GABAA Receptor Desensitization Research Articles

Physiological role for GABAA receptor desensitization in the induction of long-term potentiation at inhibitory synapses

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels distributed throughout the brain where they mediate synaptic and tonic inhibition. Following activation, these receptors undergo desensitization which involves entry into long-lived agonist-bound closed states. Although the kinetic effects of this state are recognised and its structural basis has been uncovered, the physiological impact of desensitization on inhibitory neurotransmission remains unknown. Here we describe an enduring form of long-term potentiation at inhibitory synapses that elevates synaptic current amplitude for 24 h following desensitization of GABAARs in response to agonist exposure or allosteric modulation. Using receptor mutants and allosteric modulators we demonstrate that desensitization of GABAARs facilitates their phosphorylation by PKC, which increases the number of receptors at inhibitory synapses. These observations provide a physiological relevance to the desensitized state of GABAARs, acting as a signal to regulate the efficacy of inhibitory synapses during prolonged periods of inhibitory neurotransmission.

Molecular and Regulatory Mechanisms of Desensitization and Resensitization of GABAA Receptors with a Special Reference to Propofol/Barbiturate.

It is known that desensitization of GABAA receptor (GABAAR)-mediated currents is paradoxically correlated with the slowdown of their deactivation, i.e., resensitization. It has been shown that an upregulation of calcineurin enhances the desensitization of GABAAR-mediated currents but paradoxically prolongs the decay phase of inhibitory postsynaptic currents/potentials without appreciable diminution of their amplitudes. The paradoxical correlation between desensitization and resensitization of GABAAR-mediated currents can be more clearly seen in response to a prolonged application of GABA to allow more desensitization, instead of brief pulse used in previous studies. Indeed, hump-like GABAAR currents were produced after a strong desensitization at the offset of a prolonged puff application of GABA in pyramidal cells of the barrel cortex, in which calcineurin activity was enhanced by deleting phospholipase C-related catalytically inactive proteins to enhance the desensitization/resensitization of GABAAR-mediated currents. Hump-like GABAAR currents were also evoked at the offset of propofol or barbiturate applications in hippocampal or sensory neurons, but not GABA applications. Propofol and barbiturate are useful to treat benzodiazepine/alcohol withdrawal syndrome, suggesting that regulatory mechanisms of desensitization/resensitization of GABAAR-mediated currents are important in understanding benzodiazepine/alcohol withdrawal syndrome. In this review, we will discuss the molecular and regulatory mechanisms underlying the desensitization and resensitization of GABAAR-mediated currents and their functional significances.

Enhanced lateral inhibition in the barrel cortex by deletion of phospholipase C-related catalytically inactive protein-1/2 in mice.

We previously demonstrated that the deletion of phospholipase C-related catalytically inactive protein-1/2 (PRIP-1/2) enhances the desensitization of GABAA receptors (GABAARs), while it facilitates their resensitization at the offset of GABA puff, causing a hump-like tail current (tail-I) in layer 3 (L3) pyramidal cells (PCs) of the barrel cortex. In the present study, we investigated whether inhibitory synaptic transmission in L3 PCs in the barrel cortex is altered in the PRIP-1/2 double-knockout (PRIP-DKO) mice, and if so, how the interaction between excitation and inhibition is subsequently modified. PRIP-1/2 deletion resulted in the prolongation of the decay phase of inhibitory postsynaptic currents/potentials (IPSCs/IPSPs) in L3 PCs evoked by stimulation of L3, leaving the overall features of miniature IPSCs unchanged. An optical imaging revealed that the spatiotemporal profile of a horizontal excitation spread across columns in L2/3 caused by L4 stimulation in the barrel cortex was more restricted in PRIP-DKO mice compared to the wild type, while those obtained in the presence of bicuculline were almost identical between the two genotypes. These findings suggest that PRIP-1/2 deletion enhances the lateral inhibition by prolonging inhibitory synaptic actions to limit the intercolumnar integration in the barrel cortex. Considering the present findings together with our previous study including a mathematical simulation, the prolongation of inhibitory synaptic actions is likely to result from an enhancement of desensitization followed by an enhanced resensitization in GABAARs.

The neuropharmacology of the age-old sedative/hypnotic, ethanol

Ethyl alcohol (ethanol) is produced by the process of fermentation whereby plant sugars are converted to ethanol by yeast. Products of fermentation include mead from honey, wine from grapes, sake from rice, and beer from grains. Fermented products can also be distilled, for example when whisky is distilled from grains (Herbst and Herbst, 2003). The history of voluntary ethanol use by humans dates as far back as 8000 years ago for wine and possibly earlier for beer, as barley may have been the oldest cultivated grain (Pendell, 2010). It is also documented that ethanol was an extremely important staple to ancient civilizations such as the Egyptians and the Maya (Johnson, 1994), as well as more modern societies such as the original American colonies (Pendell, 2010). It seems that modern society's interest in alcohol has certainly not waned, and from the standpoint of neuropharmacology, has probably increased. During the past several decades, there has been a concerted effort to determine the neuropharmacological mechanism of action(s) of ethanol. Ethanol has been reported to produce its effects via modulation of neural cell membrane fluidity as well as modulation of several neurotransmitter systems, including γ-amino butyric acid (GABA), glutamate, dopamine, and opioid systems (for reviews, see Koob et al., 1998; Kumar et al., 2009). GABA is an inhibitory amino acid neurotransmitter that is ubiquitously distributed in the mammalian brain, and ethanol's effects on the GABA system are thought to be mediated primarily by activating the GABAA receptor, a 5-subunit receptor that gates Cl− ions (for a review, see Kumar et al., 2009). Thus, GABA receptor activation produces CNS inhibition via Cl− influx. It has also been reported that specific GABAA receptor subunits mediate specific effects. For example, the α1 subunit is associated with sedation, whereas the α2 and α3 subunits are associated with anti-anxiety effects (Licata and Rowlett, 2008; Ator et al., 2010). Many types of sedative-hypnotic compounds (drugs producing dose-dependent sedation and ultimately sleep), including the benzodiazepines, barbiturates, and ethanol, bind to and activate the GABAA receptor, and it is reasonable to assume that the sedative effects of these agents are mediated, at least in part, by the α1 subunit. It is known that neurotransmitters, including GABA, bind to their receptors as “first messengers,” and initiate a complex cascade of intra-cellular events. In the case of GABA, part of this cellular cascade includes modulation of the “second messenger” systems Ca++ and cAMP that ultimately modulate two protein kinases, PKC and PKA, resulting in myriad effects in the cell, including changes to gene expression (Moss et al., 1992; Diamond and Gordon, 1997; Brandon et al., 2000 for a review, see Kumar et al., 2009). Leslie Morrow and colleagues have been exploring the complex cascade of downstream cellular events mediated by ethanol-induced GABAA receptor activation. In their recent Frontiers in Neuroscience report (Kumar et al., 2012), this research team determined the effects of acute ethanol exposure on PKA-mediated GABAA receptor expression. Although chronic ethanol exposure has been reported to produce down-regulation and/or desensitization of GABAA receptors, Morrow and colleagues reported an interesting finding of acute ethanol exposure. Specifically, acute intracerebroventricular administration of ethanol produced dose-dependent increases in expression of PKA and GABAA receptor α1 subunits. In addition, antagonism studies revealed that blockade of PKA blocked the ethanol-induced increases in GABAA receptor α1 subunit expression, providing further evidence for a PKA-GABAA receptor network communication. An additional manipulation showed that enhancing PKA activity actually enhanced ethanol-induced loss of righting reflex. Together with earlier reports from this research group, these data indicate that PKA (Kumar et al., 2012) and PKC (Kumar et al., 2006) may have antagonistic effects on GABAA receptor α1 subunit expression, thus delineating specific potential pathway(s) for mediation of ethanol effects on cell function and overt organismal behavior. This group's current report represents a comprehensive analysis of the specific effects of ethanol on kinase activity and receptor subunit expression levels, and adds another layer of knowledge about the neuropharmacological mechanisms of ethanol. It is quite possible that further elucidation of the complex molecular “systems level” analysis of ethanol's actions in nerve cells may yield important “systems level” approaches and advances for the treatment of alcohol abuse and dependence.

Modulation of GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila

Many lines of evidence indicate that GABA and GABA(A) receptors make important contributions to human sleep regulation. Pharmacological manipulation of these receptors has differential effects on sleep onset and sleep maintenance insomnia. Here we show that sleep is regulated by GABA in Drosophila and that a mutant GABA(A) receptor, Rdl(A302S), specifically decreases sleep latency. The drug carbamazepine (CBZ) has the opposite effect on sleep; it increases sleep latency as well as decreasing sleep. Behavioral and physiological experiments indicated that Rdl(A302S) mutant flies are resistant to the effects of CBZ on sleep latency and that mutant RDL(A302S) channels are resistant to the effects of CBZ on desensitization, respectively. These results suggest that this biophysical property of the channel, specifically channel desensitization, underlies the regulation of sleep latency in flies. These experiments uncouple the regulation of sleep latency from that of sleep duration and suggest that the kinetics of GABA(A) receptor signaling dictate sleep latency.

In vivo imaging of anaesthetic action in humans: approaches with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI)

In vivo imaging of anaesthetic action in humans: approaches with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI)

Changes in intracellular calcium concentration affect desensitization of GABAA receptors in acutely dissociated P2-P6 rat hippocampal neurons.

The whole cell configuration of the patch-clamp technique was used to study the effects of different cytosolic calcium concentrations [Ca2+]i on desensitization kinetics of gamma-aminobutyric acid (GABA)-activated receptors in acutely dissociated rat hippocampal neurons. Two different intrapipette concentrations of the calcium chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 11 and 0.9 mM, respectively) were used to yield a low (1.2 x 10(-8) M) or a high (2.2 x 10(-6) M) [Ca2+]i. In low [Ca2+]i, peak values of GABA-evoked currents (20 microM) evoked at -30 mV, were significantly larger than those recorded in high calcium [2,970 +/- 280 (SE) pA vs. 1,870 +/- 150 pA]. The extent of desensitization, assessed from steady-state to peak ratio was significantly higher in high calcium conditions (0.14 +/- 0.007 vs. 0.11 +/- 0.008). Similar effects of -Ca2+-i on desensitization were observed with GABA (100 microM). Recovery from desensitization, measured at 30 s interval with double pulse protocol was significantly slower in high [Ca2+]i than in low [Ca2+]i (54 +/- 3% vs. 68 +/- 2%). The current-voltage relationship of GABA-evoked currents was linear in the potential range between -50 and 50 mV. The kinetics of desensitization process including the rate of onset, extent of desensitization, and recovery were voltage independent. The run down of GABA-evoked currents was faster with the higher intracellular calcium concentration. The run down process was accompanied by changes in desensitization kinetics: in both high and low [Ca2+]i desensitization rate was progressively increasing with time as the slow component of the desensitization onset was converted into the fast one. In excised patches, the desensitization kinetics was much faster and more profound than in the whole cell configuration, indicating the involvement of intracellular factors in regulation of this process. In conclusion, [Ca2+]i affects the desensitization of GABAA receptors possibly by activating calcium-dependent enzymes that regulate their phosphorylation state. This may lead to modifications in cell excitability because of changes in GABA-mediated synaptic currents.

Frequency-dependent actions of benzodiazepines on GABAA receptors in cultured murine cerebellar granule cells.

1. Miniature IPSCs recorded from cultured murine cerebellar granule cells increased in half-width and amplitude following application of the benzodiazepine (BDZ) Flunitrazepam (Flu, 1 microM). The increase in the half-width was much greater than that in the amplitude. 2. Five-millisecond applications of 1 mM GABA to nucleated outside-out patches elicited rapidly rising biexponentially decaying responses that resembled IPSCs. Flu had no effect on the amplitude of such responses, but consistently slowed their deactivation by approximately 50%. This effect was reversed by Flu washout or application of the BDZ antagonist Ro15-1788. The partial inverse agonist. Ro15-4513 speeded deactivation and depressed peak current amplitude by 23 +/- 12%. 3. The EC50 for GABA was between 45 and 50 microM. At submaximally effective agonist concentrations, Flu increased response amplitude and slowed response deactivation. Both effects were present in all cells taken from young cultures (4-7 days in vitro) but the latter was absent in 55% of the neurones obtained from older cultures (14-27 days in vitro). 4. With 120 ms applications of 20 microM GABA, responses activated monoexponentially (time constant, 39.8 +/- 2.8 ms) and deactivated biexponentially (time constants, 40.4 +/- 2.1 and 251 +/- 15 ms). Application of Flu slowed both activation and deactivation. The latter effect arose from an increased contribution of the slower component of decay. 5. Desensitization of responses to 1 mM GABA was biexponential, with time constants of 47 +/- 11 and 479 +/- 49 ms. Flu speeded desensitization by decreasing both fast and slow time constants. GABAA receptor desensitization consistently slowed subsequent deactivation. No significant relationship between the level of desensitization and the amount of slowing of deactivation produced by Flu was found. 6. Responses to paired 5 ms applications of 1 mM GABA indicated that the slowing of deactivation and the speeding of desensitization produced by Flu combine to generate a marked frequency dependence in the actions of this BDZ. Thus when compared with control responses, GABA-induced charge transfer was only enhanced by Flu during the first of two successive agonist applications.

Recombinant GABAA receptor desensitization: the role of the gamma 2 subunit and its physiological significance.

1. The purpose of these investigations was to examine the role that the gamma 2 subunit plays in human GABAA receptor desensitization. Two different recombinant GABAA receptors (alpha 1 beta 3 and alpha 1 beta 3 gamma 2) were compared by measuring the relaxation of whole-cell currents during the application of GABA, isoguvacine or taurine. 2. At concentrations which trigger a maximum response (100-500 microM GABA) the current relaxation usually fitted the sum of two exponentials. For alpha 1 beta 3 subunit receptors these values were tau 1 = 145 +/- 12 ms and tau 2 = 6.3 +/- 2.1 s (means +/- S.E.M.). Receptors consisting of alpha 1 beta 3 gamma 2 subunits desensitized faster: tau 1 = 41.6 +/- 8.3 ms and tau 2 = 2.4 +/- 0.6 s. 3. The Hill slope, determined for each receptor subunit combination, was the same and greater than 1.0, implying two binding steps in the activation of both receptor subunit combinations. 4. For alpha 1 beta 3 subunit receptors the fast desensitization rates were unaltered by reducing the GABA concentration from the EC100 (100 microM) to the approximate EC50 values (10-20 microM), whereas for alpha 1 beta 3 gamma 2 subunit receptors a significant slowing was observed. The fast desensitization disappeared at agonist concentrations below the EC50 for both subunit combinations. In contrast, the slow desensitization appeared at agonist concentrations near the EC20. This rate was dependent on agonist concentration reaching a maximum near the EC60 value of GABA. 5. The fast desensitization rates were unaltered by changing the holding potential of the cell during agonist application. However, for alpha 1 beta 3 gamma 2 subunit receptors the slow desensitization rate increased by approximately 15- to 20-fold over the range of voltages of -60 to +40 mV. This indicates that the gamma 2 subunit makes GABAA receptor desensitization voltage dependent. 6. Recovery from desensitization was also biphasic. The first recovery phase was faster for alpha 1 beta 3 gamma 2 than for alpha 1 beta 3 subunit receptors (0.13 vs. 0.03 s-1, respectively). The second phase of recovery for the two receptors were the same (approximately 0.003 s-1). 7. There was only a poor correlation between agonist potency and the degree or time course of desensitization. Isoguvacine (EC50 approximately to 10 microM) induced biphasic relaxation for both alpha 1 beta 3 and alpha 1 beta 3 gamma 2 subunit receptors (tau 1 = 288.6 +/- 43.3 and 167 +/- 15 ms, and tau 2 = 8.0 +/- 1.9 and 4.4 +/- 0.4 S, respectively, for each subunit combination). Taurine (EC50 approximately 7 mM) usually induced monophasic relaxation for both subunit combinations (tau 2 = 7.1 +/- 1.6 and 23.0 +/- 6.6 s, respectively). 8. A computer model was developed to examine the effect of the gamma 2 subunit on the time course of a synaptic potential. It was found that the gamma 2 subunit theoretically prolongs the time course of a synaptic potential by inducing desensitization more rapidly. The subsequent relaxation of the desensitized receptors through the open state increases Popen (the probability that the GABAA receptor is in an open conducting state) altering the time course of the modelled potential. alpha 1 beta 3 subunit receptors do not desensitize sufficiently rapidly to induce this desensitized state and, therefore, are shorter in time course. These data imply that the physiological role of the gamma 2 subunit is to increase synaptic efficacy by prolonging Popen.

A Cholecystokinin‐B Receptor Antagonist Potentiates GABAergic and Glycinergic Inhibition in the Nucleus of the Solitary Tract of the Rat

In both rodent and primate in vivo models, cholecystokininB (CCKB) antagonists such as PD134,308 have anxiolytic effects that may involve the potentiation of GABAergic transmission. We have investigated this interaction using exogenous application of GABA and whole cell patch recording techniques in neurons of the nucleus of the solitary tract (NTS) in brainstem slice preparations. In the presence of PD143,308 the magnitude of the GABA-evoked decrease in membrane input resistance was enhanced by 41.2 +/- 3.1% and the duration of the response was prolonged by 34.8 +/- 2.2%. Also, PD134, 308 potentiated glycine-evoked decreases in membrane input resistance, increasing the amplitude of the response by 62.8 +/- 4. 85 and prolonging the duration of the response by 23.5 +/- 3.6%. The effect of PD134,308 persisted in the presence of tetrodotoxin, after reversal of the transmembrane gradient of chloride ions and under conditions of exaggerated GABAA receptor desensitization. Our results demonstrate that at least part of the functional link between PD134,308 and the GABAA response occurs postsynaptically.

Different mechanisms for use-dependent depression of two GABAA-mediated IPSCs in rat hippocampus.

1. The mechanisms involved in the use-dependent depression of GABAA,fast and GABAA,slow, two GABAA-mediated IPSCs in the rat hippocampal slice preparation, were investigated by observing the effects of paired-pulse depression and of baclofen and CGP 35348 on monosynaptic inhibitory currents recorded from CA1 pyramidal neurons. 2. The second of a pair of evoked responses that consisted of both inhibitory components was depressed and decayed more rapidly compared to the first at an interpulse interval (IpI) of 200 ms. This effect was due to a decrease in the amplitude of GABAA,slow, with no effect on the time constant of decay or on the amplitude or time constant of GABAA,fast. 3. The time course of paired-pulse depression of both components at IpIs ranging from 5 to 2560 ms was compared. GABAA,slow was depressed maximally by 55% at IpIs of 80-160 ms. GABAA,fast was depressed maximally by 38% at 5 ms, and recovered exponentially with a time constant of 130 ms. 4. GABAA,slow was more sensitive than GABAA,fast to depression by baclofen. GABAA,slow was susceptible to complete block, with an ED50 of approximately 200 nM for (+/-)-baclofen and 100 nM for the active enantiomer, (R)-(+)-baclofen. GABAA,fast was blocked by only 50% by the highest concentrations of baclofen tested (10-100 microM (R)-(+)-baclofen), with an ED50 of approximately 2 microM for (+/-)-baclofen and 1 microM for (R)-(+)-baclofen. Paired-pulse depression of GABAA,fast was not occluded by 10 or 100 microM (R)-(+)-baclofen. 5. The GABAB antagonist CGP 35348 (0.4-1 mM), prevented paired-pulse depression of GABAA,slow at IpIs of 160 to 200 ms, and reversed the depression of GABAA,fast by baclofen, but had no effect on paired-pulse depression of GABAA,fast at IpIs of 20 to 40 ms. 6. It is concluded that use-dependent depression of GABAA,slow, but not GABAA,fast, is mediated by a presynaptic GABAB receptor. It is speculated that use-dependent depression of GABAA,fast, which occurs only over a much faster time scale, may be due to rapid postsynaptic GABAA receptor desensitization.

Propofol modulates activation and desensitization of GABAA receptors in cultured murine hippocampal neurons

Propofol (2,6 di-isopropylphenol) is an alkyphenol recently introduced for use as a general anesthetic. The modulation of GABAA receptor activation and desensitization by propofol was studied using a rapid perfusion system and whole-cell voltage-clamp recordings from mouse hippocampal neurons. The effects of concentrations of propofol used clinically on single-channel and synaptic currents were also examined. Propofol evoked current responses (EC50 = 61 microM) and shifted the dose-response curve of GABA-activated current to the left without altering the maximum of the GABA response. Preincubation with propofol and GABA led to desensitization of the GABA response (EC50 = 454 microM and 23 microM, respectively). Saturating concentrations of GABA (600 microM) evoked currents that peaked and then declined in a biexponential fashion with fast and slow time constants of tau f = 1.0 sec and tau s = 3.5 sec. Propofol (10 microM) did not change the amplitude of the peak response but decreased the rates of decay approximately 1.5-fold and enhanced the steady-state current proportionately. Recovery from desensitization was also biexponential (tau f = 11 sec, tau s = 69 sec) but not influenced by propofol. Single-channel recordings from outside-out patches demonstrated that both propofol and GABA activated channels with a 30 pS and 21 pS open state. Propofol increased the frequency but not the duration or conductance of GABA-activated events. Miniature inhibitory postsynaptic currents (mlPSCs) were evoked by the application of hypertonic sucrose to the cell soma. Propofol (2 microM) prolonged the decay time of mlPSCs to an extent similar to which it increased the open probability of GABA-activated channels (2.3- vs 3-fold). A sequential model, based on a previous scheme of GABA receptor gating (Weiss and Magelby, 1989), is presented to summarize propofol's actions on GABAA receptor function. We show through simulation that the model reliably reproduced the whole-cell tracings. Our results indicate that propofol's neurodepressive actions will be associated with enhancement of inhibitory synaptic transmission.

The GABAA receptor channel mediated chloride ion translocation through the plasma membrane: new insights from 36Cl- ion flux measurements.

GABAA receptors in plasma membranes of neurons are integral oligomers which form chloride channels. The binding of GABA molecules at recognition sites for channel opening triggers a transient increase in transmembrane chloride ion flux. The multiplicity and drug specificity of GABAA receptor, kinetics of channel opening, and desensitization of GABAA receptor and its short- and long-term regulation have been investigated by the use of tracer amounts of the radioactive chloride isotope, 36Cl- ion. Results and new insights from 36Cl- ion flux measurements have been reviewed.

Na+-dependent GABA transport system scavenges endogenous external GABA and prevents desensitization of GABAA receptors in rat cerebrocortical synaptoneurosomes

Muscimol-induced 36Cl- uptake in rat cerebrocortical synaptoneurosomes was reduced upon exposure of the membrane sacs to low Na+ media. This Na+ requirement led us to examine the role of the Na(+)-dependent gamma-aminobutyric acid (GABA) transport system in 36Cl- uptake. Incubation of the synaptoneurosomes with nipecotic acid, a specific inhibitor of the GABA transport system, for 10 min increased the level of endogenous external GABA from less than 10 to 150 microM and induced the same signs of desensitization as observed with high muscimol-treated synaptoneurosomes; a marked reduction of muscimol-induced 36Cl- uptake and an appearance of a slow bicuculline-sensitive 36Cl- uptake, probably due to a continuous recovery of a population of GABAA receptors from desensitization. Similar results were obtained upon dissipation of Na+ electrochemical gradient across the membranes by inhibition of Na+, K(+)-ATPase with ouabain or by blocking energy metabolism with azide or N-ethylmaleimide. We propose that the Na(+)-dependent GABA transport system, its operation being dependent on inwardly directed Na+ electrochemical gradient, is responsible for scavenging endogenous GABA released from the synaptoneurosomes, and thus prevents desensitization of GABAA receptors.