GABA EC50 Research Articles

Charged residues in the GABA-a receptor intracellular domain impact channel activation and desensitization.

Opening and closing of GABA receptor (GABAR) channels regulate inhibitory signaling in the brain. Mechanisms underlying channel open, closed, desensitized transitions remain unclear. Although GABAR cryo-EM structures provide details about its extracellular and transmembrane domains, little is understood about its intracellular domain (ICD) and its role in regulating channel gating. We constructed a GABAR homology model based on the 5HT-type-3 receptor, which revealed a helix (MX) at the C-terminal end of M3 that is lysine rich in the α1 (6 lysines) and γ2 (8 lysines) subunits. Charged residues in the α and γ subunits were mutated to alanine. Effects on GABA EC50 were measured using two-electrode voltage clamping of oocytes expressing α1β2γ2L WT and mutant GABARs. αK327A significantly increased GABA EC50 6-fold in comparison to WT and significantly reduced GABAR macroscopic desensitization. Extent of current desensitization after 10 seconds in saturating GABA (10 mM) was 6.86 ± 2.28% (n=7) versus WT (23.7 ± 7.29%, n=10). Neutralizing charge at αK328, K330 and K335 had no functional effects. γK328A, γ2K330A and γ2K332A significantly increased GABA EC50 by 2 to 3 fold. γ2K330A reduced GABAR desensitization (extent after 10 seconds 7.36 ± 2.37%, n=2). Our data indicate structural perturbations in the α and γ MX regions of the GABAR ICD alter GABAR channel activation and desensitization. Interestingly, in our GABAR structural model, α1R312 (in MX) interacts with α1R249 (M1-M2 linker). Single charge reversals at these positions increased GABA EC50 and the rate of GABA desensitization. A charge swap (R312E - E249R) rescued receptor function to WT suggesting a functional electrostatic interaction and that these residues form a salt bridge, providing support for our homology model.

Characterization of adjacent charged residues near the agonist binding site of the nematode UNC-49 GABA receptor

The UNC-49 receptor is a Cys-loop GABA receptor that is unique to the nematode phylum. The receptor differs from mammalian GABA receptors both in amino acid sequence and pharmacology which highlights its potential as a novel anthelmintic target. Sequence differences within and near the various ligand-binding loops of the nematode receptor suggest that there could be structural differences compared to mammalian receptors that result in different pharmacological and functional features. Here we investigated three residues in the UNC-49 receptor from the parasitic nematode Haemonchus contortus: K181, E183, and T230. Analysis of these residues was conducted via site-directed mutagenesis, electrophysiology, MD simulations, and mutant cycling analysis. In the UNC-49 receptor, E183 lies in close proximity to K181 where together they appear to play a role in GABA sensitivity and pharmacology, possibly interacting via an ionic bond. While the introduction of single alanine residues at each position separately had a negative impact on GABA EC50, the double alanine mutant (K181A/E183A) exhibited wildtype-level GABA EC50 and some differences in pharmacology. Overall, this study has revealed a potentially novel role for these two residues in nematode UNC-49 GABA receptors that could aid in understanding their function.

Pharmacological and Biophysical Characteristics of Picrotoxin-Resistant, δSubunit-Containing GABAA Receptors

GABAA receptors (GABAARs) play a crucial role in inhibition in the central nervous system. GABAARs containing the δ subunit mediate tonic inhibition, have distinctive pharmacological properties and are associated with disorders of the nervous system. To explore this receptor sub-class, we recently developed mice with δ-containing receptors rendered resistant to the common non-competitive antagonist picrotoxin (PTX). Resistance was achieved with a knock-in point mutation (T269Y; T6’Y) in the mouse genome. Here we characterize pharmacological and biophysical features of GABAARs containing the mutated subunit to contextualize results from the KI mice. Recombinant receptors containing δ T6’Y plus WT α4 and WT β2 subunits exhibited 3-fold lower EC50 values for GABA but not THIP. GABA EC50 values in native receptors containing the mutated subunit were in the low micromolar range, in contrast with some published results that have suggested nM sensitivity of recombinant receptors. Rectification properties of δ-containing GABAARs were similar to γ2-containing receptors. Receptors containing δ T6’Y had marginally weaker sensitivity to positive allosteric modulators, likely a secondary consequence of differing GABA sensitivity. Overexpression of δT6’Y in neurons resulted in robust PTX-insensitive IPSCs, suggesting that δ-containing receptors are readily recruited by synaptically released GABA. Overall, our results give context to the use of δ receptors with the T6’Y mutation to explore the roles of δ-containing receptors in inhibition.

Functional Characteristics of the Lepidopteran Ionotropic GABA Receptor 8916 Subunit Interacting with the LCCH3 or the RDL Subunit.

The ionotropic γ-aminobutyric acid (iGABA) receptor is commonly considered as a fast inhibitory channel and is an important insecticide target. Since 1990, RDL, LCCH3, and GRD have been successively isolated and found to be potential subunits of the insect iGABA receptor. More recently, one orphan gene named 8916 was found and considered to be another potential iGABA receptor subunit according to its amino acid sequence. However, little information about 8916 has been reported. Here, the 8916 subunit from Chilo suppressalis was studied to determine whether it can form part of a functional iGABA receptor by co-expressing this subunit with CsRDL1 or CsLCCH3 in the Xenopus oocyte system. Cs8916 or CsLCCH3 did not form functional ion channels when expressed alone. However, Cs8916 was able to form heteromeric ion channels when expressed with either CsLCCH3 or CsRDL1. The recombinant heteromeric Cs8916/LCCH3 channel was a cation-selective channel, which was sensitive to GABA or β-alanine. The current of the Cs8916/LCCH3 channel was inhibited by dieldrin, endosulfan, fipronil, or ethiprole. In contrast, fluralaner, broflanilide, and avermectin showed little effect on the Cs8916/LCCH3 channel (IC50s > 10 000 nM). The Cs8916/RDL1 channel was sensitive to GABA, but was significantly different in EC50 and Imax for GABA to those of homomeric CsRDL1. Fluralaner, fipronil, or dieldrin showed antagonistic actions on Cs8916/RDL1. In conclusion, Cs8916 is a potential iGABA receptor subunit, which can interact with CsLCCH3 to generate a cation-selective channel that is sensitive to several insecticides. Also, as Cs8916/RDL1 has a higher EC50 than homomeric CsRDL1, Cs8916 may affect the physiological functions of CsRDL1 and therefore play a role in fine-tuning GABAergic signaling.

Influence of GABA-A Receptor Intracellular Domain on Channel Activation and Desensitization

Rapid opening and closing of GABA-A receptor (GABAR) channels regulate signaling in the brain. Mechanisms underlying channel open, closed, desensitized transitions remain unclear. GABAR cryo-EM structures provide details about its extracellular and transmembrane domains, but little information exists about its intracellular domain (ICD) and its role in regulating channel gating. We constructed a GABAR ICD homology model based on a cryo-EM 5HT3AR structure, which revealed a helix (MX) at the C-terminal end of M3 and a helix (MA) at the N-terminal end of M4 projecting intracellularly. Four residues with potential interactions between these helices were mutated to alanine (α1K327A in MX, α1K390A in MA, and γ2K409A and γ2Y413A in MA). Mutations effects on GABA-EC50 and current desensitization were measured using two-electrode voltage clamping of oocytes expressing wild-type (WT) and mutant GABARs. α1K327A increased GABA EC50 ∼6-fold compared to α1β2γ2 WT (35.98 ± 1.03 μM, n=9) and significantly reduced GABAR desensitization. Extent of current desensitization after 60 seconds in saturating GABA (10mM) was 30.16 ± 2.24 % (n=9) versus WT (62.96 ± 4.15 %, n=13). α1K390A had no effect on GABA EC50 or desensitization, and mutation of the aligned residue in γ2 subunit, γ2K409A, decreased GABA EC50 ∼2 fold without affecting desensitization. γ2Y413A significantly increased desensitization and increased GABA EC50 by 2-fold. Desensitization extent after 10 seconds was 76.57 ± 2.09 % (n=18) compared to WT (23.71 ± 2.30 %, n=10). These data indicate that structural perturbations of the α1MX and γ2MA regions in the GABAR ICD can influence macroscopic GABAR current desensitization. Interestingly, GABAR MX regions are lysine rich. We speculate that mechanisms underlying GABAR gating transitions may involve charged interactions between MX and other regions of the GABAR ICD (e.g. MA). Experiments are underway to test this idea.

GABAA Receptors Expressed in Oligodendrocytes Cultured from the Neonatal Rat Contain α3 and γ1 Subunits and Present Differential Functional and Pharmacological Properties.

Oligodendrocytes (OLs) express functional GABAA receptors (GABAARs) that are activated by GABA released at synaptic contacts with axons or by ambient GABA in extrasynaptic domains. In both instances, the receptors' molecular identity has not been fully defined. Furthermore, data on their structural diversity in different brain regions and information on age-dependent changes in their molecular composition are scant. This lack of knowledge has delayed access to a better understanding of the role of GABAergic signaling between neurons and OLs. Here, we used functional, and pharmacological analyses, as well as gene and protein expression of GABAAR subunits, to explore the subunit combination that could explain the receptor functional profile expressed in OLs from the neonate rat. We found that GABAAR composed of α3β2γ1 subunits mimicked the characteristics of the endogenous receptor when expressed heterologously in Xenopus laevis oocytes. Either α3 or γ1 subunit silencing by small interfering RNA transfection changed the GABA-response characteristics in oligodendrocyte precursor cells, indicating their participation in the endogenous receptor conformation. Thus, α3 subunit silencing shifted the mean EC50 for GABA from 75.1 to 46.6 µM, whereas γ1 silencing reduced the current amplitude response by 55%. We also observed that β-carbolines differentially enhance GABA responses in oligodendroglia as compared with those in neurons. These results contribute to defining the molecular and pharmacological properties of GABAARs in OLs. Additionally, the identification of β-carbolines as selective enhancers of GABAARs in OLs may help to study the role of GABAergic signaling during myelination. SIGNIFICANCE STATEMENT: GABAergic signaling through GABAA receptors (GABAARs) expressed in the oligodendroglial lineage contributes to the myelination control. Determining the molecular identity and the pharmacology of these receptors is essential to define their specific roles in myelination. Using GABAAR subunit expression and silencing, we identified that the GABAAR subunit combination α3β2γ1 conforms the bulk of GABAARs in oligodendrocytes from rat neonates. Furthermore, we found that these receptors have differential pharmacological properties that allow specific positive modulation by β-carbolines.

Interactions of N, N‐ diethyl‐meta‐toluamide (DEET) and Novel Insect Repellents with Mammalian GABAA and Glycine Receptors

Mosquito‐borne diseases such as malaria, dengue and yellow fever are responsible for millions of deaths annually. Repellents disrupt interactions between humans and insects, thus providing personal protection from bites through which diseases are transmitted. The most commonly used repellent today is N, N‐ diethyl‐meta‐toluamide, DEET. Despite its popularity, DEET is not effective against all species of mosquitoes, and there are widespread concerns about its possible toxicity to users. Consequently, there is a demand for new repellents which are safer, effective for longer durations, and more efficacious against a broader range of arthropods. The novel repellents tested in the following study fall under two classes of chemicals: acylpiperidines and carboxamides.In the present electrophysiological investigation, the neurotoxicity of these novel repellents was explored relative to that of DEET, focusing on the effects of the repellents on inhibitory neurotransmitter receptors. Two‐electrode voltage clamp technique was employed with Xenopus laevis oocytes expressing mammalian GABAA (α1β3γ2S) and glycine (α1 homomer) receptors. Results indicated that high concentrations of DEET negatively modulated glycine receptor activity when co‐applied with submaximal glycine concentrations. 1 mM DEET inhibited glycine receptor activity by 48.4% ± 4.0% and 10 mM DEET produced a maximal inhibition of 80.4% ± 3.2%. Interestingly, most carboxamides (including DEET) inhibited the activity of glycine receptors, while the acylpiperidines enhanced the activity of glycine receptors, acting as positive modulators. Neither acylpiperidines nor carboxamides were found to inhibit the activity of GABAA receptors, with DEET and other carboxamides acting as weak agonists at high concentrations (>1 mM) to produce directly‐activated GABA currents. The EC50 for the activation of GABAA receptors by DEET was 4760.4 ± 50.6 μM, compared to a GABA EC50 of 42.9 ± 8.0 μM. These data suggest that DEET and the novel repellents have distinct effects on mammalian GABAA and glycine receptors. Furthermore, the results highlighted a stark difference between the two classes of chemicals, whereby acylpiperidines have the potential to result in less neurotoxicity relative to carboxamides. Like strychnine, carboxamides, including DEET, act as competitive antagonists at glycine receptors albeit with considerably less potency.Support or Funding InformationBlakeslee and Holmes funding, Smith CollegeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GABAρ3 expression in lobule X of the cerebellum is reduced in the valproate model of autism

Autism spectrum disorder (ASD) is a group of developmental disorders characterized by social interaction deficits, communication impairments, and stereotyped and repetitive behaviors. Additionally, impairments in the GABAergic circuitry have been associated with ASD. Several studies have shown that dysfunction of the cerebellum is a hallmark of ASD, and postmortem studies in humans reported a reduced density of Purkinje cells (PCs) together with an abnormal expression of GABAA subunits, among which GABAρ3 is expressed in early postnatal development, forms homomeric receptors with high affinity to the agonist (GABA EC50 ∼ 3 μM) and desensitize very little upon activation. Thus, we tested if the expression of GABAρ3 was modified by prenatal exposure to valproate (VPA), a well-known murine model of autism. The latency to find the nest increased in VPA-treated mice when compared to controls at postnatal day 8 (P8). Immunofluorescence studies showed a reduced expression of GABAρ3 in Purkinje cells (PCs) and ependymal glial cells (EGCs) from lobule X of VPA-treated mice. Finally, the expression of GABAρ3 increases linearly throughout normal development of the cerebellum, but this pattern is disrupted in the VPA model of autism. We conclude that the expression of GABAρ3 is reduced in PCs and EGCs from lobule X of the cerebellum in the VPA model of autism. Thus, GABAρ3 may be a relevant marker for ASD etiology.

Molecular Characterization of Binding Loop E in the Nematode Cys-Loop GABA Receptor.

Nematodes exhibit a vast array of cys-loop ligand-gated ion channels with unique pharmacologic characteristics. However, many of the structural components that govern the binding of various ligands are unknown. The nematode cys-loop GABA receptor uncoordinated 49 (UNC-49) is an important receptor found at neuromuscular junctions that plays an important role in the sinusoidal movement of worms. The unique pharmacologic features of this receptor suggest that there are structural differences in the agonist binding site when compared with mammalian receptors. In this study, we examined each amino acid in one of the main agonist binding loops (loop E) via the substituted cysteine accessibility method (SCAM) and analyzed the interaction of various residues by molecular dynamic simulations. We found that of the 18 loop E mutants analyzed, H142C, R147C, and S157C had significant changes in GABA EC50 and were accessible to modification by a methanethiosulfonate reagent (MTSET) resulting in a change in I GABA In addition, the residue H142, which is unique to nematode UNC-49 GABA receptors, appears to play a negative role in GABA sensitivity as its mutation to cysteine increased sensitivity to GABA and caused the UNC-49 receptor partial agonist 5-aminovaleric acid (DAVA) to behave as a full agonist. Overall, this study has revealed potential differences in the agonist binding pocket between nematode UNC-49 and mammalian GABA receptors that could be exploited in the design of novel anthelmintics.

GABA beyond the synapse: defining the subtype-specific pharmacodynamics of non-synaptic GABAA receptors.

Physiologically relevant combinations of recombinant GABAA receptor (GABAR) subunits were expressed in HEK293 cells. Using whole-cell voltage clamp and rapid drug application, we measured the GABAR-subtype-specific properties to convey either synaptic or extrasynaptic signalling in a range of physiological contexts. α4βδ GABARs are optimally tuned to submicromolar tonic GABA and transient surges of micromolar GABA concentrations. α5β2γ2l GABARs are better suited to higher tonic GABA levels, but also convey robust responses to brief synaptic and perisynaptic GABA fluctuations. α1β2/3δ GABARs function well at prolonged, micromolar (>2μm) GABA levels, but not to low tonic (<1μm GABA) or synaptic/transient GABAergic signalling. These results help illuminate the context- and isoform-specific modes of GABAergic signalling in the brain. GABAA receptors (GABARs) mediate a remarkable diversity of signalling modalities in vivo. Yet most published work characterizing responses to GABA has focused on the properties needed to convey fast, phasic synaptic inhibition. We therefore aimed to characterize the most prevalent (α4βδ, α5β3γ2L) and least prevalent (α1β2δ) non-synaptic GABAR currents, using whole-cell voltage clamp recordings of recombinant GABAR expressed in HEK293 cells and drug application protocols to recapitulate the GABA concentration profiles occurring during both fast synaptic and slow extrasynaptic signalling. We found that α4βδ GABARs were very sensitive to submicromolar GABA, with a rank order potency of α4β2δ ≥ α4β1δ ≈ α4β3δ GABARs. In comparison, the GABA EC50 was up to 20 times higher for α1β2γ2L GABARs, with α1β2δ and α5β3γ2L GABARs having intermediate GABA potency. Both α4βδ and α5β3γ2L GABAR currents exhibited slow, but substantial, desensitization as well as prolonged rates of deactivation. These GABAR current properties defined distinct 'dynamic ranges' of responsiveness to changing GABA for α4β2δ (0.1-1μm), α5β3γ2L (0.5-7μm) and α1β2γ2L (0.6-9μm) GABARs. Finally, α1β2δ GABARs were notable for their relative lack of desensitization and extremely quick deactivation. In summary, our results help delineate the roles that specific GABARs may play in mediating non-synaptic GABA signals. Since ambient GABA levels may be altered during development as well as by drugs and disease states, these findings may help future efforts to understand disrupted inhibition underlying a variety of neurological illnesses, such as epilepsy.

Investigating the Role of Loop C Hydrophilic Residue 'T244' in the Binding Site of ρ1 GABAC Receptors via Site Mutation and Partial Agonism.

The loop C hydrophilic residue, threonine 244 lines the orthosteric binding site of ρ1 GABAC receptors was studied by point mutation into serine, alanine and cysteine, and tested with GABA, some representative partial agonists and antagonists. Thr244 has a hydroxyl group essential for GABA activity that is constrained by the threonine methyl group, orienting it toward the binding site. Significant decreases in activation effects of the studied ligands at ρ1 T244S mutant receptors, suggests a critical role for this residue. Results of aliphatic and heteroaromatic partial agonists demonstrate different pharmacological effects at ρ1 T244S mutant receptors when co-applied with GABA EC50 responses. ρ1 T244A and ρ1 T244C mutant receptors have minimal sensitivity to GABA at high mM concentrations, whereas, the ρ1 WT partial agonists, β-alanine and MTSEA demonstrate more efficacy and potency, respectively, than GABA at these mutant receptors. This study explores the role of Thr244 in the binding of agonists as an initial step during channel gating by moving loop C towards the ligand.

Etomidate uniquely modulates the desensitization of recombinant α1β3δ GABAA receptors

Central GABAA receptors mediate GABAergic phasic and tonic inhibition. While synaptic αβγ GABAA receptors primarily mediate phasic inhibition, extrasynaptic αβδ receptors play an important role in mediating tonic inhibition. Etomidate is a general anesthetic that produces its effects by enhancing GABAA receptor activity. We previously showed that etomidate modulates the gating of oocyte-expressed αβγ and αβδ receptors with similar overall allosteric impact, but different pharmacological patterns. In αβγ receptors, etomidate enhances apparent GABA sensitivity (reduces GABA EC50), modestly increases maximal GABA efficacy, and slows current deactivation without affecting desensitization (Zhong et al., 2008). In αβδ receptors characterized by low GABA efficacy, etomidate dramatically increases responses to both low and maximal GABA. The effects of etomidate on desensitization and deactivation of αβδ receptors are unknown. To investigate the kinetic effects of etomidate on α1β3δ receptors of defined subunit arrangement, we expressed concatenated trimer (β3-α1-δ) and dimer (β3-α1) GABAA receptor subunit assemblies in human embryonic kidney (HEK)293T cells and recorded whole-cell voltage-clamp currents during rapid external solution exchanges. As expected, etomidate substantially increased maximal GABA-induced currents and prolonged deactivation. Moreover, desensitization was significantly decreased by etomidate. During prolonged GABA applications, etomidate enhanced steady-state currents more than peak currents. Thus, etomidate enhances tonic GABAergic inhibition through extrasynaptic αβδ receptors by both augmenting gating and reducing desensitization.

An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation

GABA-activated RDL receptors are the insect equivalent of mammalian GABAA receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6′M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC50 of WT receptors, enhancing responses, but in T6′M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC50, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6′ residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.

Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16′A-, F16′D-, F16′S-, and F16′T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16′ is small. T6′S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16′S, indicating that the inhibitor binds at position 6′, as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16′ is taken into consideration.

Novel GABAA‐rho1 interactions with acid sensing ion channel ligands (1059.3)

γ‐ amino butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and targets the ionotropic GABAA receptors. GABAC, or GABAA‐rho, is a subclass of GABAA receptors composed entirely of rho (ρ) subunits and are located on the axonal terminal of retinal bipolar cells, where it not only exhibits a tonic inhibitory current, but also regulates the GABA‐A and other GABAA‐rho synaptic currents (Jones et al 2011). GABAA‐rho exhibits unique properties, such as insensitivity to select antagonists of the heteromeric GABAA receptors (Korpi et al., 2002). A group of ligands, which possess a guanidine group, have been shown to influence GABAA receptors. These compounds, such as (S)‐2‐Guanidinopropionic acid and guanidine acetic acid were competitive antagonists for the GABAA‐rho1 receptor. Other guanidine compounds that are acid sensing ion channel (ASIC) ligands, might also exhibit unique effects on the GABAA‐rho1 receptor. We hypothesize that these ASIC ligands will exhibit unique intrinsic activities on the GABAA‐rho1 receptor, which is different from that of the heteromeric GABAA receptor. The human GABAA‐rho1 receptors were expressed in HEK‐293T cells, and activity was analyzed using whole cell patch‐clamp electrophysiology. When co‐applied with GABA and compared to the GABA concentration profile, one ligand was found to decrease the maximal response, with no change in the GABA EC50. This indicates characteristics of non‐competitive inhibition, while a different ligand shifted the GABA EC50 to lower GABA concentrations, suggesting this guanidine compound is a positive allosteric modulator. When applied alone, it failed to directly activate GABA rho1 receptors. These contrasting effects suggest that these ligands act at two binding sites within the GABAA‐rho architecture. Future experiments will focus on additional characterization of these novel effects on GABA rho receptors and offer a novel chemical structure to design novel GABAA‐rho therapeutics.Grant Funding Source: Supported by: Welch Foundation Grant No: BK‐1736, NIH training grant: NBA T32 AG020494

Regulation of α3-containing GABAA receptors in guinea-pig adrenal medullary cells by adrenal steroids

GABA is thought to function as a paracrine factor in adrenal medullary (AM) cells. Thus, we electrophysiologically and immunologically examined the properties of GABAA receptors (GABAARs) in guinea-pig AM cells. Bath application of GABA produced an inward current at −60mV in a dose-dependent manner with an EC50 of 32.3μM. This GABA-induced current was enhanced by allopregnanolone at concentrations of 0.01μM and more. A prior exposure to allopregnanolone resulted in a decrease in an EC50 for GABA in activating GABAARs. The GABA-induced current was suppressed by Zn2+ in a dose-dependent manner with an IC50 of 18μM, whereas it was enhanced by 100μM La3+. The benzodiazepine analog diazepam was three times more potent than zolpidem in enhancing the GABA current, and it was also augmented by L-838,417, which has no action on α1-containing GABAARs. The GABAAR α3, but not α1, and γ2 subunits were immunologically detected at the cell periphery. The expression of α3 subunits in PC12 cells was enhanced by glucocorticoid activity. The results indicated that GABAARs in guinea-pig AM cells mainly comprise α3, β, and γ2 subunits and are enhanced by allopreganalone and glucocorticoids may play a major role in the expression of α3 subunits.

Characterising the Ion Selectivity and Calcium Permeability of the Erwinia Ligand-Gated Ion Channel

The Erwinia ligand-gated ion channel (ELIC) is a bacterial GABA-gated cation-selective channel that shares structural homology with vertebrate Cys-loop receptors[1]. Here we investigated the effects of Ca2+ on the functional properties of ELIC using wild-type and mutant receptors. Receptors were expressed in Xenopus oocytes and characterised using two electrode voltage-clamp electrophysiology. GABA concentration-response curves were recorded in ND96 buffer with 0, 1.8, 5 or 10 mM Ca2+. The data show that Ca2+ alters the rate of apparent desensitization and the GABA EC50. To probe ion selectivity the external NaCl concentration was altered and the effects on reversal potential measured by clamping cells between −60 and +100 mV in 10 mV steps. These data showed that ELIC is predominantly cation selective as previously reported[2]. The −1' (E229) residue has been identified as a critical region for controlling cation selectivity in Cys-loop receptors[3][4]. Consistent with this, an E229A mutation in ELIC decreased cation selectivity. When E229A was combined with a mutation at the N20' position (E229A/N250R) ELIC became predominantly anion selective, similar to experiments described for the 5-HT3 receptor[4]. This mutant eliminated the effects of Ca2+ on ELIC, demonstrating that Ca2+ effects are mediated either in the channel or in the intracellular domain.[1] Hilf & Dutzler., 2008, Nature, 452 (7185) 375-9.[2] Zimmermann et al., 2011 PLoS Biol, 9 (6).[3] Galzi et al., 1992 Nature, 359 (6395) 500-5.[4] Thompson & Lummis, 2003 Br J Pharmacol, 140 359-65.

Two Etomidate Sites in α1β2γ2 γ-Aminobutyric Acid Type A Receptors Contribute Equally and Noncooperatively to Modulation of Channel Gating

Etomidate is a potent hypnotic agent that acts via γ-aminobutyric acid receptor type A (GABA(A)) receptors. Evidence supports the presence of two etomidate sites per GABA(A) receptor, and current models assume that each site contributes equally and noncooperatively to drug effects. These assumptions remain untested. We used concatenated dimer (β2-α1) and trimer (γ2-β2-α1) GABA(A) subunit assemblies that form functional α1β2γ2 channels, and inserted α1M236W etomidate site mutations into both dimers (β2-α1M236W) and trimers (γ2-β2-α1M236W). Wild-type or mutant dimers (D(wt) or D(αM236W)) and trimers (T(wt) or T(αM236W)) were coexpressed in Xenopus oocytes to produce four types of channels: D(wt)T(wt), D(αM236W)T(wt), D(wt)T(αM236W), and D(αM236W)T(αM236W). For each channel type, two-electrode voltage clamp was performed to quantitatively assess GABA EC(50), etomidate modulation (left shift), etomidate direct activation, and other functional parameters affected by αM236W mutations. Concatenated wild-type D(wt)T(wt) channels displayed etomidate modulation and direct activation similar to α1β2γ2 receptors formed with free subunits. D(αM236W)T(αM236W) receptors also displayed altered GABA sensitivity and etomidate modulation similar to mutated channels formed with free subunits. Both single-site mutant receptors (D(αM236W)T(wt) and D(wt)T(αM236W)) displayed indistinguishable functional properties and equal gating energy changes for GABA activation (-4.9 ± 0.48 vs. -4.7 ± 0.48 kJ/mol, respectively) and etomidate modulation (-3.4 ± 0.49 vs. -3.7 ± 0.38 kJ/mol, respectively), which together accounted for the differences between D(wt)T(wt) and D(αM236W)T(αM236W) channels. These results support the hypothesis that the two etomidate sites on α1β2γ2 GABA(A) receptors contribute equally and noncooperatively to drug interactions and gating effects.

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABAC ρ1 Receptors

Designing potent and subtype-selective ligands with therapeutic value requires knowledge about how endogenous ligands interact with their binding site. 4-Amino-3-hydroxybutanoic acid (GABOB) is an endogenous ligand found in the central nervous system in mammals. It is a metabolic product of GABA, the major inhibitory neurotransmitter. Homology modeling of the GABA(C) ρ(1) receptor revealed a potential H-bond interaction between the hydroxyl group of GABOB and threonine 244 (T244) located on loop C of the ligand binding site of the ρ(1) subunit. Using site-directed mutagenesis, we examined the effect of mutating T244 on the efficacy and pharmacology of GABOB and various ligands. It was found that mutating T244 to amino acids that lacked a hydroxyl group in their side chains produced GABA insensitive receptors. Only by mutating ρ(1)T244 to serine (ρ(1)T244S) produced a GABA responsive receptor, albeit 39-fold less sensitive to GABA than ρ(1)wild-type. We also observed changes in the activities of the GABA(C) receptor partial agonists, muscimol and imidazole-4-acetic acid (I4AA). At the concentrations we tested, the partial agonists antagonized GABA-induced currents at ρ(1)T244S mutant receptors (Muscimol: ρ(1)wild-type, EC(50) = 1.4 μM; ρ(1)T244S, IC(50) = 32.8 μM. I4AA: ρ(1)wild-type, EC(50) = 8.6 μM; ρ(1)T244S, IC(50) = 21.4 μM). This indicates that T244 is predominantly involved in channel gating. R-(-)-GABOB and S-(+)-GABOB are full agonists at ρ(1)wild-type receptors. In contrast, R-(-)-GABOB was a weak partial agonist at ρ(1)T244S (1 mM activates 26% of the current produced by GABA EC(50) versus ρ(1)wild-type, EC(50) = 19 μM; I(max) 100%), and S-(+)-GABOB was a competitive antagonist at ρ(1)T244S receptors (ρ(1)wild-type, EC(50) = 45 μM versus ρ(1)T244S, IC(50) = 417.4 μM, K(B) = 204 μM). This highlights that the interaction of GABOB with T244 is enantioselective. In contrast, the potencies of a range of antagonists tested, 3-aminopropyl(methyl)phosphinic acid (3-APMPA), 3-aminopropylphosphonic acid (3-APA), S- and R-(3-amino-2-hydroxypropyl)methylphosphinic acid (S-(-)-CGP44532 and R-(+)-CGP44533), were not altered. This suggests that T244 is not critical for antagonist binding. Receptor gating is dynamic, and this study highlights the role of loop C in agonist-evoked receptor activation, coupling agonist binding to channel gating.

Clobazam and Its Active Metabolite, N-Desmethylclobazam, Are Partial Benzodiazepine Receptor Agonists at Cloned GABAA Receptors Expressed in Xenopus leavis Oocytes (P05.085)

Objective: We investigated functional effects of clobazam and its active metabolite, N-desmethylclobazam (N-CLB), on recombinant GABA A receptors by electrophysiology. Background Clobazam, a 1,5-benzodiazepine, is in FDA review for Lennox-Gastaut syndrome. Design/Methods: To better understand the observed clinical profile of clobazam, we first characterized its GABA A -receptor-subtype–dependent positive allosteric modulation. Using two electrode voltage clamps, we determined that clobazam and N-CLB were both positive allosteric modulators at Xenopus leavis oocytes injected with cRNA encoding different human GABA A -receptor subunits. GABA concentration-response curves were generated at combinations of α (α 1 , α 2 , α 3 , and α 5 ), β 2, and γ 2 subunits injected at 1:1:1 ratios. At each oocyte, maximum response to GABA and EC 50 and EC 20 values were detemined. Modulatory effects of the two 1,5-benzodiazepines to GABA EC 20 response was determined by co-application of different concentrations of the individual benzodiazepines with a GABA EC 20 concentration. Potentiation of GABA EC 20 response exerted by the respective benzodiazepines was related to maximum responses elicited by GABA on the GABA A -receptor–expressing oocyte. Results: Clobazam and N-CLB both acted as potent partial agonists at α 1 β 2 γ 2 and α 2 β 2 γ 2 receptors. Clobazam exhibited an EC 50 value of 120 nM and a maximum effect of 51% of that of the maximum response of GABA at α 1 β 2 γ 2 receptors, while it displayed an EC 50 value of 110 nM and a maximum effect of 50% at α 2 β 2 γ 2 receptors. Similarly, N-CLB displayed an EC 50 value of 130 nM and maximum effect of 39% at α 1 β 2 γ 2 , and an EC 50 value of 110 nM and maximum effect of 51% at α 2 β 2 γ 2 . Conclusions: Clobazam and N-desmethylclobazam were shown to be partial agonists acting through the benzodiazepine site of the GABA A receptor. This partial agonism may contribute to clobazam9s overall clinical effects and raises the possibility that clobazam9s tolerance profile in animal models may be different than those of 1,4-benzodiazepines such as diazepam. Supported by: H. Lundbeck A/S. Disclosure: Dr. Jensen has received research support from Lundbeck A/S. Dr. Hammer has received research support from Lundbeck Research USA, Inc. Dr. Ebert has received personal compensation for activities with Lundbeck Research USA as an employee. Dr. Sindal Jensen has received personal compensation for activities with H. Lundbeck A/S as an employee.