RESUMEN
Tonic GABA currents mediated by high-affinity extrasynaptic GABAA receptors, are increasingly recognized as important regulators of cell and neuronal network excitability. Dysfunctional GABAA receptor signaling that results in modified tonic GABA currents is associated with a number of neurological disorders. Consequently, developing compounds to selectively modulate the activity of extrasynaptic GABAA receptors underlying tonic inhibition is likely to prove therapeutically useful. Here, we examine the GABAA receptor subtype selectivity of the weak partial agonist, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), as a potential mechanism for modulating extrasynaptic GABAA receptor-mediated tonic currents. By using recombinant GABAA receptors expressed in HEK293 cells, and native GABAA receptors of cerebellar granule cells, hippocampal neurons, and thalamic relay neurons, 4-PIOL evidently displayed differential agonist and antagonist-type profiles, depending on the extrasynaptic GABAA receptor isoforms targeted. For neurons, this resulted in differential modulation of GABA tonic currents, depending on the cell type studied, their respective GABAA receptor subunit compositions, and critically, on the ambient GABA levels. Unexpectedly, 4-PIOL revealed a significant population of relatively low-affinity γ2 subunit-containing GABAA receptors in the thalamus, which can contribute to tonic inhibition under specific conditions when GABA levels are raised. Together, these data indicate that partial agonists, such as 4-PIOL, may be useful for modulating GABAA receptor-mediated tonic currents, but the direction and extent of this modulation is strongly dependent on relative expression levels of different extrasynaptic GABAA receptor subtypes, and on the ambient GABA levels. SIGNIFICANCE STATEMENT: A background level of inhibition (tonic) is important in the brain for controlling neuronal excitability. Increased levels of tonic inhibition are associated with some neurological disorders but there are no specific ligands capable of selectively reducing tonic inhibition. Here we explore the use of a GABA partial agonist as a selective chemical tool in three different brain regions. We discover that the activity of a partial agonist is heavily dependent upon the GABAA receptor subunit composition underpinning tonic inhibition, and on the ambient levels of GABA in the brain.
Asunto(s)
Potenciales de la Membrana/fisiología , Inhibición Neural/fisiología , Receptores de GABA-A/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/citología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , GABAérgicos/farmacología , Humanos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Isoxazoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Piperidinas/farmacología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
GABA-mediated tonic and phasic inhibition of thalamic relay neurons of the dorsal lateral geniculate nucleus (dLGN) was studied after ablating tyrosine (Y) phosphorylation of receptor γ2-subunits. As phosphorylation of γ2 Y365 and Y367 reduces receptor internalization, to understand their importance for inhibition we created a knock-in mouse in which these residues are replaced by phenylalanines. On comparing wild-type (WT) and γ2(Y365/367F)+/- (HT) animals (homozygotes are not viable in utero), the expression levels of GABAA receptor α4-subunits were increased in the thalamus of female, but not male mice. Raised δ-subunit expression levels were also observed in female γ2(Y365/367F) +/- thalamus. Electrophysiological analyses revealed no difference in the level of inhibition in male WT and HT dLGN, while both the spontaneous inhibitory postsynaptic activity and the tonic current were significantly augmented in female HT relay cells. The sensitivity of tonic currents to the δ-subunit superagonist THIP, and the blocker Zn(2+), were higher in female HT relay cells. This is consistent with upregulation of extrasynaptic GABAA receptors containing α4- and δ-subunits to enhance tonic inhibition. In contrast, the sensitivity of GABAA receptors mediating inhibition in the female γ2(Y356/367F) +/- to neurosteroids was markedly reduced compared with WT. We conclude that disrupting tyrosine phosphorylation of the γ2-subunit activates a sex-specific increase in tonic inhibition, and this most likely reflects a genomic-based compensation mechanism for the reduced neurosteroid sensitivity of inhibition measured in female HT relay neurons.
Asunto(s)
Cuerpos Geniculados/citología , Inhibición Neural/fisiología , Neuronas/fisiología , Receptores de GABA-A/metabolismo , Tirosina/metabolismo , Anestésicos/farmacología , Animales , Animales Recién Nacidos , Línea Celular Transformada , Femenino , Cuerpos Geniculados/fisiología , Humanos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Fosforilación , Receptores de GABA-A/genética , Receptores de GABA-B/química , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Tirosina/genéticaRESUMEN
Brain inhibition is a vital process for controlling and sculpting the excitability of the central nervous system in healthy individuals. This level of control is provided over several timescales and involves the neurotransmitter GABA acting at inhibitory synapses to: rapidly inhibit neurons by activating the GABAA receptor; over a slower timescale, to tonically activate extrasynaptic GABAA receptors to provide a low level of background inhibition; and finally, to activate G-protein coupled GABAB receptors to control transmitter release by inhibiting presynaptic Ca2+ channels whilst providing postsynaptic inhibition via K+ channel activation. From this plethora of roles for GABA and its receptors, the GABAA receptor isoform is of major interest due to its dynamic functional plasticity, which in part, is due to being targeted by modulatory brain neurosteroids derived from sex and stress hormones. This family of neurosteroids can, depending on their structure, potentiate, activate and also inhibit the activity of GABAA receptors to affect brain inhibition. This review tracks the methods that have been deployed in probing GABAA receptors, and charts the sterling efforts made by several groups to locate the key neurosteroid binding sites that affect these important receptors. Increasing our knowledge of these binding sites will greatly facilitate our understanding of the physiological roles of neurosteroids and will help to advance their use as novel therapeutics to combat debilitating brain diseases.
RESUMEN
Tonic inhibition mediated by extrasynaptic GABA(A) receptors (GABA(A) Rs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA(A) Rs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA(A) R-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA(A) R activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4ß2δ GABA(A) Rs, which represent a key extrasynaptic GABA(A) R isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA(A) Rs. The inhibitory effects of PKC activation on α4ß2δ GABA(A) R activity appeared to be mediated by direct phosphorylation at a previously identified site on the ß2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABA(A) R-mediated inhibition.
Asunto(s)
Hipocampo/fisiología , Inhibición Neural/fisiología , Proteína Quinasa C/fisiología , Receptores de GABA-A/fisiología , Tálamo/fisiología , Animales , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Humanos , Ratones , Inhibición Neural/efectos de los fármacos , Técnicas de Cultivo de Órganos , Receptores de GABA-A/metabolismo , Tálamo/efectos de los fármacos , Tálamo/enzimología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Neurosteroids are important endogenous modulators of GABAA receptor-mediated neurotransmission within the CNS and play a vital role in maintaining normal healthy brain function. Research has mainly focussed on neurosteroids such as allopregnanolone and tetrahydro-deoxycorticosterone (THDOC) which are allosteric potentiators of GABAA receptors, whilst the sulphated steroids, including pregnenolone sulphate (PS), which inhibit GABAA receptor function, have been relatively neglected. Importantly, a full description of PS effects on inhibitory synaptic transmission, at concentrations that are expected to inhibit postsynaptic GABAA receptors, is lacking. Here, we address this deficit by recording inhibitory postsynaptic currents (IPSCs) from rat hippocampal neurons both in culture and in acute brain slices and explore the impact of PS at micromolar concentrations. We reveal that PS inhibits postsynaptic GABAA receptors, evident from reductions in IPSC amplitude and decay time. Concurrently, PS also causes an increase in synaptic GABA release which we discover is due to the activation of presynaptic TRPM3 receptors located close to presynaptic GABA release sites. Pharmacological blockade of TRPM3 receptors uncovers a PS-evoked reduction in IPSC frequency. This second presynaptic effect is caused by PS activation of inwardly-rectifying Kir2.3 channels on interneurons, which act to depress synaptic GABA release. Overall, we provide a comprehensive characterisation of pre- and postsynaptic modulation by PS of inhibitory synaptic transmission onto hippocampal neurons which elucidates the diverse mechanisms by which this understudied neurosteroid can modulate brain function.
Asunto(s)
Neuroesteroides , Canales Catiónicos TRPM , Ratas , Animales , Receptores de GABA-A/metabolismo , Neuroesteroides/farmacología , Transmisión Sináptica , Pregnenolona/farmacología , Hipocampo , Potenciales Postsinápticos Inhibidores , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
High-affinity extrasynaptic GABA(A) receptors (GABA(A)Rs) are a prominent feature of cerebellar granule neurons and thalamic relay neurons. In both cell types, the presence of synaptic glomeruli would be expected to promote activation of these GABA(A)Rs, contributing to phasic spillover-mediated currents and tonic inhibition. However, the precise role of different receptor subtypes in these two phenomena is unclear. To address this question, we made recordings from neurons in acute brain slices from mice, and from tsA201 cells expressing recombinant GABA(A)Rs. We found that δ subunit-containing GABA(A)Rs of both cerebellar granule neurons and thalamic relay neurons of the lateral geniculate nucleus contributed to tonic conductance caused by ambient GABA but not to spillover-mediated currents. In the presence of a low "ambient" GABA concentration, recombinant "extrasynaptic" δ subunit-containing GABA(A)Rs exhibited profound desensitization, rendering them insensitive to brief synaptic- or spillover-like GABA transients. Together, our results demonstrate that phasic spillover and tonic inhibition reflect the activation of distinct receptor populations.
Asunto(s)
Receptores de GABA-A/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Línea Celular , Cerebelo/citología , Cerebelo/fisiología , Humanos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Técnicas de Placa-Clamp , Subunidades de Proteína/fisiología , Ratas , Receptores de GABA-A/genética , Sinapsis/fisiología , Tálamo/citología , Tálamo/fisiología , TransfecciónRESUMEN
GABAA receptors are vital for controlling neuronal excitability and can display significant levels of constitutive activity that contributes to tonic inhibition. However, the mechanisms underlying spontaneity are poorly understood. Here we demonstrate a strict requirement for ß3 subunit incorporation into receptors for spontaneous gating, facilitated by α4, α6 and δ subunits. The crucial molecular determinant involves four amino acids (GKER) in the ß3 subunit's extracellular domain, which interacts with adjacent receptor subunits to promote transition to activated, open channel conformations. Spontaneous activity is further regulated by ß3 subunit phosphorylation and by allosteric modulators including neurosteroids and benzodiazepines. Promoting spontaneous activity reduced neuronal excitability, indicating that spontaneous currents will alter neural network activity. This study demonstrates how regional diversity in GABAA receptor isoform, protein kinase activity, and neurosteroid levels, can impact on tonic inhibition through the modulation of spontaneous GABAA receptor gating.
Asunto(s)
Hipocampo/fisiología , Activación del Canal Iónico/fisiología , Neuronas/fisiología , Receptores de GABA-A/fisiología , Algoritmos , Secuencia de Aminoácidos , Animales , Células Cultivadas , Células HEK293 , Hipocampo/citología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Ratones , Modelos Moleculares , Modelos Neurológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Ratas Sprague-Dawley , Receptores de GABA-A/química , Receptores de GABA-A/genética , Homología de Secuencia de Aminoácido , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The time-dependent integration of excitatory and inhibitory synaptic currents is an important process for shaping the input-output profiles of individual excitable cells, and therefore the activity of neuronal networks. Here, we show that the decay time course of GABAergic inhibitory synaptic currents is considerably faster when recorded with physiological internal Cl(-) concentrations than with symmetrical Cl(-) solutions. This effect of intracellular Cl(-) is due to a direct modulation of the GABA(A) receptor that is independent of the net direction of current flow through the ion channel. As a consequence, the time window during which GABAergic inhibition can counteract coincident excitatory inputs is much shorter, under physiological conditions, than that previously measured using high internal Cl(-). This is expected to have implications for neuronal network excitability and neurodevelopment, and for our understanding of pathological conditions, such as epilepsy and chronic pain, where intracellular Cl(-) concentrations can be altered.
Asunto(s)
Cloruros/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Líquido Intracelular/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Animales Recién Nacidos , Línea Celular , Humanos , Inhibición Neural/fisiología , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Tonic inhibition has emerged as a key regulator of neuronal excitability in the CNS. Thalamic relay neurons of the dorsal lateral geniculate nucleus (dLGN) exhibit a tonic GABA(A) receptor (GABA(A)R)-mediated conductance that is correlated with delta-subunit expression. Indeed, consistent with the absence of delta-subunit expression, no tonic conductance is found in the adjacent ventral LGN. We show that, in contrast to the situation in cerebellar granule cells, thalamic delta-subunit-containing GABA(A)Rs (delta-GABA(A)Rs) do not contribute to a spillover component of IPSCs in dLGN. However, tonic activation of thalamic delta-GABA(A)Rs is sensitive to the global level of inhibition, showing an absolute requirement on the synaptic release of GABA. Thus, the tonic conductance is abolished when transmitter release probability is reduced or action potential-evoked release is blocked. We further show that continuous activation of delta-GABA(A)Rs introduces variability into the timing of low-threshold rebound bursts. Hence, activation of delta-GABA(A)Rs could act to destabilize thalamocortical oscillations and therefore have an important impact on behavioral state.
Asunto(s)
Neuronas/fisiología , Receptores de GABA-A/fisiología , Sinapsis/fisiología , Tálamo/fisiología , Animales , Cloruros/metabolismo , Umbral Diferencial , Conductividad Eléctrica , Cuerpos Geniculados/citología , Cuerpos Geniculados/fisiología , Homeostasis/fisiología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibición Neural/fisiología , Isoformas de Proteínas/fisiología , Tiempo de Reacción , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
We have discovered that adult thalamocortical relay neurones exhibit a sustained enhancement of synaptic inhibition triggered by transient action potential firing of a single thalamic relay neurone. The sustained activity-dependent increase in IPSC frequency (+48.3 +/- 11.4%, n = 32) was blocked by chelating calcium inside an individual cell, by scavenging nitric oxide or by blocking NMDA receptor activation in the thalamus. Surprisingly, the tonic inhibition that is known to result from extrasynaptic GABA(A) receptor activation in these cells was unaffected by this local form of plasticity. However, tonic inhibition was increased (+131.9 +/- 56.5%, n = 13) following widespread changes in GABA release across the thalamus. These data suggest that thalamocortical sleep-state oscillations requiring membrane hyperpolarization will be influenced by global sensing of GABA release acting through extrasynaptic GABA(A) receptors. In contrast, local changes in GABA release of the type observed following this novel form of activity-dependent plasticity will influence local integration of sensory information without changing levels of tonic inhibition.
Asunto(s)
Plasticidad Neuronal/fisiología , Neuronas/efectos de los fármacos , Sinapsis/fisiología , Tálamo/citología , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal/efectos de los fármacos , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Penicilamina/análogos & derivados , Penicilamina/farmacología , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Tálamo/fisiologíaRESUMEN
γ-aminobutyric acid type A receptors (GABAARs) are important components of the central nervous system and they are functionally tasked with controlling neuronal excitability. These receptors are subject to post-translational modification and also to modulation by endogenous regulators, such as the neurosteroids. These modulators can either potentiate or inhibit GABAAR function. Whilst the former class of neurosteroids are considered to bind to and act from the transmembrane domain of the receptor, the domains that are important for the inhibitory neurosteroids remain less clear. In this study, we systematically compare a panel of recombinant synaptic-type and extrasynaptic-type GABAARs expressed in heterologous cell systems for their sensitivity to inhibition by the classic inhibitory neurosteroid, pregnenolone sulphate. Generally, peak GABA current responses were inhibited less compared to steady-state currents, implicating the desensitised state in inhibition. Moreover, pregnenolone sulphate inhibition increased with GABA concentration, but showed minimal voltage dependence. There was no strong dependence of inhibition on receptor subunit composition, the exception being the ρ1 receptor, which is markedly less sensitive. By using competition experiments with pregnenolone sulphate and the GABA channel blocker picrotoxinin, discrete binding sites are proposed. Furthermore, by assessing inhibition using site-directed mutagenesis and receptor chimeras comprising α, ß or γ subunits with ρ1 subunits, the receptor transmembrane domains are strongly implicated in mediating inhibition and most likely the binding location for pregnenolone sulphate in GABAARs. This article is part of the "Special Issue Dedicated to Norman G. Bowery".
Asunto(s)
Neurotransmisores/farmacología , Receptores de GABA-A/metabolismo , Animales , Sitios de Unión , Línea Celular , Humanos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Pentobarbital/farmacología , Picrotoxina/farmacología , Pregnenolona/farmacología , Dominios Proteicos , Proteínas Recombinantes/metabolismoRESUMEN
Tonic inhibitory conductances mediated by GABAA receptors have now been identified and characterized in many different brain regions. Most experimental studies of tonic GABAergic inhibition have been carried out using acute brain slice preparations but tonic currents have been recorded under a variety of different conditions. This diversity of recording conditions is likely to impact upon many of the factors responsible for controlling tonic inhibition and can make comparison between different studies difficult. In this review, we will firstly consider how various experimental conditions, including age of animal, recording temperature and solution composition, are likely to influence tonic GABAA conductances. We will then consider some technical considerations related to how the tonic conductance is measured and subsequently analyzed, including how the use of current noise may provide a complementary and reliable method for quantifying changes in tonic current.
Asunto(s)
Encéfalo/fisiología , Electrofisiología/métodos , Inhibición Neural/fisiología , Técnicas de Placa-Clamp/métodos , Receptores de GABA-A/fisiología , Animales , Potenciales de la Membrana/fisiologíaRESUMEN
We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(+) enantiomer being significantly more potent than the S(-) enantiomer. Its effects on alpha1beta2gamma2s GABA(A) receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (beta2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds approximately 75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABA(A) receptor or other putative targets.
Asunto(s)
Anestésicos/farmacología , Etomidato/análogos & derivados , Etomidato/farmacología , Albúmina Sérica/química , Animales , Azidas/farmacología , Sitios de Unión , Relación Dosis-Respuesta a Droga , Etomidato/química , Humanos , Masculino , Modelos Químicos , Unión Proteica , Rana temporaria , Ratas , Ratas Sprague-Dawley , EstereoisomerismoRESUMEN
Nitrous oxide, xenon, and cyclopropane are anesthetic gases that have a distinct pharmacological profile. Whereas the molecular basis for their anesthetic actions remains unclear, they behave very differently to most other general anesthetics in that they have little or no effect on GABAA receptors, yet strongly inhibit the N-methyl-d-aspartate subtype of glutamate receptors. Here we show that certain members of the two-pore-domain K+ channel superfamily may represent an important new target for these gaseous anesthetics. TREK-1 is markedly activated by clinically relevant concentrations of nitrous oxide, xenon, and cyclopropane. In contrast, TASK-3, a member of this family that is very sensitive to volatile anesthetics, such as halothane, is insensitive to the anesthetic gases. We demonstrate that the C-terminal cytoplasmic domain is not an absolute requirement for the actions of the gases, although it clearly plays an important modulatory role. Finally, we show that Glu306, an amino acid that has previously been found to be important in the modulation of TREK-1 by arachidonic acid, membrane stretch and internal pH, is critical for the activating effects of the anesthetic gases.