Α1β2γ2 Receptors Research Articles

Rat α6β2δ GABAA receptors exhibit two distinct and separable agonist affinities

The onset of motor learning in rats coincides with exclusive expression of GABAA receptors containing α6 and δ subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through α6‐containing GABAA receptors, known as tonic inhibition. Here we report that the coexpression of α6, β2, and δ subunits produced receptor–channels which possessed two distinct and separable states of agonist affinity, one exhibiting micromolar and the other nanomolar affinities for GABA. The high‐affinity state was associated with a significant level of spontaneous channel activity. Increasing the level of expression or the ratio of β2 to α6 and δ subunits increased the prevalence of the high‐affinity state. Comparative studies of α6β2δ, α1β2δ, α6β2γ2, α1β2γ2 and α4β2δ receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to α6β2δ receptors. The pharmacology of spontaneous channel activity arising from α6β2δ receptor expression corresponded to that of tonic inhibition. For example, GABAA receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5α‐THDOC and classical glycine receptor agonists β‐alanine and taurine directly activated α6β2δ receptors with high potency. Specific mutation within the GABA‐dependent activation domain (βY157F) impaired both low‐ and high‐affinity components of GABA agonist activity in α6βY157Fδ receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the δ subunit (δR287M) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the α6β2δ receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.

Identification of the Sites for CaMK-II-dependent Phosphorylation of GABAA Receptors

Phosphorylation can affect both the function and trafficking of GABA(A) receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABA(A) receptor beta and gamma subunits thereby modulating receptor function in heterologous expression systems and in neurons (1, 2). Specifically, CaMK-II has been demonstrated to phosphorylate the M3-4 loop of GABA(A) receptor subunits expressed as GST fusion proteins (3, 4). It also increases the amplitude of GABA(A) receptor-mediated currents in a number of neuronal cell types (5-7). To identify which substrate sites CaMK-II might phosphorylate and the consequent functional effects, we expressed recombinant GABA(A) receptors in NG108-15 cells, which have previously been shown to support CaMK-II modulation of GABA(A) receptors containing the beta3 subunit (8). We now demonstrate that CaMK-II mediates its effects on alpha1beta3 receptors via phosphorylation of Ser(383) within the M3-4 domain of the beta subunit. Ablation of beta3 subunit phosphorylation sites for CaMK-II revealed that for alphabetagamma receptors, CaMK-II has a residual effect on GABA currents that is not mediated by previously identified sites of CaMK-II phosphorylation. This residual effect is abolished by mutation of tyrosine phosphorylation sites, Tyr(365) and Tyr(367), on the gamma2S subunit, and by the tyrosine kinase inhibitor genistein. These results suggested that CaMK-II is capable of directly phosphorylating GABA(A) receptors and activating endogenous tyrosine kinases to phosphorylate the gamma2 subunit in NG108-15 cells. These findings were confirmed in a neuronal environment by expressing recombinant GABA(A) receptors in cerebellar granule neurons.

Synthesis, 3D-QSAR, and docking studies of 1-phenyl-1 H-1,2,3-triazoles as selective antagonists for β3 over α1β2γ2 GABA receptors

A series of 16 1-phenyl-1 H-1,2,3-triazoles with substituents at both the 4- and 5-positions of the triazole ring were synthesized, and a total of 49 compounds, including previously reported 4- or 5-monosubstituted analogues, were examined for their ability to inhibit the specific binding of [ 3H]4′-ethynyl-4- n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist, to human homo-oligomeric β3 and hetero-oligomeric α1β2γ2 γ-aminobutyric acid (GABA) receptors. Among all tested compounds, the 4- n-propyl-5-chloromethyl analogue of 1-(2,6-dichloro-4-trifluoromethylphenyl)-1 H-1,2,3-triazole showed the highest level of affinity for both β3 and α1β2γ2 receptors, with K i values of 659 pM and 266 nM, respectively. Most of the tested compounds showed selectivity for β3 over α1β2γ2 receptors. Among all 1-phenyl-1 H-1,2,3-triazoles, the 4- n-propyl-5-ethyl analogue exhibited the highest (>1133-fold) selectivity, followed by the 4- n-propyl-5-methyl analogue of 1-(2,6-dibromo-4-trifluoromethylphenyl)-1 H-1,2,3-triazole with a >671-fold selectivity. The 2,6-dichloro plus 4-trifluoromethyl substitution pattern on the benzene ring was found to be important for the high affinity for both β3 and α1β2γ2 receptors. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) provided similar contour maps, revealing that an electronegative substituent at the 4-position of the benzene ring, a compact, hydrophobic substituent at the 4-position of the triazole ring, and a small, electronegative substituent at the 5-position of the triazole ring play significant roles for the high potency in β3 receptors. Molecular docking studies suggested that the putative binding sites for 1-phenyl-1 H-1,2,3-triazole antagonists are located in the channel-lining 2′-6′ region of the second transmembrane segment of β3 and α1β2γ2 receptors. A difference in the hydrophobic environment at the 2′ position might underlie the selectivity of 1-phenyl-1 H-1,2,3-triazoles for β3 over α1β2γ2 receptors. The compounds that had high affinity for β3 receptors with homology to insect GABA receptors showed insecticidal activity against houseflies with LD 50 values in the pmol/fly range. The information obtained in the present study should prove helpful for the discovery of selective insect control chemicals.

Desensitization and binding properties determine distinct α1β2γ2 and α3β2γ2 GABAA receptor‐channel kinetic behavior

GABAA receptor subtypes comprising the α1 and α3 subunits change with development and have a specific anatomical localization in the adult brain. These receptor subtypes have been previously demonstrated to greatly differ in deactivation kinetics but the underlying gating mechanisms have not been fully elucidated. Therefore, we expressed rat α1β2γ2 and α3β2γ2 receptors in human embryonic kidney 293 cells and recorded current responses to ultrafast GABA applications at macroscopic and single-channel levels. We found that the slow deactivation of α3β2γ2-mediated currents is associated with a relatively small rate and extent of apparent desensitization. In contrast, responses mediated by α1β2γ2 receptors had faster deactivation and stronger desensitization. α3β2γ2 receptors had faster recovery in the paired-pulse agonist applications than α1β2γ2 channels. The onset of currents mediated by α3β2γ2 receptors was slower than that of α1β2γ2 for a wide range of GABA concentrations. Single-channel analysis did not reveal differences in the opening/closing kinetics of α1β2γ2 and α3β2γ2 channels but burst durations were longer in α3β2γ2 receptors. Simulation with a previously reported kinetic model was used to explore the differences in respective rate constants. Reproduction of major kinetic differences required a smaller desensitization rate as well as smaller binding and unbinding rates in α3β2γ2 compared with α1β2γ2 receptors. Our work describes the mechanisms underlying the kinetic differences between two major GABAA receptor subtypes and provides a framework to interpret data from native GABA receptors.

Status epilepticus alters zolpidem sensitivity of [3H]flunitrazepam binding in the developing rat brain

GABA, the main inhibitory neurotransmitter in the adult brain, exerts its effects through multiple GABAA receptor subtypes with different pharmacological profiles, the α subunit variant mainly determining the binding properties of benzodiazepine site on the receptor protein. In adult experimental epileptic animals and in humans with epilepsy, increased excitation, i.e. seizures, alters GABAA receptor subunit expression leading to changes in the receptor structure, function, and pharmacology. Whether this also occurs in the developing brain, in which GABA has a trophic, excitatory effect, is not known. We have now applied autoradiography to study properties of GABAA/benzodiazepine receptors in 9-day-old rats acutely (6 h) and sub-acutely (7 days) after kainic acid–induced status epilepticus by analyzing displacement of [3H]flunitrazepam binding by zolpidem, a ligand selective for the α1β2γ2 receptor subtype. Regional changes in the binding properties were further corroborated at the cellular level by immunocytochemistry. The results revealed that status epilepticus significantly decreased displacement of [3H]flunitrazepam binding by zolpidem 6 h after the kainic acid-treatment in the dentate gyrus of the hippocampus, parietal cortex, and thalamus, and in the hippocampal CA3 and CA1 cell layers 1 week after the treatment. Our results suggest that status epilepticus modifies region-specifically the pharmacological properties of GABAA receptors, and may thus disturb the normal, strictly developmentally-regulated maturation of zolpidem-sensitive GABAA receptors in the immature rat brain. A part of these changes could be due to alterations in the cell surface expression of receptor subtypes.

GABA Increases both the Conductance and Mean Open Time of Recombinant GABAA Channels Co-expressed with GABARAP

The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.

When is Hot Not So Hot? Fever Reduces Brain Inhibition

Why Does Fever Trigger Febrile Seizures? GABAA Receptor γ2 Subunit Mutations Associated with Idiopathic Generalized Epilepsies Have Temperature-Dependent Trafficking Deficiencies Kang JQ, Shen W, Macdonald RL J Neurosci 2006;26:2590–2597 With a worldwide incidence as high as 6.7% of children, febrile seizures are one of the most common reasons for seeking pediatric care, but the mechanisms underlying generation of febrile seizures are poorly understood. Febrile seizures have been suspected to have a genetic basis, and recently, mutations in GABAA receptor and sodium channel genes have been identified that are associated with febrile seizures and generalized seizures with febrile seizures plus pedigrees. Pentameric GABAA receptors mediate the majority of fast synaptic inhibition in the brain and are composed of combinations of α( 1 – 6 ), β( 1 – 3 ), and γ( 1 – 3 ) subunits. In αβγ2 GABAA receptors, the γ2 subunit is critical for receptor trafficking, clustering, and synaptic maintenance, and mutations in the γ2 subunit have been monogenically associated with autosomal dominant transmission of febrile seizures. Here, we report that whereas trafficking of wild-type α1 β2 γ2 receptors was slightly temperature dependent, trafficking of mutant α1 β2 γ2 receptors containing γ2 subunit mutations [ γ2(R43Q), γ2(K289M),and γ2(Q351X)] associated with febrile seizures was highly temperature dependent. In contrast, trafficking of mutant α1 β2 γ2 receptors containing an α1 subunit mutation [ α1(A322D)] not associated with febrile seizures was not highly temperature dependent. Brief increases in temperature from 37 to 40°C rapidly (<10 min) impaired trafficking and/or accelerated endocytosis of heterozygous mutant α1 β2 γ2 receptors containing γ2 subunit mutations associated with febrile seizures but not of wild-type α1 β2 γ2 receptors or heterozygous mutant α1(A322D) β2 γ2 receptors, suggesting that febrile seizures may be produced by a temperature-induced dynamic reduction of susceptible mutant surface GABAA receptors in response to fever.

An Asymmetric Contribution to γ-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of α1, β2, and γ2 Subunits

An Asymmetric Contribution to γ-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of α1, β2, and γ2 Subunits

Effect of extracellular pH on recombinant α1β2γ2 and α1β2 GABA A receptors

Recently, we have reported that extracellular protons allosterically modulated neuronal GABA A receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981–7992]. However, GABA ARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, γ2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate γ2-containing and γ2-free GABA ARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for α1β2γ2 and α1β2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for α1β2 receptors. In the case of α1β2γ2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of α1β2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABA A receptors and that, depending on the presence of the γ2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.

Why Does Fever Trigger Febrile Seizures? GABAAReceptor γ2 Subunit Mutations Associated with Idiopathic Generalized Epilepsies Have Temperature-Dependent Trafficking Deficiencies

With a worldwide incidence as high as 6.7% of children, febrile seizures are one of the most common reasons for seeking pediatric care, but the mechanisms underlying generation of febrile seizures are poorly understood. Febrile seizures have been suspected to have a genetic basis, and recently, mutations in GABAA receptor and sodium channel genes have been identified that are associated with febrile seizures and generalized seizures with febrile seizures plus pedigrees. Pentameric GABAA receptors mediate the majority of fast synaptic inhibition in the brain and are composed of combinations of alpha(1-6), beta(1-3), and gamma(1-3) subunits. In alphabetagamma2 GABAA receptors, the gamma2 subunit is critical for receptor trafficking, clustering, and synaptic maintenance, and mutations in the gamma2 subunit have been monogenically associated with autosomal dominant transmission of febrile seizures. Here, we report that whereas trafficking of wild-type alpha1beta2gamma2 receptors was slightly temperature dependent, trafficking of mutant alpha1beta2gamma2 receptors containing gamma2 subunit mutations [gamma2(R43Q), gamma2(K289M), and gamma2(Q351X)] associated with febrile seizures was highly temperature dependent. In contrast, trafficking of mutant alpha1beta2gamma2 receptors containing an alpha1 subunit mutation [alpha1(A322D)] not associated with febrile seizures was not highly temperature dependent. Brief increases in temperature from 37 to 40 degrees C rapidly (<10 min) impaired trafficking and/or accelerated endocytosis of heterozygous mutant alpha1beta2gamma2 receptors containing gamma2 subunit mutations associated with febrile seizures but not of wild-type alpha1beta2gamma2 receptors or heterozygous mutant alpha1(A322D)beta2gamma2 receptors, suggesting that febrile seizures may be produced by a temperature-induced dynamic reduction of susceptible mutant surface GABAA receptors in response to fever.

Tandem Subunits Effectively Constrain GABAAReceptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABAAReceptor-Associated Protein

GABAergic synapses likely contain multiple GABAA receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of alpha, beta, and gamma subunits can be confounded by receptors expressed from alpha and beta subunits alone. To produce recombinant GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a "tandem" alpha1 subunit linked to a beta2 subunit. Cotransfection of the gamma2 subunit with alphabeta-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the gamma subunit with nonlinked alpha1 and beta2 subunits. Similarly, expression of alpha subunits together with alphabeta-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of alpha with beta subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of alphabeta-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of alpha1beta2 and alpha1beta2gamma2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in alpha1beta2gamma2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of alphabetagamma receptors.

Endoplasmic Reticulum Retention and Associated Degradation of a GABAA Receptor Epilepsy Mutation That Inserts an Aspartate in the M3 Transmembrane Segment of the α1 Subunit

A GABA(A) receptor alpha1 subunit epilepsy mutation (alpha1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha1 subunit. We reported previously that heterologous expression of alpha1(A322D)beta2gamma2 receptors in mammalian cells resulted in reduced total and surface alpha1 subunit protein. Here we demonstrate the mechanism of this reduction. Total alpha1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 +/- 6%) or when coexpressed with beta2 and gamma2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha1beta2gamma2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha1 subunits, consistent with substantial protein maturation, but in alpha1(A322D)beta2gamma2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha1 subunit was tagged with yellow (alpha1-YFP) or cyan (alpha1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha1-YFPbeta2gamma2 but only 5 +/- 1% alpha1(A322D)-YFPbeta2gamma2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha1-YFPbeta2gamma2S, 86 +/- 3% alpha1(A322D)-YFP). Heterozygous expression of alpha1-CFPbeta2gamma2S and alpha1(A322D)-YFPbeta2gamma2S or alpha1-YFPbeta2gamma2S and alpha1(A322D)-CFPbeta2gamma2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha1 subunits. These data demonstrate that the A322D mutation reduces alpha1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha1 subunits that are almost exclusively wild type subunits.

Spontaneous Thermal Motion of the GABAA Receptor M2 Channel-lining Segments

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

The epilepsy mutation, γ2(R43Q) disrupts a highly conserved inter-subunit contact site, perturbing the biogenesis of GABA A receptors

Given the association of a γ2 mutation (R43Q) with epilepsy and the reduced cell surface expression of mutant receptors, we investigated a role for this residue in α1β2γ2 receptor assembly when present in each subunit. Regardless of which subunit contained the mutation, mutant GABA A receptors assembled poorly into functional cell surface receptors. The low level of functional expression gives rise to reduced GABA EC 50s (α1(R43Q)β2γ2 and α1β2(R43Q)γ2) or reduced benzodiazepine potentiation of GABA-evoked currents (α1β2γ2(R43Q)). We determined that a 15-residue peptide surrounding R43 is capable of subunit binding, with a profile that reflected the orientation of subunits in the pentameric receptor. Subunit binding is perturbed when the R43Q mutation is present suggesting that this residue is critical for the formation of inter-subunit contacts at (+) interfaces of GABA A subunits. Rather than being excluded from receptors, γ2(R43Q) may form non-productive subunit interactions leading to a dominant negative effect on other receptor subtypes.

Differential Protein Mobility of the γ-Aminobutyric Acid, Type A, Receptor α and β Subunit Channel-lining Segments

The gamma-aminobutyric acid, type A (GABAA), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn(2+)-binding sites. Disulfide bonds formed in alpha1beta1E270Cgamma2 but not in alpha1N275Cbeta1gamma2 or alpha1beta1gamma2K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha1beta1E270Cgamma2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-A resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is approximately 19 A. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn(2+)-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha1beta1E270Cgamma2 was 34 nm. In contrast, it was greater than 100 microM in alpha1N275Cbeta1gamma2 and alpha1beta1gamma2K285C receptors. The high Zn2+ affinity in alpha1beta1E270Cgamma2 implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alpha M2 segments may have important implications for GABA-induced conformational changes during channel gating.

Compounds exhibiting selective efficacy for different beta subunits of human recombinant gamma-aminobutyric acid A receptors.

Inhibitory GABA(A) receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk(-) cells stably expressing human recombinant GABA(A) subunits (alpha1beta1-3gamma2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl(-) buffer using a voltage/ion probe reader. The influence of different betasubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at alpha1beta1gamma2, alpha1beta2gamma2, and alpha1beta3gamma2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC(50) values of 2.4 +/- 0.2, 2.5 +/- 0.2, and 1.3 +/- 0.1 microM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC(50) values of 8.7 +/- 0.9, 9.2 +/- 0.5, and 11.7 +/- 1.2, and 43.7 +/- 6.4, 24.8 +/- 1.6, and 26.1 +/- 2.4 microM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, pentobarbital, and steroids, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one, were unaffected by the beta isoform present in the receptor complex. However, several compounds displayed beta2/3 subunit selectivity, notably loreclezole, R(-)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at beta2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at alpha1beta1gamma2. Diflunisal was the most efficacious compound, eliciting greater potentiation than loreclezole (90 +/- 14% and 109 +/- 14% at beta3 and beta2, respectively, compared with 62 +/- 6% and 56 +/- 3%), whereas niflumic acid exhibited the lowest efficacy. An additional agent, olsalazine, weakly potentiated responses at all three receptors without any selectivity. This study identifies and characterizes a variety of allosteric modulators for which betasubunits are an important determinant of efficacy and potency.

Molecular basis for modulation of recombinant alpha1beta2gamma2 GABAA receptors by protons.

We have previously shown that extracellular protons inhibit recombinant and native GABA(A) receptors. In this report, we studied the site(s) and mechanism by which protons modulate the GABA(A) receptor. Whole cell GABA-activated currents were recorded from human embryonic kidney (HEK) 293 cells expressing recombinant alpha1beta2gamma2 GABA(A) receptors. Protons competitively inhibited the response to GABA and bicuculline. In contrast, change in pH did not influence direct gating of the channel by pentobarbital, and it did not influence spontaneous channel openings in alpha1(L264T)beta2gamma2 receptors, suggesting pH does not modulate channel activity by affecting the channel gating process directly. To test the hypothesis that protons modulate GABA(A) receptors at the ligand binding site, we systemically mutated N-terminal residues known to be involved in GABA binding and assessed effects of pH on these mutant receptors. Site-specific mutation of beta2 Y205 to F or alpha1 F64 to A, both of which are known to influence GABA binding, significantly reduced pH sensitivity of the GABA response. These mutations did not affect Zn(2+) sensitivity, suggesting that H(+) and Zn(2+) do not share a common site of action. Additional experiments further tested this possibility. Treatment with the histidine-modifying reagent diethylpyrocarbonate (DEPC) reduced Zn(2+)-mediated inhibition of GABA(A) receptors but had no effect on proton-induced inhibition of GABA currents. In addition, mutation of residues known to be involved in Zn(2+) modulation had no effect on pH modulation of GABA(A) receptors. Our results support the hypothesis that protons inhibit GABA(A) receptor function by direct or allosteric interaction with the GABA binding site. In addition, the sites of action of H(+) and Zn(2+) in GABA(A) receptors are distinct.

Inhibition of type a GABA receptors by L-type calcium channel blockers

Modulation of type A GABA receptors (GABA A) by L-type Ca ++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat α1β2γ2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC 50 for GABA current inhibition was lowest for nitrendipine (17.3±1.3 μM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC 50 was 10-fold lower in α1β2 receptors. Nitrendipine's effect in recombinant human α1β2γ2 receptors was similar (IC 50=23.0±1.3 μM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABA A receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (β2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist α-isopropyl-α-methyl-γ-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABA A receptor. Possible interaction of nitrendipine with the Zn ++ site was also eliminated, as mutation of β2 H267 to A, which confers resistance to Zn ++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABA A receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABA A receptor.

Neurosteroids Shift Partial Agonist Activation of GABAAReceptor Channels from Low- to High-Efficacy Gating Patterns

Although GABA activates synaptic (alphabetagamma) GABA(A) receptors with high efficacy, partial agonist activation of alphabetagamma isoforms and GABA activation of the primary extrasynaptic (alphabetadelta) GABA(A) receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of alphabetadelta receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the delta subunit or the low-efficacy channel function that it confers. We show that the isoform specificity (alphabetadelta > alphabetagamma) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of beta-alanine to achieve increased efficacy with alphabetadelta receptors and taurine to render alphabetagamma receptors low efficacy. We suggest that neurosteroids preferentially enhance low-efficacy GABA(A) receptor activity independent of subunit composition. Allosteric conversion of partial to full agonism may be a general mechanism for reversibly scaling the efficacy of GABA(A) receptors to endogenous partial agonists.

Recombinant α1β2γ2 GABAA receptors expressed in HEK293 and in QT6 cells show different kinetics

Neuronal γ-aminobutyric acid (GABA)A receptors are extremely heterogeneous and therefore GABAergic currents represent responses of unknown mixture of receptor subtypes. Expression of recombinant receptors in foreign cells allows to investigate a defined receptor subtype but its properties can be altered due to, e.g., differences in the endogenous modulators. In the present study the α1β2γ2 receptors were expressed in HEK293 and QT-6 cells and current responses to ultrafast GABA applications were recorded. Rise time and rapid deactivation component were faster in responses recorded from QT-6 cells. Moreover, in QT-6 cells desensitization was faster and more profound. Recovery in the paired pulse experiments was faster in HEK293 cells. In conclusion, we provide evidence that recombinant receptors may show functional differences when expressed in different cells.