Subunit Composition and Function of GABAA Receptors of Rat Spermatozoa

GABA triggers mammalian sperm acrosome reaction (AR). Here, evidence is presented, showing that rat spermatozoa contain GABA_A receptors, composed of ₅, ₁ and ₃ subunits. The effects of GABA_A receptor agonist and antagonist on the induction of AR in rat spermatozoa were assessed using the chlortetracycline assay. Muscimol, a GABA_A receptor agonist, triggered AR; whereas bicuculline, a GABA_A receptor antagonist and picrotoxin, a GABA_A receptor/Cl^– channel blocker, inhibited the ability of GABA or progesterone to induce AR. In conclusion, GABA_A receptors appear to mediate the action of progesterone in inducing AR in rat spermatozoa.     

Differential Drug Responses on Native GABAA Receptors Revealing Heterogeneity in Extrasynaptic Populations in Cultured Hippocampal Neurons

Hippocampal pyramidal neurons potentially express multiple subtypes of GABA_A receptors at extrasynaptic locations that could therefore respond to different drugs. We activated extrasynaptic GABA_A receptors in cultured rat hippocampal pyramidal neurons and measured single-channel currents in order to compare the actions of two drugs that potentially target different GABA_A receptor subtypes. Despite the possible difference in receptor targets of etomidate and diazepam, the two drugs were similar in their actions on native extrasynaptic GABA_A receptors. Each drug produced three distinct responses that differed significantly in current magnitude, implying heterogeneous GABA_A receptor populations. In the majority of patches, drug application increased both the single-channel conductance (>40 pS) and the open probability of the channels. By contrast, in the minority of patches, drug application caused an increase in open probability only. In the third group high-conductance channels were observed upon GABA activation and drug application increased their open probability only. The currents potentiated by etomidate or diazepam were substantially larger in patches displaying high-conductance GABA channels compared to those displaying only low-conductance channels. Factors contributing to the large magnitude of these currents were the long mean open time of high-conductance channels and the presence of multiple channels in these patches. In conclusion, we suggest that the local density of extrasynaptic GABA_A receptors may influence their single-channel properties and may be an additional regulating factor for tonic inhibition and, importantly, differential drug modulation. This work is dedicated to the memory of Professor P. W. Gage.     

Clozapine's Antipsychotic Effects do not Depend on Blockade of 5-HT3 Receptors

Sixteen known 5-HT₃ receptor blockers, including clozapine, fully or partially reverse the inhibitory effect of 1 M GABA on [³⁵S]TBPS binding, indicating that they are also GABA_A antagonists, some of them selective for subsets of GABA_A receptors. The 5-HT₃ receptor blocker, ondansetron, has been reported to produce some antipsychotic and anxiolytic effects. However, no antipsychotic effects have been reported for a large number of highly potent 5-HT₃ receptor blockers. Like clozapine, ondansetron partially reverses the inhibitory effect of GABA on [³⁵S]TBPS binding. Additivity experiments suggest that ten 5-HT₃ receptor blockers tested at low concentrations preferentially block subtypes of GABA_A receptors that are among those blocked by clozapine. Wiley and Porter (29) reported that MDL-72222, the most potent GABA_A antagonist decribed here, partially generalizes (71%) with clozapine in rats trained to discriminate an interoceptive clozapine stimulus, but only at a dose that severly decreases responding. Tropisetron (ICS-205,930) exhibits both GABA-positive and GABA-negative effects. R-(+)-zacopride is 6-fold more potent than S-(–)-zacopride as a GABA_A antagonist. We conclude that the observed antipsychotic and, possibly, anxiolytic effects of some 5-HT₃ receptor blockers are due to selective antagonism of certain GABA_A receptors, and not to blockade of 5-HT₃ receptors. We speculate that the anxiolytic and sedative effects of clozapine and several other antipsychotic drugs may be due to selective blockade of 122 GABA_A receptors which are preferentially located on certain types of GABAergic interneurons (probably parvalbumin positive). Blockade of these receptors will increase the inhibitory output of these interneurons. So far, no highly potent GABA_A antagonists with clozapine-like selectivity have been identified. Such compounds may exhibit improved clozapine-like antipsychotic activity.     

Effect of flumazenil on GABAA receptors in isolated rat hippocampal neurons

Using whole cell patch-clamp recordings from pyramidal cells acutely dissociated from rat hippocampal slices, Ro-15 1788 (flumazenil, FLU) was shown to enhance the GABA_A-receptor mediated currents evoked by application of -aminobutyric acid (GABA) and to antagonize the enhancing effect of the benzodiazepine agonist flurazepam (FZP) on the GABA_A response. Both FLU and FZP increased the peak and the steady-state components of the responses and accelerated the current decay. This suggests that both agents act via a common mechanism on GABA transmission. It is concluded that FLU possesses high affinity for the binding site, but low efficacy on the GABA_A-benzodiazepine receptor. This suggests that FLU acts as a partial agonist on GABA_A receptors.     

GABA Analogues Derived from 4-Aminocyclopent-1-enecarboxylic Acid

The incorporation of extra binding groups onto known ligands is a powerful tool for the development of more potent and selective agents at target sites such as the GABA receptors. In the present work we have attempted to build on the activity of the know potent GABA_A agonist 4-ACP-3-CA and its cis and trans saturated analogues CACP and TACP. We have investigated reactions to add thiol substituents to the α,β-unsaturated system of 4-ACP-3-CA. The reaction was successful with a limited number of thiols but gave products of mixed stereochemistry. The resultant thioether amino acids were screened for activity at human recombinant α₁β₂ γ_2L GABA_A receptors. The most interesting derivative was the benzylthioether which acted as an antagonist with an IC₅₀ of 42 μM for the inhibition of a GABA EC₅₀ dose (50 μM). This study has shown that GABA analogues derived by thiol addition to 4-aminocyclopent-1-enecarboxylic acid display interesting antagonist activity at the α₁β₂γ_2L GABA_A receptor. Special issue article in honour of Dr. Graham Johnston.     

GABA-mediated effects of some taurine derivatives injected i.c.v. on rabbit rectal temperature and gross motor behavior

Summary. Some synthetic taurine analogues, namely ethanolamine-O-sulphate (EOS), N,N-dimethyltaurine (DMT), N,N,N-trimethyltaurine (TMT) and 2-aminoethylphosphonic acid (AEP) were shown to interact with rabbit brain GABA_A- or GABA_B-receptors, while (±)piperidine-3-sulfonic acid (PSA) inhibited the activity of rabbit brain 4-aminobutyrate transaminase. This suggests that they behave like direct/indirect GABA agonists or GABA antagonists and affect thermoregulation and gross motor behaviour (GMB) which are under GABA control. In the present study micromole (1.2–48) amounts of these compounds were i.c.v. injected in conscious, restrained rabbits while monitoring rectal temperature (RT), ear skin temperature (EST) and GMB. AEP, EOS, DMT and TMT induced a dose-related hyperthermia, ear vasoconstriction and excitation of GMB, while PSA induced a dose-related hypothermia, ear vasodilation and inhibition of GMB. EOS antagonized in a dose-related fashion hypothermia induced by 60 nmol THIP, a GABA_A agonist, while AEP, DMT and TMT counteracted that induced by 8 nmol R(-)Baclofen, a GABA_B agonist. In conclusion, EOS and AEP, DMT, TMT seem to act as GABA_A and GABA_B antagonists, respectively, while PSA behaves like an indirect GABA agonist, all affecting the central mechanisms which drive rabbit thermoregulation.     

GABAA receptors on rat cerebellar granule cells are potently activated by muscimol but only slightly modulated by the benzodiazepine agonist flunitrazepam

Summary GABA_A receptors present on rat cerebellar granule cells in culture were studied by the whole cell patch clamp technique. Muscimol appeared to be more potent than GABA itself in activating Cl^– currents. A benzodiazepine, flunitrazepam, only slightly (10%) potentiated the GABA action.These results support the previous suggestion that GABA_A receptors containing the subunit, such as those in the cerebellum granule cells, are potently activated by muscimol. The present results also bear out the concept that GABA action on receptors containing the subunit is not potentiated by benzodiazepines.     

Suppression of sustained and transient ON signals of amacrine cells by GABA is mediated by different receptor subtypes

Intracellular recordings were made from amacrine cells in the isolated, superfused carp retina, and the effects of γ-aminobutyric acid (GABA) on sustained and transient ON signals of these cells were studied. Exogenous GABA application partially suppressed the sustained response of ON amacrine cells, which could be completely reversed by picrotoxin (PTX), a chloride channel blocker, and by bicuculline (BCC), a specific GABA_A receptor antagonist. On the other hand, suppression by GABA of the ON response which was predominantly driven by rod signals in a certain portion of transient ON-OFF amacrine cells was completely blocked by PTX, but not by BCC, indicating that GABA_C receptors may be involved in the effect. These results suggest that GABA_A and GABA_C receptors may be respectively involved in mediating the transmission of sustained and transient signals in the carp inner retina.     

GABA-Dopamine Receptor-Receptor Interactions in Neostriatal Membranes of the Rat

Recent evidence has shown in membrane preparations that the binding of one ligand to its receptor is able to modify the binding parameters of a second receptor (receptor-receptor interactions), allowing the modulation of incoming signals onto a neuron. To further understand the -amino-butyric acid (GABA)-dopamine (DA) interactions in the neostriatum we have carried out experiments to explore whether an activation of the GABA_A receptor could affect the binding characteristics of the D₂ DA receptor in membrane preparations of the rat neostriatum. The results show that GABA (30–100 nM) significantly increases the dissociation constant of the high affinity (K_H) D₂ DA binding site (labelled with the selective D₂ DA receptor antagonist [³H]raclopride and that such an effect is fully counteracted by the GABA_A receptor antagonist bicuculline (1 M). It is suggested that such putative GABA_A/D₂ receptor-receptor interactions may take place in the somato-dendritic membrane of the striato-pallidal GABA neurons and that it may modulate the inhibitory effects of DA on these neurons, mediated via D₂ receptors.     

Excitatory GABAA receptor in autonomic pelvic ganglion neurons innervating bladder

Major pelvic ganglia (MPG) are relay centers for autonomic reflexes such as micturition and penile erection. MPG innervate the urogenital system, including bladder. γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and may also play an important role in some peripheral autonomic ganglia, including MPG. However, the electrophysiological properties and function of GABA_A receptor in MPG neurons innervating bladder remain unknown. This study examined the electrophysiological properties and functional roles of GABA_A receptors in bladder-innervating neurons identified by retrograde Dil tracing. Neurons innervating bladder showed previously established parasympathetic properties, including small membrane capacitance, lack of T-type Ca²⁺ channel expression, and tyrosine-hydroxylase immunoreactivity. GABA_A receptors were functionally expressed in bladder innervating neurons, but GABA_C receptors were not. GABA elicited strong depolarization followed by increase of intracellular Ca²⁺ in neurons innervating bladder, supporting the hypothesis GABA may play an important role in bladder function. These results provide useful information about the autonomic function of bladder in physiological and pathological conditions.     

Assembly of GABAA receptors (Review)

GABA_A receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by γ-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABA_A receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABA_A receptor subtypes in the brain. The majority of GABA_A receptors seems to be composed of two α, two β and one γ subunit and the occurrence of a defined subunit stoichiometry and arrangement in αβγ receptors strongly indicates that assembly of GABA_A receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABA_A receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABA_A receptors are discussed in the light of recent publications.     

Differential Effects of Thiopental and Pentobarbital on Spinal GABAA Receptors

General anesthetics thiopental and pentobarbital possess very similar chemical structures whereas their clinical potency is quite different. The underlying molecular mechanism is not fully understood. This study was designed to assess the differential effects of thiopental and pentobarbital on GABA_A receptors of mechanically dissociated rat spinal dorsal horn neurons by using whole-cell patch-clamp technique. Pentobarbital, at a concentration of 30 μM, which markedly enhanced sub-saturated GABA-induced current (I _GABA), had no effect on thiopental-induced maximal current. Similarly, the pentobarbital-induced maximal current was also not affected by 30 μM thiopental. Moreover, a linear summation of thiopental-induced maximal current and pentobarbital-induced sub-maximal current was observed. In addition, pentobarbital failed to further enhance I _GABA in the presence of thiopental at a concentration with maximal modulatory effects on I _GABA, and vice versa. Our results thus suggest that thiopental and pentobarbital might exert the GABA mimetic effects independently, but share a common mechanism to produce the GABA modulatory effects. Special issue article in honor of Dr. Ji-Sheng Han.     

Brain GABAA receptors studied with subunit-specific antibodies

Brain GABA_A/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl⁻ channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl⁻ channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl⁻ channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABA_A receptor subunits and the relationships between subunit composition, ligand binding, and Cl⁻ channel properties. Nevertheless, little is known about the complete subunit composition of the native GABA_A receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (not reconstituted) brain GABA_A receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABA_A receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABA_A receptors and that have fewer side effects.     

Dipeptide Tyr-Leu (YL) exhibits anxiolytic-like activity after oral administration via activating serotonin 5-HT1A, dopamine D1 and GABAA receptors in mice

We found that Tyr-Leu (YL) dose-dependently exhibits potent anxiolytic-like activity (0.1-1 mg/kg, i.p.) comparable to diazepam in the elevated plus-maze test in mice. YL was orally active (0.3-3 mg/kg). A retro-sequence peptide or a mixture of Tyr and Leu was inactive. The anxiolytic-like activity of YL was inhibited by antagonists for serotonin 5-HT_1A, dopamine D₁ and GABA_A receptors; however, YL had no affinity for them. We also determined the order of their activation is 5-HT_1A, D₁ and GABA_A receptors using selective agonists and antagonists. Taken together, YL may exhibit anxiolytic-like activity via activation of 5-HT_1A, D₁ and GABA_A receptors.     

Evidence for a Reduction of Coupling between GABA<Subscript>A</Subscript> Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[³⁵S]TBPS binding to the GABA_A receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [³⁵S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [³⁵S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [³H]flunitrazepam binding to benzodiazepine site of recombinant α1β2γ2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [³⁵S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABA_A receptors. Special issue dedicated to Simo S. Oja     

Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors

GABA_A receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α₅ has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α₅ subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α₅ subunit-containing GABA_A receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α_4/6 subunit-containing GABA_A receptors, but is sensitive to L-655708, an antagonist of the GABA_A receptors containing α₅ subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α₅ subunit in the turtle spinal cord. Together, these results suggest that GABA_A receptors containing the α₅ subunit mediate the tonic inhibitory currents observed in VHIs.     

GABA<Subscript>B</Subscript> Receptors in Neuroendocrine Regulation

Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA_A and GABA_C (ionotropic receptors) and GABA_B (metabotropic receptor). Here we reviewed the participation of GABA_B receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA_B1 knock-out mice, that lack functional GABA_B receptors. Our general conclusion indicates that GABA_B receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA_B receptor activation, as demonstrated in the rat and also in the GABA_B1 knock-out mouse. In addition, hypothalamic and pituitary GABA_B receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA_B receptors depends on physiological conditions, being age and sex critical factors. These results indicate that patients receiving GABA_B agonists/antagonists should be monitored for possible endocrine side effects.     

mtDNA m.3635G>A may be classified as a common primary mutation for Leber hereditary optic neuropathy in the Chinese population

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via ionotropic (GABA_A and GABA_C) and metabotropic (GABA_B) receptors. The GABA_B receptor is a dimer composed of R1 and R2 components. In addition to their location on neurons, GABA and functional GABA_B receptors also have been detected in some peripheral tissues. In the present study, we combined immunohistochemistry, immunoblot and tension recording to determine if the human fallopian tube express glutamic acid decarboxylase (GAD65/67), two isoforms for synthesis of GABA and functional GABA_B receptors. Immunoblots showed that the human fallopian tube tissue contained GABA_BR1 protein which was localized in the epithelial cells and smooth muscle cells by immunohistochemistry. In addition, epithelial cells also expressed GAD65/67. Tension recording found that both GABA and baclofen, a GABA_B receptor agonist increased the spontaneous activity of human fallopian tube. The expressions of GABA_BR and GAD65/67 were significantly upregulated in the ectopic pregnancy group than in the intrauterine pregnancy group. We conclude that the human fallopian tube is capable of synthesizing GABA and expresses functionally active GABA_B receptors. An upregulation of GABA synthesis and corresponding GABA_B receptors may involve in ectopic pregnancy.     

GABAC receptors in the vertebrate retina

In the central nervous system (CNS), the inhibitory transmitter GABA interacts with three subtypes of GABA receptors, type A, type B, and type C. Historically, GABA receptors have been classified as either the inotropic GABA_A receptors or the metabotropic GABA_B receptors. Over the past 10 yr, studies have shown that a third class, called the GABA_C receptor, also exists. GABA_C receptors are found primarily in the vertebrate retina and to some extent in other parts of the CNS. Although GABA_A and GABA_C receptors both gate chloride channels, they are pharmacologically, molecularly, and functionally distinct. The ρ subunit of the GABA_C receptor, which has about 35% amino acid homology to GABA_A receptor subunits, was cloned from the retina and, when expressed inXenopus oocytes, has properties similar to retinal GABA_C receptors. There are probably distinct roles for GABA_C receptors in the retina, because they are found on only a subset of neurons, whereas GABA_A receptors are ubiquitous. This article reviews recent electrophysiological and molecular studies that have characterized the unique properties of GABA_C receptors and describes the roles that these receptors may play in visual information processing in the retina.     

GABAA receptor plasticity in Jurkat T cells

GABA_A receptors (GABA_AR) mediate inhibitory neurotransmission in the human brain. Neurons modify subunit expression, cellular distribution and function of GABA_AR in response to different stimuli, a process named plasticity. Human lymphocytes have a functional neuronal-like GABAergic system with GABA_AR acting as inhibitors of proliferation. We here explore if receptor plasticity occurs in lymphocytes. To this end, we analyzed human T lymphocyte Jurkat cells exposed to different physiological stimuli shown to mediate plasticity in neurons: GABA, progesterone and insulin. The exposure to 100 μM GABA differently affected the expression of GABA_AR subunits measured at both the mRNA and protein level, showing an increase of α1, β3, and γ2 subunits but no changes in δ subunit. Exposure of Jurkat cells to different stimuli produced different changes in subunit expression: 0.1 μM progesterone decreased δ and 0.5 μM insulin increased β3 subunits. To identify the mechanisms underlying plasticity, we evaluated the Akt pathway, which is involved in the phosphorylation of β subunits and receptor translocation to the membrane. A significant increase of phosphorylated Akt and on the expression of β3 subunit in membrane occurred in cells exposed 15 h to GABA. To determine if plastic changes are translated into functional changes, we performed whole cell recordings. After 15 h GABA-exposure, a significantly higher percentage of cells responded to GABA application when compared to 0 and 40 h exposure, thus indicating that the detected plastic changes may have a role in GABA-modulated lymphocyte function.