Endogenous neurosteroids influence synaptic GABAA receptors during postnatal development.

GABA plays a key role in both embryonic and neonatal brain development. For example, during early neonatal nervous system maturation, synaptic transmission, mediated by GABAA receptors (GABAA Rs), undergoes a temporally specific form of synaptic plasticity to accommodate the changing requirements of maturing neural networks. Specifically, the duration of miniature inhibitory postsynaptic currents (mIPSCs), resulting from vesicular GABA activating synaptic GABAA Rs, is reduced, permitting neurones to appropriately influence the window for postsynaptic excitation. Conventionally, programmed expression changes to the subtype of synaptic GABAA R are primarily implicated in this plasticity. However, it is now evident that, in developing thalamic and cortical principal- and inter-neurones, an endogenous neurosteroid tone (eg, allopregnanolone) enhances synaptic GABAA R function. Furthermore, a cessation of steroidogenesis, as a result of a lack of substrate, or a co-factor, appears to be primarily responsible for early neonatal changes to GABAergic synaptic transmission, followed by further refinement, which results from subsequent alterations of the GABAA R subtype. The timing of this cessation of neurosteroid influence is neurone-specific, occurring by postnatal day (P)10 in the thalamus but approximately 1 week later in the cortex. Neurosteroid levels are not static and change dynamically in a variety of physiological and pathophysiological scenarios. Given that GABA plays an important role in brain development, abnormal perturbations of neonatal GABAA R-active neurosteroids may have not only a considerable immediate, but also a longer-term impact upon neural network activity. Here, we review recent evidence indicating that changes in neurosteroidogenesis substantially influence neonatal GABAergic synaptic transmission. We discuss the physiological relevance of these findings and how the interference of neurosteroid-GABAA R interaction early in life may contribute to psychiatric conditions later in life.

Role of GABAA receptor subtypes in the behavioural effects of intravenous general anaesthetics.

Since the introduction of general anaesthetics into clinical practice, researchers have been mystified as to how these chemically disparate drugs act to produce their dramatic effects on central nervous system function and behaviour. Scientific advances, particularly during the last 25 years, have now begun to reveal the molecular mechanisms underpinning their behavioural effects. For certain i.v. general anaesthetics, such as etomidate and propofol, a persuasive case can now be made that the GABAA receptor, a major inhibitory receptor in the mammalian central nervous system, is an important target. Advances in molecular pharmacology and in genetic manipulation of rodent genes reveal that different subtypes of the GABAA receptor are responsible for mediating particular aspects of the anaesthetic behavioural repertoire. Such studies provide a better understanding of the neuronal circuitry involved in the various anaesthetic-induced behaviours and, in the future, may result in the development of novel therapeutics with a reduced propensity for side-effects.

GABAA receptor subtype involvement in addictive behaviour.

GABAA receptors form the major class of inhibitory neurotransmitter receptors in the mammalian brain. This review sets out to summarize the evidence that variations in genes encoding GABAA receptor isoforms are associated with aspects of addictive behaviour in humans, while animal models of addictive behaviour also implicate certain subtypes of GABAA receptor. In addition to outlining the evidence for the involvement of specific subtypes in addiction, we summarize the particular contributions of these isoforms in control over the functioning of brain circuits, especially the mesolimbic system, and make a first attempt to bring together evidence from several fields to understanding potential involvement of GABAA receptor subtypes in addictive behaviour. While the weight of the published literature is on alcohol dependency, the underlying principles outlined are relevant across a number of different aspects of addictive behaviour.

A study of subunit selectivity, mechanism and site of action of the delta selective compound 2 (DS2) at human recombinant and rodent native GABA(A) receptors.

BACKGROUND AND PURPOSE: Most GABA(A) receptor subtypes comprise 2α, 2ß and 1γ subunit, although for some isoforms, a δ replaces a γ-subunit. Extrasynaptic δ-GABA(A) receptors are important therapeutic targets, but there are few suitable pharmacological tools. We profiled DS2, the purported positive allosteric modulator (PAM) of δ-GABA(A) receptors to better understand subtype selectivity, mechanism/site of action and activity at native δ-GABA(A) receptors. EXPERIMENTAL APPROACH: Subunit specificity of DS2 was determined using electrophysiological recordings of Xenopus laevis oocytes expressing human recombinant GABA(A) receptor isoforms. Effects of DS2 on GABA concentration-response curves were assessed to define mechanisms of action. Radioligand binding and electrophysiology utilising mutant receptors and pharmacology were used to define site of action. Using brain-slice electrophysiology, we assessed the influence of DS2 on thalamic inhibition in wild-type and δ(0/0) mice. KEY RESULTS: Actions of DS2 were primarily determined by the δ-subunit but were additionally influenced by the α, but not the ß, subunit (α4/6ßxδ > α1ßxδ >> γ2-GABA(A) receptors > α4ß3). For δ-GABA(A) receptors, DS2 enhanced maximum responses to GABA, with minimal influence on GABA potency. (iii) DS2 did not act via the orthosteric, or known modulatory sites on GABA(A) receptors. (iv) DS2 enhanced tonic currents of thalamocortical neurones from wild-type but not δ(0/0) mice. CONCLUSIONS AND IMPLICATIONS: DS2 is the first PAM selective for α4/6ßxδ receptors, providing a novel tool to investigate extrasynaptic δ-GABA(A) receptors. The effects of DS2 are mediated by an unknown site leading to GABA(A) receptor isoform selectivity.

Regulation of AMPA receptor surface trafficking and synaptic plasticity by a cognitive enhancer and antidepressant molecule.

The plasticity of excitatory synapses is an essential brain process involved in cognitive functions, and dysfunctions of such adaptations have been linked to psychiatric disorders such as depression. Although the intracellular cascades that are altered in models of depression and stress-related disorders have been under considerable scrutiny, the molecular interplay between antidepressants and glutamatergic signaling remains elusive. Using a combination of electrophysiological and single nanoparticle tracking approaches, we here report that the cognitive enhancer and antidepressant tianeptine (S 1574, [3-chloro-6-methyl-5,5-dioxo-6,11-dihydro-(c,f)-dibenzo-(1,2-thiazepine)-11-yl) amino]-7 heptanoic acid, sodium salt) favors synaptic plasticity in hippocampal neurons both under basal conditions and after acute stress. Strikingly, tianeptine rapidly reduces the surface diffusion of AMPA receptor (AMPAR) through a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent mechanism that enhances the binding of AMPAR auxiliary subunit stargazin with PSD-95. This prevents corticosterone-induced AMPAR surface dispersal and restores long-term potentiation of acutely stressed mice. Collectively, these data provide the first evidence that a therapeutically used drug targets the surface diffusion of AMPAR through a CaMKII-stargazin-PSD-95 pathway, to promote long-term synaptic plasticity.

Novel compounds selectively enhance delta subunit containing GABA A receptors and increase tonic currents in thalamus.

Inhibition in the brain is dominated by the neurotransmitter gamma-aminobutyric acid (GABA); operating through GABA(A) receptors. This form of neural inhibition was presumed to be mediated by synaptic receptors, however recent evidence has highlighted a previously unappreciated role for extrasynaptic GABA(A) receptors in controlling neuronal activity. Synaptic and extrasynaptic GABA(A) receptors exhibit distinct pharmacological and biophysical properties that differentially influence brain physiology and behavior. Here we used a fluorescence-based assay and cell lines expressing recombinant GABA(A) receptors to identify a novel series of benzamide compounds that selectively enhance, or activate alpha4beta3delta GABA(A) receptors (cf. alpha4beta3gamma2 and alpha1beta3gamma2). Utilising electrophysiological methods, we illustrate that one of these compounds, 4-chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS1) potently (low nM) enhances GABA-evoked currents mediated by alpha4beta3delta receptors. At similar concentrations DS1 directly activates this receptor and is the most potent known agonist of alpha4beta3delta receptors. 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS2) selectively potentiated GABA responses mediated by alpha4beta3delta receptors, but was not an agonist. Recent studies have revealed a tonic form of inhibition in thalamus mediated by the alpha4beta2delta extrasynaptic GABA(A) receptors that may contribute to the regulation of thalamocortical rhythmic activity associated with sleep, wakefulness, vigilance and seizure disorders. In mouse thalamic relay cells DS2 enhanced the tonic current mediated by alpha4beta2delta receptors with no effect on their synaptic GABA(A) receptors. Similarly, in mouse cerebellar granule cells DS2 potentiated the tonic current mediated by alpha6betadelta receptors. DS2 is the first selective positive allosteric modulator of delta-GABA(A) receptors and such compounds potentially offer novel therapeutic opportunities as analgesics and in the treatment of sleep disorders. Furthermore, these drugs may be valuable in elucidating the physiological and pathophysiological roles played by these extrasynaptic GABA(A) receptors.

Novel structural determinants of single-channel conductance in nicotinic acetylcholine and 5-hydroxytryptamine type-3 receptors.

Nicotinic ACh (acetylcholine) and 5-HT3 (5-hydroxytryptamine type-3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Numerous lines of evidence indicate that the channel lining domain of such receptors is formed by the alpha-helical M2 domain (second transmembrane domain) contributed by each of five subunits present within the receptor complex. Specific amino acid residues within the M2 domain have accordingly been demonstrated to influence both single-channel conductance (gamma) and ion selectivity. However, it is now clear from work performed on the homomeric 5-HT3A receptor, heteromeric 5-HT3A/5-HT3B receptor and 5-HT3A/5-HT3B receptor subunit chimaeric constructs that an additional major determinant of gamma resides within a cytoplasmic domain of the receptor termed the MA-stretch (membrane-associated stretch). The MA-stretch, within the M3-M4 loop, is not traditionally thought to be implicated in ion permeation and selection. Here, we describe how such observations extend to a representative neuronal nicotinic ACh receptor composed of alpha4 and beta2 subunits and, by inference, probably other members of the Cys-loop family. In addition, we will attempt to interpret our results within the context of a recently developed atomic scale model of the nicotinic ACh receptor of Torpedo marmorata (marbled electric ray).

Neuroactive steroids and inhibitory neurotransmission: mechanisms of action and physiological relevance.

Dysfunction of GABA(A) receptor-mediated inhibition is implicated in a number of neurological and psychiatric conditions including epilepsy and affective disorders. Some of these conditions have been associated with abnormal levels of certain endogenously occurring neurosteroids, which potently and selectively enhance the function of the brain's major inhibitory receptor, the GABA(A) receptor. Consistent with their ability to enhance neuronal inhibition, such steroids exhibit in animals and humans anxiolytic, anticonvulsant and anesthetic actions. Neurosteroids, exemplified by the potent progesterone metabolite, 5alpha-pregnan-3alpha-ol-20-one can be synthesized de novo in the CNS both in neurones and glia in levels sufficient to modulate GABA(A) receptor function. Neurosteroid levels are not static, but are subject to dynamic fluctuations, for example during stress, or the later stages of pregnancy. These observations suggest that these endogenous modulators may refine the function of the brain's major inhibitory receptor and thus, play an important physiological and pathophysiological role. However, given the ubiquitous expression of GABA(A) receptors throughout the mammalian CNS, changes in neurosteroid levels should be widely experienced, causing a generalized enhancement of neuronal inhibition. Such a non-specific action would seem incompatible with a physiological role. However, neurosteroid action is both brain region and neurone selective. This specificity results from a variety of molecular mechanisms including receptor subunit composition, local steroid metabolism and phosphorylation. This paper will evaluate the relative contribution these mechanisms play in defining the interaction of neurosteroids with synaptic and extra-synaptic GABA(A) receptors.

The 5-hydroxytryptamine type 3 (5-HT3) receptor reveals a novel determinant of single-channel conductance.

5-HT3 (5-hydroxytryptamine type 3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Two 5-HT3 receptor subunits, 5-HT3A and 5-HT3B, have been characterized in detail, although additional putative 5-HT3 subunit genes (HTR3C, HTR3D and HTR3E) have recently been reported. 5-HT3 receptors function as homopentameric assemblies of the 5-HT3 subunit, or heteropentamers of 5-HT3A and 5-HT3B subunits of unknown stoichiometry. The single-channel conductances of human recombinant homomeric and heteromeric 5-HT3 receptors are markedly different, being <1 and approx. 16 pS respectively. Paradoxically, from the results of studies performed on the closely related nicotinic acetylcholine receptor, the channel-lining M2 domain of the 5-HT3A subunit is predicted to enhance cation conduction, whereas that of the 5-HT3B subunit would not. The present study describes a novel determinant of single-channel conductance, out with the M2 domain, which accounts for this anomaly. Utilizing a panel of chimaeric 5-HT3A and 5-HT3B subunits, a profound determinant of single-channel conductance was traced to a putative amphipathic helix (the 'HA stretch') within the large cytoplasmic loop of the receptor. Replacement of three arginine residues (R432, R436 and R440) unique to the HA stretch of the 5-HT3A subunit with the aligned residues (Q395, D399 and A403) of the 5-HT3B subunit increased the single-channel conductance 28-fold. Significantly, from ultrastructural studies of the Torpedo nicotinic acetylcholine receptor, the key residues may be components of narrow openings within the inner vestibule of the channel, located in the cytoplasm, which contribute to the permeation pathway. Our findings indicate an important and hitherto unappreciated function for the HA stretch in the Cys-loop family of transmitter-gated ion channels.

The interaction of anaesthetic steroids with recombinant glycine and GABAA receptors.

BACKGROUND: Anaesthetic steroids are established positive allosteric modulators of GABAA receptors, but little is known concerning steroid modulation of strychnine-sensitive glycine receptors, the principal mediators of fast, inhibitory neurotransmission in the brain stem and spinal cord. This study compared the modulatory actions of five anaesthetic pregnane steroids and two non-anaesthetic isomers at human recombinant alpha1 glycine and alpha1beta2gamma2L GABAA receptors. METHODS: Recombinant alpha1 glycine or alpha1beta2gamma2L GABAA receptors were expressed in Xenopus laevis oocytes and agonist-evoked currents recorded under voltage-clamp. Steroid modulation of currents evoked by GABA, or glycine, was quantified by determining the potency (EC50) and maximal effect of the compounds. RESULTS: The anaesthetics minaxolone (EC50=1.3 microM), Org20599 (EC50=1.1 microM) and alphaxalone (EC50=2.2 microM) enhanced currents mediated by GABAA receptors. The anaesthetics also enhanced currents mediated by glycine receptors, although with higher EC50 values (minaxolone 13.1 microM; Org20599=22.9 microM and alphaxalone=27.8 microM). The maximal enhancement (to 780-950% of control) produced by the three steroids acting at the GABAA receptor was similar, but currents evoked by glycine were potentiated with increasing effectiveness by alphaxalone (199%)

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system.

Without medical progress, dementing diseases such as Alzheimer's disease will become one of the main causes of disability. Preventing or delaying them has thus become a real challenge for biomedical research. Steroids offer interesting therapeutical opportunities for promoting successful aging because of their pleiotropic effects in the nervous system: they regulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination and influence cognitive processes, in particular learning and memory. Preclinical research has provided evidence that the normally aging nervous system maintains some capacity for regeneration and that age-dependent changes in the nervous system and cognitive dysfunctions can be reversed to some extent by the administration of steroids. The aging nervous system also remains sensitive to the neuroprotective effects of steroids. In contrast to the large number of studies documenting beneficial effects of steroids on the nervous system in young and aged animals, the results from hormone replacement studies in the elderly are so far not conclusive. There is also little information concerning changes of steroid levels in the aging human brain. As steroids present in nervous tissues originate from the endocrine glands (steroid hormones) and from local synthesis (neurosteroids), changes in blood levels of steroids with age do not necessarily reflect changes in their brain levels. There is indeed strong evidence that neurosteroids are also synthesized in human brain and peripheral nerves. The development of a very sensitive and precise method for the analysis of steroids by gas chromatography/mass spectrometry (GC/MS) offers new possibilities for the study of neurosteroids. The concentrations of a range of neurosteroids have recently been measured in various brain regions of aged Alzheimer's disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimer's patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimer's disease, the phosphorylated tau protein and the beta-amyloid peptides. The metabolism of dehydroepiandrosterone has also been analyzed for the first time in the aging brain from Alzheimer patients and non-demented controls. The conversion of dehydroepiandrosterone to Delta5-androstene-3beta,17beta-diol and to 7alpha-OH-dehydroepiandrosterone occurred in frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimer's patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

GABAA receptor modulation by the novel intravenous general anaesthetic E-6375.

E-6375 (4-butoxy-2-[4-(2-cyanobenzoyl)-1-piperazinyl] pyrimidine hydrochloride) is a new intravenous general anaesthetic with an anaesthetic potency, in mice, comparable to propofol, or etomidate. Here, we examined the effect of E-6375 upon the GABAA receptor, a putative target of intravenous anaesthetic action. E-6375 reversibly enhanced GABA-evoked currents mediated by recombinant GABAA (alpha1beta2gamma2L) receptors expressed in Xenopus laevis oocytes, with little effect on NMDA- and kainate-evoked currents mediated by NR1a/NR2A and GluR1o/GluR2o glutamate receptors, respectively. E-6375 prolonged the decay of GABA-evoked miniature inhibitory postsynaptic currents recorded from rat Purkinje neurones demonstrating the anaesthetic also enhanced the activity of synaptic GABAA receptors. The GABA enhancing action of E-6375 on recombinant GABAA receptors was unaffected by the subtype of the alpha isoform (i.e. alphaxbeta2gamma2L; x=1-3) within the receptor, but was increased by the omission of the gamma2L subunit. Receptors incorporating beta2, or beta3, subunits were more sensitive to modulation by E-6375 than those containing the beta1 subunit. The selectivity of E-6375 was largely governed by the identity (serine or asparagine) of a single amino acid residue within the second transmembrane domain of the beta-subunit. The various in vivo actions of general anaesthetics may be mediated by GABAA receptor isoforms that have a differential distribution within the CNS. The identification of agents, such as E-6375, that discriminate between GABAA receptor subtypes may augur the development of general anaesthetics with an improved therapeutic profile.

Expression of cell cycle-related gene products in Langerhans cell histiocytosis.

BACKGROUND The pathogenesis of Langerhans cell histiocytosis (LCH), a disease characterized by an abnormal accumulation of the dendritic Langerhans cells, is still unknown. Based on the monoclonality of the CD1a+ cell and reports of familial clustering, it is hypothesized that a genetic alteration at a cellular level may be causative. This genetic change may have an effect on the cellular mechanisms controlling proliferation and apoptosis. MATERIALS AND METHODS LCH-lesions were studied for the expression of Ki-67, present in the nucleus of proliferating cells. Furthermore, the expression of cell cycle-related gene products TGF-beta receptor I and II, MDM2, p53, p21, p16, Rb, and Bcl2 were studied. The TGF-betaR genes play a role in tumor suppression, whereas Bcl2 inhibits apoptosis. The remaining genes are part of either the p53-p21 and/or p16-Rb pathways, which induce cell cycle arrest or apoptosis in response to DNA damage. RESULTS In 30 biopsies the diagnosis of LCH could be confirmed on the basis of CD1a positivity (27 bone and 3 skin). All cases showed scattered nuclear-positive staining for the proliferation marker Ki-67. In more than 90% (n >/=27) of these cases, expression of TGFbeta receptor I and II, MDM2, p53, p21, p16, Rb, and Bcl2 was detected in lesional LCH cells. The overexpression was in general heterogeneous, ranging from limited focal staining of scattered cells within the lesion to strong diffuse staining. CONCLUSIONS These findings suggest that the cellular mechanisms that sense and respond to DNA-damage, namely the p53-p21 pathway and the p16-Rb pathway, are activated. The expression of Ki-67 indicates that the cells in LCH are proliferating. The observed overexpression of Bcl2 may play a role in the activation of p53 and p16 and/or the arrest of apoptosis.

Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors: commentary on Brown et al.

British Journal of Pharmacology (2000) 136, 957-959

Modulation of native and recombinant GABA(A) receptors by endogenous and synthetic neuroactive steroids.

Upon administration, certain pregnane steroids produce clear behavioural effects including, anxiolysis, sedation, analgesia, anaesthesia and are anti-convulsant. This behavioural profile is characteristic of compounds that act to enhance the actions of GABA acting at the GABA(A) receptor. In agreement, numerous studies have now demonstrated these steroids to be potent, positive allosteric modulators of the GABA(A) receptor. The pregnane steroids are synthesized in the periphery by endocrine glands such as the adrenals and the ovaries, but are also made by neurons and glial cells in the central nervous system itself. Hence, these compounds could play both an endocrine and a paracrine role to influence neuronal excitability by promoting inhibition. Here we review evidence that the pregnane steroids are highly selective and extremely potent GABA(A) receptor modulators and that their effects at 'physiological' concentrations (low nanomolar) may be influenced by the subunit composition of the GABA(A) receptor. This feature may underlie recent findings demonstrating the effects of the neurosteroids on inhibitory synaptic transmission to be brain region dependent, although recent reports suggest that phosphorylation mechanisms may additionally influence neurosteroid sensitivity of the GABA(A) receptor. Numerous synthetic steroids have been synthesized in an attempt to therapeutically exploit the behavioural effects of the pregnane steroids and progress with this approach will be discussed. However, the demonstration that the steroids may be made within the central nervous system offers the alternative strategy of targeting the enzymes that synthesize/metabolise the neurosteroids to exploit this novel endocrine/paracrine interaction.

Neurosteroid modulation of recombinant and synaptic GABAA receptors.

Certain pregnane steroids are now established as potent, positive allosteric modulators of the gamma-aminobutyric acid type A (GABAA) receptor. These compounds are known to be synthesized in the periphery by endocrine glands, such as the ovaries and the adrenal glands, and can rapidly cross the blood-brain barrier. Therefore, such steroids could act as endogeneous modulators of the major inhibitory receptor in the mammalian central nervous system. However, the demonstration that certain neurons and glia can synthesize the pregnane steroids (i.e., neurosteroids) additionally suggests that they may serve a paracrine role by influencing GABAA-receptor function through their local release in the brain itself. Here, we demonstrate that these neurosteroids are highly selective and extremely potent modulators of the GABAA receptor. The subunit composition of the GABAA receptor may influence the actions of the neurosteroids, particularly when considering concentrations of these agents thought to occur physiologically, which may underlie their reported differential effects at certain inhibitory synapses. However, recent work suggests that the phosphorylation status of either the synaptic GABAA receptor or its associated proteins may also influence neurosteroid sensitivity; these findings are discussed. Upon administration, the neurosteroids exhibit clear behavioral effects, including sedation, anticonvulsant actions, and behaviors predictive of anxiolysis; when given at high doses, they induce general anesthesia. Numerous synthetic steroids have been synthesized in an attempt to therapeutically exploit these properties, and these data are reviewed in this chapter. However, targeting the brain enzymes that synthesize and metabolize the neurosteroids may offer a new approach to exploit this novel endocrine-paracrine neurotransmitter interaction.

Alpha-amino acid phenolic ester derivatives: novel water-soluble general anesthetic agents which allosterically modulate GABA(A) receptors.

In the search for a novel water-soluble general anesthetic agent the activity of an alpha-amino acid phenolic ester lead, identified from patent literature, was markedly improved. In addition to improving in vivo activity in mice, good in vitro activity at GABA(A) receptors was also conferred. Within the series of compounds good enantioselectivity for both in vitro and in vivo activity was found, supporting a protein-mediated mechanism of action for anesthesia involving allosteric modulation of GABA(A) receptors. alpha-Amino acid phenolic ester 19, as the hydrobromide salt Org 25435, was selected for clinical evaluation since it retained the best overall anesthetic profile coupled with improved stability and water solubility. In the clinic it proved to be an effective intravenous anesthetic in man with rapid onset of and recovery from anesthesia at doses of 3 and 4 mg/kg.

Developmental profile of excitatory GABA(A) responses in cultured rat cerebellar granule cells.

GABA induced a transient increase in cytosolic free Ca2+ in cerebellar granule cells, which decreased from 3 to 8 days in vitro (DIV). Cytosolic Ca2+ changes induced by glutamate/glycine were comparable at 3 and 7 DIV. The GABA response was ascribed to GABA(A)-receptor mediated depolarization activating L-type Ca2+ channels since the response was inhibited by bicuculline or nifedipine. GABA-mediated Ca2+ rise at 4 DIV was potentiated by pentobarbital or by the neurosteroid 5beta-pregnan-3alpha-ol-20-one, or by decreasing the extracellular Cl- concentration. Neurons cultured for > 7 DIV showed no rise in intracellular Ca2+ in response to GABA regardless of the Cl- gradient. GABA(A) receptor-mediated cytosolic Ca2+ rise suggests an important role for the excitatory activity of GABA in developing cerebellar granule neurons.

The 4'lysine in the putative channel lining domain affects desensitization but not the single-channel conductance of recombinant homomeric 5-HT3A receptors.

The 5-HT3 receptor is a transmitter-gated ion channel of the Cys-loop superfamily. Uniquely, 5-HT3 receptor subunits (5-HT3A and 5-HT3B) possess a positively charged lysine residue within the putative channel lining M2 domain (4' position). Using whole cell recording techniques, we examined the role of this residue in receptor function using wild-type (WT) and mutant 5-HT3A receptor subunits of murine origin transiently expressed in human embryonic kidney (HEK 293) cells. WT 5-HT3A receptors mediated rapidly activating currents in response to 5-HT (10-90 % rise time, 103 ms; EC50, 2.34 microM; Hill coefficient, nH, 2.87). The currents rectified inwardly, reversed in sign at a potential of -9 mV and desensitized in the continuous presence of agonist (half-time of desensitization, t(1/2), 2.13 s). 5-HT3A receptor subunits in which the 4'lysine was mutated to arginine, glutamine, serine or glycine formed functional receptors. 5-HT EC50 values were approximately 2-fold lower than for WT 5-HT3A receptors, but Hill coefficients, kinetics of current activation, rectification, and reversal potentials were unaltered. Each of the mutants desensitized more slowly than the WT 5-HT3A receptor, with the arginine and glycine mutations exhibiting the greatest effect (5-fold reduction). The rank order of effect was arginine > glycine > serine > glutamine. The single-channel conductance of the WT 5-HT3A receptor, as assessed by fluctuation analysis of macroscopic currents, was 390 fS. A similar value was obtained for the 4'lysine mutant receptors. Thus it appears unlikely that 4'lysine is exposed to the channel lumen. Mutation of residues immediately adjacent to 4'lysine to glutamate or lysine resulted in lack of receptor expression or function. We conclude that 4'lysine does not form part of the channel lining, but may play an important role in 5-HT3 receptor desensitization.

General anaesthetic action at transmitter-gated inhibitory amino acid receptors.

Research within the past decade has provided compelling evidence that anaesthetics can act directly as allosteric modulators of transmitter-gated ion channels. Recent comparative studies of the effects of general anaesthetics across a structurally homologous family of inhibitory amino acid receptors that includes mammalian GABAA, glycine and Drosophila RDL GABA receptors have provided new insights into the structural basis of anaesthetic action at transmitter-gated channels. In this article, the differential effects of general anaesthetics across inhibitory amino acid receptors and the potential relevance of such actions to general anaesthesia will be discussed.