Taurine Interaction with Neurotransmitter Receptors in the CNS: An Update

Taurine appears to have multiple functions in the brain participating both in volume regulation and neurotransmission. In the latter context it may exert its actions by serving as an agonist at receptors of the GABAergic and glycinergic neurotransmitter systems. Its interaction with GABA_A and GABA_B receptors as well as with glycine receptors is reviewed and the physiological relevance of such interactions is evaluated. The question as to whether local extracellular concentrations of taurine are likely to reach the threshold level for the pertinent receptor populations cannot presently be answered satisfactorily. Hence more sophisticated analytical methods are warranted in order to obtain a definite answer to this important question. Special issue dedicated to Dr. Simo S. Oja     

Synthesis and evaluation of highly potent GABA(A) receptor antagonists based on gabazine (SR-95531)

A selection of highly potent analogues based on the gabazine structure is described. Their syntheses are carried out in just four steps, and their potencies for antagonism at the GABA_A receptor were measured. All antagonists showed significantly higher potencies compared to the parent competitive antagonist, gabazine.     

An Electrogenic Ionic Pump Derived from an Ionotropic Receptor: Assessment of a Candidate

1.Data obtained studying permeability characteristics of single Deiters' membranes in a microchamber system show that intracellular GABA can activate chloride in out passage with a GABA_A pharmacology.2.The overall data suggest the presence of a chloride extrusion pump in these neurons based on intracellular GABA activated chloride channels.3.This conclusion takes up a previous theoretical suggestion that ionic channels could work as ionic pumps provided an energy input modifies the energy profile along the permeation path.4.According to our quantitative evaluation, this pumping mechanism works with a low yield and along a cycle with a strongly asymmetric behavior, being far from equilibrium due to powerful leakage pathways for chloride in these neurons.     

Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

GABA_A-receptors were localized in explant cultures of rat cerebellum and in dissociated primary cultures of rat cerebellar granule cells and rat cerebellar astrocytes using the monoclonal antibody bd-17 directed against the -subunit of the GABA_A/benzodiazepine/chloride channel complex. At the light microscope level specific staining of GABA_A-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling of GABA_A-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations to be performed. Quantification of the labeling intensity revealed a higher concentration of GABA_A-receptors per m plasma membrane in the cell bodies than in the processes. In discrete areas an extremely high density of the GABA_A-receptors was observed. No specific labeling of GABA_A-receptors was observed in dissociated primary cultures of cerebellar astrocytes.Special issue dedicated to Dr. Eugene Roberts.     

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.     

Subcellular Distribution of α1 and β2/3 GABAA Receptor Subunits in Sensory Neurons of the Bovine Trigeminal Mesencephalic Nucleus: Evidence Suggesting Their Axoplasmic Transport

Neurons were free-hand isolated under the stereomicroscope from the bovine trigeminal mesencephalic nucleus. These neurons were challenged with monoclonal antibodies against the alpha1 and the beta2/3 subunits of the GABA(A) receptor. The neurons showed a strong reaction to both antibodies. The reaction was mainly intracellular in the round cell bodies and in the axoplasm of the axon emerging from these pseudounipolar cells. This suggests the synthesis of these subunits in the cell body and their transport along the axon.     

Alcohol effects on gamma-aminobutyric acid type A receptors: are extrasynaptic receptors the answer?

GABA(A) receptors have long been implicated in mediating at least part of the actions of ethanol in mammalian brain. However, until very recently, reports of the actions of EtOH on recombinant receptors have required very high doses of ethanol and animals lacking receptor subunits shown to be important for ethanol actions in vitro did not support the view that these subunits are crucial in ethanol actions. Recombinant alpha4beta3delta and alpha6beta3delta GABA(A) receptors are uniquely sensitive to ethanol, with a dose-response relationship mirroring the well known effects of alcohol consumption on the human brain. Receptors containing the delta subunit are thought to be located extrasynaptically and it will be important to determine if these extrasynaptic GABA(A) receptor subunit combinations mediate low dose alcohol effects in vivo.     

Hypoxia Differentially Reduces GABAA Receptor Density During Embryonic Chick Optic Lobe Development

It has been demonstrated that the CNS is severely affected by hypoxic-ischemic insults during the prenatal-perinatal period, including imbalance in excitatory and inhibitory neurotransmitter release. Using a previously developed model of acute normobaric hypoxic hypoxia on chick embryos, we studied alterations observed both on [3H]GABA binding saturation parameters and on lactate concentration on successive embryonic days (ED). While maximal density of GABA binding sites (Bmax) from the low-affinity site was significantly reduced in an age-dependent manner, earlier stages of development (ED12 and 16) proving more vulnerable (ED12: control = 5.48 +/- 0.20, hypoxia = 3.90 +/- 0.39 pmol/mg prot, P < .05; ED16: control = 3.89 +/- 0.26, hypoxia = 2.80 +/- 0.28 pmol/mg prot, P < .05), ligand affinity (Kd) values and kinetic constants of the high-affinity site remained unaltered. Not unlikely, a physiological hypoxic state prevailing from ED17 up to hatching time rendered the whole embryo less sensitive to an externally induced hypoxic state (ED17: control = 2.93 +/- 0.06, hypoxia = 2.38 +/- 0.04 pmol/mg prot, P < .05; ED18: control = 2.97 +/- 0.12, hypoxia = 2.87 +/- 0.27 pmol/mg prot). Lactate levels in chick optic lobe homogenates were constant during development. The increase observed after hypoxic treatment compared to control value was significant at all stages studied, but increased percentage changes proved similar, indicating that all days of development equally perceive externally induced hypoxia. In conclusion, the present work demonstrates that after normobaric hypoxic hypoxia at different embryonic days, the embryo senses the externally induced hypoxic state as from ED12, but the GABA(A) receptor is differentially affected. It may be speculated that a different subunit composition of GABA(A) receptor is assembled in order to build a more stable receptor capable of resisting the physiological hypoxic state observed during the last few days before hatching.     

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.     

Stimulation of Chloride In→Out Permeation Across the Deiters' Neuron Membrane by Pentobarbital on the Cytoplasmic Side: Additional Evidence of GABAA Receptors Acting as Chloride Extrusion Pumps

Pentobarbital stimulates ³⁶Cl^– permeation across single Deiters' membranes in a microchamber system, acting on classical, extracellularly facing, GABA_A receptors. However, when applied on the membrane cytoplasmic side it activates per se labeled chloride inout permeation. No effect was found on chloride outin permeation. Similarly, at lower concentrations it facilitates the increase of ³⁶Cl^– inout permeation by application of GABA on the membrane inside, again via asymmetric chloride channels allowing inout but not outin passage. These data confirm that on the Deiters' membrane cytoplasmic side there are structures behaving pharmacologically as GABA_A receptors whose function is that of a Cl^– extrusion pump. This mechanism involves a cycle of activation-phosphorylation/desensitization-reactivation of the receptor complexes     

Identification of inosine as an endogenous modulator for the benzodiazepine binding site of the GABAA receptors

Previously we have reported the presence of endogenous ligands that are involved in the regulation of the binding of muscimol to the GABA binding site of the GABA_A receptors. Here, we report the presence of multiple forms of endogenous ligands in the brain which modulate the binding of flunitrazepam (FNZP) to the benzodiazepine (BZ) binding site of the GABA_A receptor. Furthermore, one of the endogenous ligands for the BZ receptors, referred to as EBZ, has been identified as inosine based on the following observations: (1) standard inosine and the EBZ have identical NMR and UV spectra; (2) the elution profile of inosine and the EBZ from a HPLC column are indistinguishable, and (3) inosine and the EBZ show identical activity in inhibiting [³H]FNZP binding.