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Dopamine D-3 receptors regulate GABA(A) receptor function through a phospho-dependent endocytosis mechanism in nucleus accumbens
No full textThe dopamine D-3 receptor, which is highly enriched in nucleus accumbens (NAc), has been suggested to play an important role in reinforcement and reward. To understand the potential cellular mechanism underlying D-3 receptor functions, we examined the effect of D-3 receptor activation on GABA(A) receptor (GABA(A)R)-mediated current and inhibitory synaptic transmission in medium spiny neurons of NAc. Application of PD128907 [(4aR, 10bR)-3,4a, 4,10b-tetrahydro-4-propyl-2H, 5H-[1] benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride], a specific D-3 receptor agonist, caused a significant reduction of GABAAR current in acutely dissociated NAc neurons and miniature IPSC amplitude in NAc slices. This effect was blocked by dialysis with a dynamin inhibitory peptide, which prevents the clathrin/activator protein 2 (AP2)-mediated GABA(A) receptor endocytosis. In addition, the D-3 effect on GABA(A)R current was prevented by agents that manipulate protein kinase A (PKA) activity. Infusion of a peptide derived from GABA(A) beta subunits, which contains an atypical binding motif for the clathrin AP2 adaptor complex and the major PKA phosphorylation sites and binds with high affinity to AP2 only when dephosphorylated, diminished the D-3 regulation of IPSC amplitude. The phosphorylated equivalent of the peptide was without effect. Moreover, PD128907 increased GABAAR internalization and reduced the surface expression of GABA(A) receptor beta subunits in NAc slices, which was prevented by dynamin inhibitory peptide or cAMP treatment. Together, our results suggest that D-3 receptor activation suppresses the efficacy of inhibitory synaptic transmission in NAc by increasing the phospho-dependent endocytosis of GABA(A) receptors
GABA(A) receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase 1 anchoring protein
No full textGABA(A) receptors are critical in controlling neuronal activity. Here, we examined the role for phospholipase C-related inactive protein type 1 (PRIP-1), which binds and inactivates protein phosphatase 1alpha (PP1alpha) in facilitating GABA(A) receptor phospho-dependent regulation using PRIP-1(-/-) mice. In wild-type animals, robust phosphorylation and functional modulation of GABA(A) receptors containing beta3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP-1(-/-) mice. PRIP-1(-/-) mice exhibited enhanced PP1alpha activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABA(A) receptor beta subunits, and moreover, these proteins were found to be PP1alpha substrates. Finally, phosphorylation of PRIP-1 on threonine 94 facilitated the dissociation of PP1alpha-PRIP-1 complexes, providing a local mechanism for the activation of PP1alpha. Together, these results suggest an essential role for PRIP-1 in controlling GABA(A) receptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors
Experimental GABA A Receptor Agonists and Allosteric Modulators for the Treatment of Focal Epilepsy
No full textSlobodan M Janković,1 Miralem Dješević,2 Snežana V Janković1 1Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia; 2Cardiology Department, Private Policlinic Center Eurofarm, Sarajevo, Bosnia and HercegovinaCorrespondence: Slobodan M JankovićUniversity of Kragujevac, Faculty of Medical Sciences, Svetozara Markovića 69 Street, Kragujevac, 34000, SerbiaEmail [email protected]: GABA A receptors are ubiquitous in the central nervous system and there is a huge diversity of receptor subtypes in almost all regions of the brain. However, the expression of GABA A receptor subtypes is altered in both the gray and white matter of patients with focal epilepsy. Although there is a number of anticonvulsants with marketing authorization for the treatment of focal epilepsy which act through GABA A receptors, potentiating the inhibitory effects of GABA, it is necessary to develop more potent and more specific GABAergic anticonvulsants that are effective in drug-resistant patients with focal epilepsy. There are three orthosteric and at least seven allosteric agonist binding sites at the GABA A receptor. In experimental and clinical studies, full agonists of GABA A receptors showed a tendency to cause desensitization of the receptors, tolerance, and physical dependence; therefore, partial orthosteric agonists and positive allosteric modulators of GABA A receptors were further developed. Preclinical studies demonstrated the anticonvulsant efficacy of positive allosteric modulators with selective action on GABA A receptors with α2/α3 subunits, but only a handful of them were further tested in clinical trials. The best results were obtained for clobazam (already marketed), ganaxolone (in phase III trials), CVL-865 (in phase II trials), and padsevonil (in phase III trials). Several compounds with more selective action on GABA A receptors, perhaps only in certain brain regions, have the potential to become effective drugs against specific subtypes of focal-onset epilepsy. However, their development needs time, and in the near future we can expect only one or two new GABA A agonists to obtain marketing authorization for focal epilepsy, an advance that would be of use for just a fraction of patients with drug-resistant epilepsy.Keywords: GABA A receptors, positive allosteric modulators, focal-onset epileps
A novel open channel blocker of GABA-A receptors
Get PDFGABA-A receptors are chloride ion channels composed of five subunits,
mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit
isoforms have been identified, with the major receptor type in mammalian adult brain
consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous
sedating and anxiolytic drugs such as benzodiazepines. The currently known
endogenous ligands are GABA, neurosteroids and the endocannabinoid 2-
arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion
channel strictly depend on receptor subunit composition and arrangement. GABA-A
receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and
cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions
with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited
currents elicited by GABA with comparable potency as picrotoxin. This
inhibition showed all characteristics of an open channel block. The GABA-A receptor ion
channel is lined by residues from the M2 membrane-spanning segment. To identify
important residues of the pore involved in the interaction with the blocking molecules
PCCP-, a mutation scan was performed in combination with subsequent analysis of the
expressed mutant proteins using electrophysiological techniques.
In a second project we characterised a light-switchable modulator of GABA-A
receptors based on propofol. It was my responsibility to investigate the switching
kinetics in patch clamp experiments. After its discovery in 1980, propofol has become
the most widely used intravenous general anaesthetic. It is commonly accepted that
the anaesthesia induced by this unusually lipophilic drug mostly results from
potentiation of GABA induced currents. While GABA-A receptors respond to a variety of
ligands, they are normally not sensitive towards light. This light sensitivity could be
indirectly achieved by using modulators that can be optically switched between an
active and an inactive form. We tested an azobenzene derivative of propofol where an
aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed
azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with
electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus
oocytes and HEK-cells.
In the third project we wanted to investigate the functional role of GABA-A
receptors in the liver, and their possible involvement in cell proliferation. GABA-A
receptors are also found in a wide range of peripheral tissues, including parts of the
peripheral nervous system and non-neural tissues such as smooth muscle, the female
reproductive system, liver and several cancer tissues. However their precise function in
non neuronal or cancerous cells is still unknown. For this purpose we investigated
expression, localization and function of the hepatocytes GABA-A receptors in model cell
lines and healthy and cancerous hepatocytes
Structure, function, and modulation of GABA(A) receptors
No full textThe GABA(A) receptors are the major inhibitory neurotransmitter receptors in mammalian brain. Each isoform consists of five homologous or identical subunits surrounding a central chloride ion-selective channel gated by GABA. How many isoforms of the receptor exist is far from clear. GABA(A) receptors located in the postsynaptic membrane mediate neuronal inhibition that occurs in the millisecond time range; those located in the extrasynaptic membrane respond to ambient GABA and confer long-term inhibition. GABA(A) receptors are responsive to a wide variety of drugs, e.g. benzodiazepines, which are often used for their sedative/hypnotic and anxiolytic effects
Control of neuronal nitric oxide synthase and brain-derived neurotrophic factor levels by GABA-A receptors in the developing rat cortex
No full textGamma-aminobutyric acid (GABA) plays an important morphogenetic role, acting through GABA-A receptors, which are depolarizing in the developing rat brain. Other molecules with major morphogenetic roles are the nitric oxide free radical (NO·) and brain-derived neurotrophic factor (BDNF), both of which are involved in the control of synaptic plasticity and apoptosis. In the present work, we investigated the effect of GABA-A receptor activation on neuronal NO· synthase (nNOS) and BDNF immunoreactivity in the developing cortex of 5-day-old rats. We also determined the effect of GABA-A receptor activation on phosphorylated cAMP-response element binding protein (pCREB) immunoreactivity in an effort to elucidate the molecular mechanisms involved. Our results show that activation of GABA-A receptors leads to increased numbers of nNOS, BDNF and pCREB, as well as nNOS-pCREB and BDNF-pCREB doubly immunopositive cells. This effect is abolished when L-type Ca2+ channels are blocked. These results indicate that the following mechanism could be operating: depolarization following GABA-A receptor activation leads to opening of L-type voltage-gated calcium channels, resulting in an increased Ca2+ influx, which in turn leads to phosphorylation and, thus, activation, of the transcription factor CREB; the phosphorylated CREB can then induce BDNF, as well as nNOS
The major central endocannabinoid directly acts at GABA(A) receptors
No full textGABA(A) receptors are the major ionotropic inhibitory neurotransmitter receptors. The endocannabinoid system is a lipid signaling network that modulates different brain functions. Here we show a direct molecular interaction between the two systems. The endocannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABA(A) receptors at low concentrations of GABA. Two residues of the receptor located in the transmembrane segment M4 of β(2) confer 2-AG binding. 2-AG acts in a superadditive fashion with the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates δ-subunit-containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB(1)/CB(2) cannabinoid receptor double-KO, whereas β(2)-KO mice show hypermotility. The identification of a functional binding site for 2-AG in the GABA(A) receptor may have far-reaching consequences for the study of locomotion and sedation
Development of GABAergic and glycinergic transmission in the neonatal rat dorsal horn
No full textCutaneous spinal sensory transmission appears to lack inhibitory control in the newborn spinal cord, but the properties of GABAergic and glycinergic synapses in the neonatal dorsal horn have not been characterized. Whole-cell patch-clamp recordings from rat superficial dorsal horn neurons in spinal cord slices at postnatal day 0 (P0) to P2, P6 - P7, and P13 - P14 revealed an age-dependent increase in the frequency of spontaneous IPSCs, which were abolished by the GABA(A) receptor (GABA(A)R) antagonist bicuculline between P0 and P7 but not at P14. GABA(A)R-mediated miniature IPSCs (mIPSCs), but not glycinergic mIPSCs, were present at birth, and GABA mIPSCs remained more frequent than glycine mIPSCs at all ages. Sciatic nerve stimulation resulted in IPSCs with both GABAergic and glycinergic components, although a larger contribution arose from GABAA receptors at all ages. In gramicidin perforated patch-clamp recordings, exogenous GABA applications produced depolarization in 40% of neurons at P0 - P2, but the reversal potential of GABA-evoked currents (E-GABA) was consistently more negative than action potential threshold at this age. By P6 - P7, GABA evoked only membrane hyperpolarization. The GABA(B)R agonist baclofen elicited an outward current in all neurons with peak amplitudes observed by P6 - P7 and abolished sciatic nerve-evoked monosynaptic glutamatergic EPSCs in all groups. The results show considerable postnatal development of inhibitory processing in the dorsal horn with GABAergic mechanisms initially dominant over glycinergic events. GABA(A)R-mediated depolarizations during the first postnatal week are likely to be important for the maturation of spinal networks but do not provide a major excitatory drive to the newborn dorsal horn
Evidence for a GABAergic system in rodent and human testis: Local GABA production and GABA receptors
Get PDFThe major neurotransmitter of the central nervous system, gamma-aminobutyric acid (GABA), exerts its actions through GABA(A), GABA(B) and GABA(C) receptors. GABA and GABA receptors are, however, also present in several non-neural tissues, including the endocrine organs pituitary, pancreas and testis. In the case of the rat testis, GABA appears to be linked to the regulation of steroid synthesis by Leydig cells via GABA(A) receptors, but neither testicular sources of GABA, nor the precise nature of testicular GABA receptors are fully known. We examined these points in rat, mouse, hamster and human testicular samples. RT-PCR followed by sequencing showed that the GABA-synthesizing enzymes glutamate decarboxylase (GAD) 65 and/or GAD67, as well as the vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT) are expressed. Testicular GAD in the rat was shown to be functionally active by using a GAD assay, and Western blot analysis confirmed the presence of GAD65 and GAD67. Interstitial cells, most of which are Leydig cells according to their location and morphological characteristics, showed positive immunoreaction for GAD and VIAAT/VGAT proteins. In addition, several GABA(A) receptor subunits (alpha1-3, beta1-3, gamma1-3), as well as GABAB receptor subunits R1 and R2, were detected by RT-PCR. Western blot analysis confirmed the results for GABA(A) receptor subunits beta2/3 in the rat, and immunohistochemistry identified interstitial Leydig cells to possess immunoreactive GABA(A) receptor subunits beta2/3 and alpha1. The presence of GABA(A) receptor subunit alpha1 mRNA in interstitial cells of the rat testis was further shown after laser microdissection followed by RT-PCR analysis. In summary, these results describe molecular details of the components of an intratesticular GABAergic system expressed in the endocrine compartment of rodent and human testes. While the physiological significance of this peripheral neuroendocrine system conserved throughout species remains to be elucidated, its mere presence in humans suggests the possibility that clinically used drugs might be able to interfere with testicular function. Copyright (C) 2003 S. Karger AG, Basel
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