GABA receptors on the cell-body membrane of an identified insect motor neuron

The pharmacology of a gamma-aminobutyric acid (GABA) receptor on the cell body of an identified motor neuron of the cockroach (Periplaneta americana) was investigated by current-clamp and voltage-clamp methods. Iontophoretic application of GABA increased membrane conductance to chloride ions, and prolonged application resulted in desensitization. Hill coefficients, determined from dose-response data, indicated that binding of at least two GABA molecules was required to activate the chloride channel. Differences between vertebrate GABAA receptors and insect neuronal GABA receptors were detected. For the GABA receptor of motor neuron Df, the following rank order of potency was observed: isoguvacine greater than muscimol greater than or equal to GABA greater than 3-aminopropanesulphonic acid. The GABAB receptor agonist baclofen was inactive. Of the potent vertebrate GABA receptor antagonists (bicuculline, pitrazepin, RU5135 and picrotoxin), only picrotoxin (10(-7) M) produced a potent, reversible block of the response to GABA of motor neuron Df. Both picrotoxinin and picrotin also blocked GABA-induced currents. Bicuculline hydrochloride (10(-4) M) and bicuculline methiodide (10(-4) M) were both ineffective when applied at resting membrane potential (-65 mV), although at hyperpolarized levels partial block of GABA-induced current was sometimes observed. Pitrazepin (10(-4) M) caused a partial, voltage-independent block of GABA-induced current. The steroid derivative RU5135 was inactive at 10(-5) M. In contrast to the potent competitive blockade of vertebrate GABAA receptors by bicuculline, pitrazepin and RU5135, none of the weak antagonism caused by these drugs on the insect GABA receptor was competitive. Flunitrazepam (10(-6) M) potentiated GABA responses, providing evidence for a benzodiazepine site on an insect GABA-receptor-chloride-channel complex.     

Pharmacology of GABA-mediated inhibition of spinal cord neurons in vivo and in primary dissociated cell culture

Summary In this paper it is shown that the postsynaptic GABA-receptor chloride ion channel complex is composed of several functional subunits. There are probably at least two stereospecific locations on the receptor for GABA-binding and both must be occupied to obtain an increase in chloride conductance. The interaction between these sites is uncertain but there could be either positive cooperativity between the sites or only a requirement that both sites are occupied without occupation of either site affecting the affinity for GABA of the other site. There is a chloride conductance channel coupled to the GABA receptor which opens for an average of 20 msec and has an average conductance of 18 pS. The GABA-coupled chloride channel may or may not have the same composition as the glycine coupled chloride channel.In addition to the GABA-recognition site and the chloride ion channel, GABA-receptors must have additional binding sites or modulator sites where drugs can bind to modify GABA activation of the GABA-receptor. The convulsant PICRO binds to a site which is independent of the GABA-recognition site and PICRO reduces GABA responses. Barbiturates and benzodiazepines augment GABA-responses without reducing GABA-binding and thus they must bind to a modulator site independent of the GABA recognition site. Whether or not this is the same site as the PICRO binding site is uncertain. Thus, the GABA-receptorchloride ion channel complex is composed of at least: 1) two GABA-binding sites; 2) a chloride ion channel; 3) a convulsant binding site (PICRO-binding site) and 4) an anticonvulsant binding site. This organization serves several obvious purposes. First, since two GABA-molecules are required to activate GABA-coupled chloride ion channels, the dose-response relationship for GABA is sigmoidal and steep. Thus minor shifts in GABA affinity will produce large alterations in GABA-responses and the GABA receptor can be easily modulated. Second, since the receptor has binding sites for convulsant and anticonvulsant compounds which decrease and increase GABA-responses, GABAergic inhibition can easily be modulated.     

Cyclic AMP-dependent protein kinase decreases GABAA receptor current in mouse spinal neurons

GABA, the major inhibitory neurotransmitter in the mammalian brain, binds to GABAA receptors, which form chloride ion channels. The predicted structure of the GABAA receptor places a consensus phosphorylation site for cAMP-dependent protein kinase (PKA) on an intracellular domain of the channel. Phosphorylation by various protein kinases has been shown to alter the activity of certain ligand- and voltage-gated ion channels. We have examined the role of phosphorylation by the catalytic subunit of PKA in the regulation of GABAA receptor channel function using whole-cell and excised outside-out patch-clamp techniques. Inclusion of the catalytic subunit of PKA in the recording pipettes significantly reduced GABA-evoked whole-cell and single-channel chloride currents. Both heat inactivation of PKA and addition of the specific protein kinase inhibitor peptide prevented the reduction of GABA-evoked currents by PKA. Neither mean channel open time nor channel conductance was affected by PKA. The reduction in GABA receptor current by PKA was primarily due to a reduction in channel opening frequency.     

Antagonist actions of bicuculline methiodide and picrotoxin on extrasynaptic gamma-aminobutyric acid receptors

The effects of picrotoxin and bicuculline methiodide to block depolarizing responses of extrasynaptic receptors for gamma-aminobutyric acid (GABA) are compared using excitability testing of myelinated axons in amphibian peripheral nerve. The actions of the antagonists appear both complex and dissimilar. Picrotoxin (10-1000 microM) produces large reversible depressions of the maximal response to GABA (0.01-10mM) and increases the EC50 from 0.33 to 12.6 mM. With high concentrations of agonist and antagonist an insensitive component is apparent. The action of picrotoxin is not classically noncompetitive: it may represent a mixed antagonism (competitive and noncompetitive) or a noncompetitive one, masked by the presence of receptor reserve and (or) secondary depolarizing influences (e.g., GABA-evoked [K+] o accumulation). Bicuculline methiodide (10-200 microM) shifts the GABA concentration-response curve to the right; maximal responses persist and are even enhanced. The impression that bicuculline methiodide has a competitive action is supported by analysis of its inhibition of responses to low concentrations of the agonist. It is suggested that the enhancement of GABA responses by bicuculline methiodide and their apparent resistance to block by picrotoxin may be due to a common secondary effect of the antagonists such as a decrease in membrane conductance to K+ and (or) block of transmitter uptake.     

Ionic mechanisms of action of GABA on dorsal and ventral root myelinated fibers: effects of K+ channel blockers

Excitability changes evoked by the inhibitory neurotransmitter, GABA (gamma-aminobutyric acid) in myelinated axons of dorsal and ventral roots of the isolated bullfrog sciatic nerve were compared in the absence and presence of K+ channel blockers. Half-maximal A-fiber responses to a 0.5-Hz stimulation of the whole nerve were recorded from individual roots. Direct applications of Ringer with raised K+ levels to the site of stimulation caused increases in excitability of both dorsal and ventral root fibers, which resembled those evoked in the ventral root by the GABA agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]ol). The increases in dorsal root fiber responses produced by GABA were depressed by tetraethylammonium (TEA) (3 mM), 4-aminopyridine (4-AP) (50 microM), Cs (2 mM), and Ba (1 mM). Ventral root fibers were less consistently affected. The early component of GABA-evoked excitability increases was depressed by 4-AP, Cs, and Ba, but greatly augmented by TEA. THIP-evoked changes in the excitability of the dorsal and ventral root fibers were, respectively, depressed and enhanced by TEA. The augmenting effect of TEA on the early component of GABA agonist effects on the ventral root fibers is attributed to their high resting K+ conductance and the presence of a slowly inactivating, fast K+ current (If1). The depressant effects of K+ channel blockade on depolarizing components of agonist-evoked changes in dorsal and ventral root responses indicate interference with release and (or) sensitivity to K+ and a possible contribution from a mechanism involving voltage-dependent delayed rectifier K+ currents.     

Pharmacology of GABA receptors on skeletal muscle fibres of the locust (Schistocerca gregaria)

GABA and the trans isomer of 4-aminocrotonic acid are equally potent at inducing increases in Cl- conductance when applied to distal extensor tibia muscle fibres of the locust (Schistocerca gregaria). beta-Alanine, norvaline, glycine and norleucine induced conductance increases of less than 5% of GABA responses. C9 and meso-di-GABA did not alter input conductance in a manner consistent with actions on a GABA receptor Cl- channel complex. Picrotoxin and anisatin were equally potent GABA antagonists, however bicuculline and penicillin G did not reduce GABA-induced changes in input conductance. Pentobarbitone, in addition to inducing an increase in K+ conductance, potentiated GABA-induced increases in Cl- permeability.     

Use-dependent block of GABA-activated chloride channels in crayfish muscle fibers by picrate

In crayfish muscle fibers studied with intracellular microelectrodes the protein-binding agent, picrate (2,4,6-trinitrophenolate; 10(-5)-2 X 10(-4) M) was found to have a specific and dose-dependent inhibitory effect on the chloride conductance activated by bath-applied gamma-aminobutyric acid (GABA). A kinetic analysis showed that picrate did not interfere with GABA binding to its receptor. The blocking action of picrate was not increased by lowering the extracellular Cl- concentration which indicates that picrate is not likely to bind to the ionic selectivity site of the postsynaptic Cl- channel. In fibers first exposed to picrate (1-2 X 10(-4) M) and then, in the continuous presence of this drug, to GABA (5 X 10(-4) M), the latter induced a transient increase in the chloride conductance with an apparent rate constant of decay of about 40 sec. It is tentatively suggested that the site of action of picrate is a positively charged amino acid residue that is exposed through the action of GABA and critically involved in the chemical gating of the postsynaptic chloride channel.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster.

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage-current relationship of the membrane. By including gamma-aminobutyrate (GABA) in the bathing medium, the voltage-current relationship was made linear in the depolarizing direction over a range of 15-20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and aspartate functioning synergistically.     

Amoxapine inhibition of GABA-stimulated chloride conductance: investigations of potential sites of activity

Amoxapine inhibits GABA-stimulated chloride conductance by acting on the GABAA-receptor chloride-ionophore complex which can be studied using membrane vesicles prepared from rat cerebral cortex. Amoxapine produces a right shift in the GABA concentration-response curve for the stimulation of 36Cl- uptake into these vesicles with no apparent change in the maximum response. Schild analysis of these data gave a pA2 value of 5.52 with a slope of 0.79. Amoxapine inhibits the binding of the GABAA receptor selective antagonist [3H]SR 95531 with an IC50 value of 3.45 microM and a pseudo Hill coefficient of 0.83. In contrast, 10 microM amoxapine inhibits [3H]flunitrazepam binding by less than 25% while the benzodiazepine antagonist Ro 15-1788 reduces the amoxapine inhibition of GABA-stimulated chloride conductance only at high concentrations. These data suggest that amoxapine does not inhibit chloride conductance by acting as a benzodiazepine inverse agonist and either acts directly on the GABAA receptor as an antagonist or blocks GABA activity at a site closely coupled to it. The ability of amoxapine to inhibit GABA-stimulated chloride conductance is a likely explanation for its proconvulsant activity observed at high doses.     

The anticonvulsant valproate increases the turnover rate of gamma-aminobutyric acid transporters

Valproate is an important anticonvulsant currently in clinical use for the treatment of seizures. We used electrophysiological and tracer uptake methods to examine the effect of valproate on a gamma-aminobutyric acid (GABA) transporter (mouse GAT3) expressed in Xenopus laevis oocytes. In the absence of GABA, valproate (up to 50 mm) had no noticeable effect on the steady-state electrogenic properties of mGAT3. In the presence of GABA, however, valproate enhanced the GABA-evoked steady-state inward current in a dose-dependent manner with a half-maximal concentration of 4.6 +/- 0.5 mm. Maximal enhancement of the GABA-evoked current was 275 +/- 10%. Qualitatively similar observations were obtained for human GAT1 and mouse GAT4. The valproate enhancement did not alter the Na(+) or Cl(-) dependence of the steady-state GABA-evoked currents. Uptake experiments under voltage clamp suggested that the valproate enhancement of the GABA-evoked current was matched by an enhancement in GABA uptake. Thus, despite the increase in GABA-evoked current, ion/GABA co-transport remained tightly coupled. Uptake experiments indicated that valproate is not transported by mouse GAT3 in the absence or presence of GABA. Valproate also enhanced the rate of the partial steps involved in transporter presteady-state charge movements. We propose that valproate increases the turnover rate of GABA transporters by an allosteric mechanism. The data suggest that at its therapeutic concentration, valproate may enhance the activity of neuronal and glial GABA transporters by up to 10%.     

The GABA dose/conductance relationship on lobster muscle

A quantitative study was made of the action of GABA, some structurally-related agonists and antagonists on the dactyl opener muscle fibres of the lobster. It was concluded that the GABA dose/conductance relationship was better described by a two independent binding-site receptor model (with KII = 30 microM) than by a single-site or a two-site high co-operativity model. The dose/conductance curves for gamma-amino-beta-hydroxybutyric acid (GABOB), delta-aminovaleric acid (DAV) and piperazine indicated 'full' agonist behaviour, whereas those for guanidoacetic acid (GuAc) indicated a partial agonist action. beta-guanidinopropionic acid (beta-GP) and gamma-guanidinobutyric acid (gamma-GB) behaved as weak competitive GABA antagonists. Bicuculline was found to antagonize GABA non-competitively on the lobster as in the crayfish, whereas picrotoxin appeared to act in a 'mixed' antagonistic fashion.     

Measurement of GABA-evoked conductance changes of lobster muscle fibres by a three-microelectrode voltage clamp technique

The effective membrane conductance and capacity of lobster muscle fibres was measured by a three-intracellular-microelectrode voltage clamp technique. Conductance values agreed well with those determined under current clamp, by means of the 'short' cable equations. Reversible increases in conductance evoked by gamma-aminobutyric acid (GABA) were reflected by differences (delta V) in electrotonic potential amplitude recorded at the centre, and midway between the centre and fibre end respectively. GABA dose--conductance curves derived from cable theory or from delta V measurements were virtually identical. The effective capacity (ceff), determined from the area beneath the 'on' delta V capacity transient, yielded values of the membrane time constant consistently lower than those obtained by the graphical method of E. Stefani & A.B. Steinbach (J. Physiol., London. 203, 383-401 (1969)); one possible explanation for this discrepancy is discussed. In the presence of GABA, the effective capacity was reduced in a dose-related manner. The results were interpreted in terms of an equivalent circuit in which surface membrane was arranged in parallel with cleft-tubular membrane of finite conductance, charged through an access resistance. GABA was though to be decreasing ceff by selectively increasing the conductance of the cleft-tubular membranes.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

Stimulating effect of gamma-aminobutyric acid on rat hippocampal neurons in vitro

Effects of GABA on the background and electrically stimulated activity of single neurons and population spikes were investigated in isolated hippocampal slices. Application of relatively large GABA concentrations (10(-3) mol/l and more) depressed an antidromic population spike, field EPSP and neuronal background activity. Low concentrations of GABA (less than 10(-3) mol/l) added to the bath increased the population spikes amplitude and the late component of field EPSP, facilitated single neurone responses, their background activity and epileptiform discharges. GABA-evoked depolarization was observed in the majority of the studied neurons. The duality of the GABA action on central neurons are discussed.     

Biochemical pharmacology of the gamma-aminobutyric acid receptor/ionophore protein

The synaptic receptor sites for the neurotransmitter gamma-aminobutyric acid (GABA) can be assayed in vitro with several radiolabeled agonists and one antagonist. Numerous criteria of specificity have been met for these binding sites. All of the ligands show heterogeneity in binding affinities. The subpopulations thus defined have a remarkably similar specificity for GABA analogs, which suggests an intimate relationship and possible interconvertibility. Modulation of GABA receptor binding by barbiturates, anions, and other membrane treatments that affect agonists and antagonists in an opposite manner suggests a three-state model of interconvertible affinities. The complex of GABA receptor and chloride ion channel contains modulatory sites for barbiturates and benzodiazepines, drugs that enhance GABA responses in neurons. The receptor complex can be solubilized in detergent with the three mutually interacting receptor activities intact. The complex has an apparent molecular weight of 355,000 and has been partially purified. GABA agonist function has been assayed at the biochemical level by measuring the activation of 36Cl- efflux from preloaded hippocampal slices by GABA, muscimol, and barbiturates. This response is blocked by the antagonists of the GABA site (bicuculline) and the barbiturate site (picrotoxin). Comparison of binding and function on the same tissue should be useful in analyzing the mechanism of action of GABA.     

GABA-mediated inhibition of primary olfactory receptor neurons

Applying GABA (1 microM-1 mM) to the soma of cultured lobster olfactory receptor neurons evokes an inward current (V(m) = -60 mV) accompanied by an increase in membrane conductance, with a half-effect of 487 microM GABA. The current-voltage relationship of this current is linear between -100 and 100 mV and reverses polarity at the equilibrium potential for Cl(-). The current is blocked by picrotoxin and bicuculline methiodide, and is evoked by trans-aminocrotonic acid, isoguvacine, muscimol, imidazole-4-acetic acid, and 3-amino-1-propanesulfonic acid, but not by the GABA(C)-receptor agonist cis-4-aminocrotonic acid and the GABA(B)-receptor agonist 3-aminopropylphosphonic. Applying GABA to the soma of the cells in situ reversibly suppresses the spontaneous discharge and substantially decreases the odor-evoked discharge. The effects of GABA on the cell soma in situ are antagonized by both picrotoxin and bicuculline methiodide. Taken together with evidence that GABA directly activates a chloride channel in outside-out patches excised from the soma of these neurons, we conclude that lobster olfactory receptor neurons express an ionotropic GABA receptor that can potentially regulate the output of these cells. Copyright Copyright 1999 S. Karger AG, Basel     

Physiological and pharmacological studies on annelid and nematode body wall muscle

1. This review covers the pharmacology and physiology of the body wall muscle systems of nematodes and annelids.2. Both acetylcholine and gamma-aminobutyric acid (GABA) play important roles in the control of body wall muscle in both phyla. In annelids and nematodes, acetylcholine is the excitatory neuromuscular transmitter while GABA is the inhibitory neuromuscular transmitter. In addition, 5-hydroxytryptamine (5-HT) has a modulatory role at annelid body wall muscle but little if any effect on nematode body wall muscle.3. The acetylcholine receptor of the body wall muscle can be classified as nicotinic-like in both phyla though the annelid receptor has not been analysed in detail. In nematodes, vertebrate ganglionic nicotinic agonists were the most effective of those so far examined while mecamylamine and benzoquinonium were the most effective antagonists. Both neuronal bungarotoxin and neosurugatoxin were potent antagonists of acetylcholine excitation at the nematode receptor.4. The GABA receptor of the body wall muscle exhibits similarities with the vertebrate GABA-A receptor in both phyla. Picrotoxin is a very weak or inactive antagonist at leech and nematode GABA receptors, while bicuculline methiodide blocks leech GABA receptors but is inactive on nematode GABA receptors. Picrotoxin does block GABA responses of earthworm body wall muscle. All these GABA responses are chloride mediated.5. Neuroactive peptides of the RFamide family occur m both phyla and FMRFamide has been identified in leeches. RFamides probably have an important role in heart regulation in leeches and in modulation of their body wall muscles. RFamides also modulate nematode body wall muscle activity with KNEFIRFamide raising muscle tone while SDPNFLRFamide relaxes the muscle. It is likely that this family and other neuroactivc peptides play an important role in the physiology of body wall muscle throughout both phyla.     

m-Sulfonate Benzene Diazonium Chloride: A Powerful Affinity Label for the γ-Aminobutyric Acid Binding Site from Rat Brain

m-Sulfonate benzene diazonium chloride (MSBD) was used to affinity-label the gamma-aminobutyric acid (GABA) binding site from rat brain membranes. To assess the irreversibility of the labeling reaction, we used an efficient ligand dissociation procedure combined to a rapid [3H]muscimol binding assay, both steps being performed on filter-adsorbed membranes. Inactivation of specific [3H]-muscimol binding sites by MSBD and its prevention by GABA were both time- and concentration-dependent. The time course of MSBD labeling was shortened as the pH of the incubation medium was increased from 6.2 to 8. These data suggest that MSBD can efficiently label the GABA binding site through alkylation of a residue having an apparent dissociation constant around neutrality.     

Histamine action on vertebrate GABAA receptors: direct channel gating and potentiation of GABA responses

Histamine is not only a crucial cytokine in the periphery but also an important neurotransmitter and neuromodulator in the brain. It is known to act on metabotropic H1-H4 receptors, but the existence of directly histamine-gated chloride channels in mammals has been suspected for many years. However, the molecular basis of such mammalian channels remained elusive, whereas in invertebrates, genes for histamine-gated channels have been already identified. In this report, we demonstrated that histamine can directly open vertebrate ion channels and identified beta subunits of GABA(A) receptors as potential candidates for histamine-gated channels. In Xenopus oocytes expressing homomultimeric beta channels, histamine evoked currents with an EC(50) of 212 microm (beta(2)) and 174 microm (beta(3)), whereas GABA is only a very weak partial agonist. We tested several known agonists and antagonists for the histamine-binding site of H1-H4 receptors and described for beta channels a unique pharmacological profile distinct from either of these receptors. In heteromultimeric channels composed of alpha(1)beta(2) or alpha(1)beta(2)gamma(2) subunits, we found that histamine is a modulator of the GABA response rather than an agonist as it potentiates GABA-evoked currents in a gamma(2) subunit-controlled manner. Despite the vast number of synthetic modulators of GABA(A) receptors widely used in medicine, which act on several distinct sites, only a few endogenous modulators have yet been identified. We show here for the first time that histamine modulates heteromultimeric GABA(A) receptors and may thus represent an endogenous ligand for an allosteric site.     

Modulation of GABA(A) receptor phosphorylation and membrane trafficking by phospholipase C-related inactive protein/protein phosphatase 1 and 2A signaling complex underlying brain-derived neurotrophic factor-dependent regulation of GABAergic inhibition

Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABA(A) receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABA(A) receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABA(A) receptor beta3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABA(A) receptors. We demonstrate that PRIP associates with phosphatases as well as with beta subunits. PRIP was found to colocalize with GABA(A) receptor clusters in cultured neurons and with recombinant GABA(A) receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to beta subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABA(A) receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABA(A) receptors by mediating the specific association between beta subunits of these receptors with protein phosphatases.