) mutation lowers the steady state apparent affinity for glutamate by 9000‐fold in comparison to GluK2 homomeric wildtype receptors. When this mutant subunit was expressed with GluK4, the rising phase of the glutamate steady state concentration–response curve overlapped with the wildtype curve, whereas the declining phase was right‐shifted toward lower affinity. Taken together, these data are consistent with a scheme whereby high‐affinity agonist binding to a non‐desensitizing GluK4 subunit opens the heteromeric channel, whereas low‐affinity agonist binding to GluK2 desensitizes the whole channel complex.

The effects of stiripentol on GABAA receptors

The anticonvulsant stiripentol (Diacomit™) has been shown to have a positive impact on control of seizures for many patients with Dravet syndrome. As with most antiepileptic drugs, stiripentol has multiple mechanisms of action. Its direct anticonvulsant activity is likely due to enhancement of inhibitory, γ‐aminobutyric acid (GABA)ergic neurotransmission. Stiripentol was shown to increase the activity of both neuronal and recombinant GABAA receptors at clinically relevant concentrations. At recombinant receptors, stiripentol was found to act through a unique site in a subunit‐dependent manner. Positive modulation by stiripentol was most effective at GABAA receptors containing an α3 subunit. The expression of the α3 subunit is developmentally regulated, with highest levels in the immature brain. This subunit selectivity may explain the greater clinical efficacy of stiripentol in childhood‐onset epilepsies, including Dravet syndrome.

Identification of Structures within GABAA Receptor α Subunits That Regulate the Agonist Action of Pentobarbital

Barbiturates act on GABAA receptors (GABARs) through three distinct mechanisms, resulting in positive allosteric modulation, direct activation, and inhibition. These effects are observed at different concentrations and are differentially affected by some mutations and by the receptors subunit composition. Mammalian GABARs can be formed from a combination of 16 different subunit subtypes. Although the effect of barbiturates depends largely on the β subunit, their agonist activity is substantially influenced by the α subunit subtype. Pentobarbital is a more effective agonist than GABA only when receptors contain an α6 subunit. Results from chimeric α1/α6 subunits suggested that structural differences within the extracellular N-terminal domain were responsible for this characteristic. Within this domain, we examined 15 amino acid residues unique to the α6 subtype. Each of these sites was individually mutated in the α6 subunit to the corresponding residue of the α1 subunit. The effect of the mutation on direct activation by pentobarbital was determined with whole-cell electrophysiological recordings. Our results indicate that only one of these mutations, α6(T69K), altered pentobarbital efficacy. This single mutation reduced the response to pentobarbital to a level intermediate to the wild-type α1β1γ2L and α6β1γ2L isoforms. The mutation did not affect the sensitivity of the receptor to GABA but did reduce the efficacy of etomidate, another i.v. anesthetic with activity similar to pentobarbital. The reverse mutation in the α1 subunit (K70T) did not alter the response to pentobarbital. This is the first identification of a structural difference in GABAR α subtypes that regulates direct activation by barbiturates.

Microscopic kinetic determinants of macroscopic currents: insights from coupling and uncoupling of GABAA receptor desensitization and deactivation.

The time course of inhibitory postsynaptic currents (IPSCs) reflects GABAA receptor deactivation, the process of current relaxation following transient activation. Fast desensitization has been demonstrated to prolong deactivation, and these processes have been described as being ‘coupled’. However, the relationship between desensitization and deactivation remains poorly understood. We investigated the ‘uncoupling’ of GABAA receptor macroscopic desensitization and deactivation using experimental conditions that affected these two processes differently. Changing agonist affinity preferentially altered deactivation, changing agonist concentration preferentially altered macroscopic desensitization, and a pore domain mutation prolonged deactivation despite blocking fast desensitization. To gain insight into the mechanistic basis for coupling and uncoupling, simulations were used to systematically evaluate the interplay between agonist affinity, gating efficacy, and desensitized state stability in shaping macroscopic desensitization and deactivation. We found that the influence of individual kinetic transitions on macroscopic currents depended not only on model connectivity, but also on the relationship among transitions within a given model. In addition, changing single rate constants differentially affected macroscopic desensitization and deactivation, thus providing parsimonious kinetic explanations for experimentally observed uncoupling. Finally, these findings permitted development of an algorithmic framework for kinetic interpretation of experimental manipulations that alter macroscopic current properties.

The Auxiliary Subunits Neto1 and Neto2 Reduce Voltage-Dependent Inhibition of Recombinant Kainate Receptors

Kainate receptors can be subject to voltage-dependent block by intracellular polyamines, which causes inward rectification of the current–voltage relationship. Sensitivity to polyamine block is largely determined by the identity of a residue within the pore domain that can be altered through RNA editing. This process causes replacement of the encoded glutamine(Q) with a positively charged arginine(R), eliminating polyamine inhibition and thus inward rectification. In neurons, kainate receptors can associate with the auxiliary subunits Neto1 or Neto2. These transmembrane proteins alter the trafficking, channel kinetics, and pharmacology of the receptors in a subunit-dependent manner. We found that coexpression of Neto subunits with recombinant GluK2(Q) kainate receptors greatly reduced inward rectification without altering calcium permeability. This effect was separate from modulation of channel kinetics, as mutations within the extracellular LDLa domain of the Neto proteins completely eliminated their effects on desensitization but only reduced their effects on rectification. Conversely, deletion of the intracellular C-terminal domain of Neto1 or Neto2 or neutralization of positively charged residues within this domain prevented the reduction in rectification but did not alter effects on channel kinetics. These results demonstrate new roles for Neto1 and Neto2 in regulating kainate receptor function and identify domains within these auxiliary subunits important for mediating their effects.