GABA-activated Currents Research Articles

Enantioselectivity of alpha-benzyl-alpha-methyl-gamma-butyrolactone-mediated modulation of anticonvulsant activity and GABA(A) receptor function.

Alkyl-substituted butyrolactones have both inhibitory and stimulatory effects on GABA(A) receptors. Lactones with small alkyl substitutions at the alpha-position positively modulate the channel, whereas beta-substituted lactones tend to inhibit the GABA(A) receptor. These compounds mediate inhibition through the picrotoxin site of the receptor. A distinct binding site that mediates the stimulatory actions of lactones is presumed to exist, although no definitive evidence to support this claim exists. In the present study, we used in vivo and in vitro assays to evaluate the effects of the enantiomers of a novel lactone, alpha-benzyl-alpha-methyl-gamma-butyrolactone (alpha-BnMeGBL), on the GABA(A) receptor. R-(-)-alpha-BnMeGBL was 2-fold more potent than the S-(+)-alpha-BnMeGBL in blocking pentylenetetrazol-induced seizures in CF-1 mice. The (+)-enantiomer inhibited binding of t-butylbicyclophosporothionate with a higher affinity than the (-)-enantiomer (IC(50) of 0.68 and 1.1 mM, respectively). Whole cell patch-clamp recordings from recombinant alpha1beta2gamma2 receptors stably expressed in HEK293 cells demonstrated that both compounds stimulated GABA-activated current. The maximal stimulation was approximately 2-fold greater with (+)-alpha-BnMeGBL than that seen with (-)-alpha-BnMeGBL. Both enantiomers of alpha-BnMeGBL directly gated the GABA(A) receptor at mM concentrations, in a nonstereoselective manner. Our data demonstrate the stimulatory actions of alpha-BnMeGBL on GABA(A) receptor function display enantioselectivity and provide strong evidence for the existence of a true "lactone site" on the receptor.

Effects of pentobarbital on GABA-activated currents in acutely-isolated rat dentate gyrus granule neurons

Granule neurons from the rat dentate gyrus were acutely isolated and whole-cell currents recorded. Maximal enhancement of 7 μM γ-aminobutyric acid (GABA; EC 30) evoked currents was obtained with 100 μM pentobarbital where the peak-current was 2.1±0.2 of control. One hundred μM pentobarbital alone evoked no current response whereas 1 mM pentobarbital elicited a current response that was 0.4±0.2 of the 100 μM GABA-activated peak current. In 100 μM pentobarbital, the GABA EC 50 value shifted from 14 to 3 μM but the peak-saturating-current value was not altered. An off-current was recorded on removal of 100 μM and higher pentobarbital concentrations. Ten mM pentobarbital abolished the peak-current response to 7 and 100 μM GABA. The results show that in the granule neurons the drug potency differs for the different effects of pentobarbital at GABA A receptors with the modulatory and inhibitory effects requiring lower concentrations than the direct activation of the receptors.

Voltage‐Dependent Block of N‐Methyl‐d‐Aspartate Receptors by the Novel Anticonvulsant Dibenzylamine, a Bioactive Constituent of l‐(+)‐β‐Hydroxybutyrate

Previously we demonstrated that L-(+)-beta-hydroxybutyrate (L-BHB), acetoacetate (ACA), acetone, and dibenzylamine (DBA) were anticonvulsant in an audiogenic seizure-susceptible model, and that DBA was a bioactive contaminant identified in commercial lots of L-BHB. In the present study, we asked whether these effects could be mediated by ionotropic glutamate or gamma-aminobutyric acidA (GABAA) receptors. We studied the effects of both stereoisomers of BHB (as well as the racemate), ACA, and DBA on N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5methyl-4-isoxazole-proprionic acid (AMPA), and GABAA receptors in cultured rodent neocortical neurons by using whole-cell voltage-clamp recording techniques. Only L-BHB and DBA exerted a concentration- and voltage-dependent block of NMDA-evoked currents, whereas none of the tested substrates affected AMPA- or GABA-activated currents. The kinetics of whole-cell block by L-BHB and DBA were similar, providing additional evidence that DBA is responsible for the anticonvulsant activity of L-BHB. BHB and ACA do not exert direct actions on GABAA or ionotropic glutamate receptors in cultured neocortical neurons. In addition, we provide additional evidence that DBA is responsible for the anticonvulsant activity of L-BHB, and that this action may be mediated in part by voltage-dependent blockade of NMDA receptors.

Two neuropharmacological types of rabbit ON-alpha ganglion cells express GABAC receptors.

The major inhibitory neurotransmitters GABA and glycine provide the bulk of input to large-field ganglion cells in the retina. Whole-cell patch-clamp recordings were used to characterize the glycine- and GABA-activated currents for morphologically identified ON-alpha ganglion cells in the rabbit retina. Cells identified as ON-alpha cells by light evoked currents were intracellularly stained and examined by light microscopy which revealed dendritic stratification in the vitreal half of the inner plexiform layer and confirmed their physiological identity. All Ca(2+)-mediated synaptic influences were abolished with Co(2+), revealing two types of ON-alpha cell characterized by their different inhibitory current profiles. One group exhibited larger glycine- than GABA-activated currents, while the other group had larger GABA- than glycine-activated currents. Both cell types demonstrated strychnine-sensitive glycine-activated currents and bicuculline-sensitive GABAA-activated currents. Surprisingly, both cell types expressed functional GABAC receptors demonstrated by their sensitivity to TPMPA. In addition, the cells with larger glycine-activated currents also possessed GABAB receptors, whereas those with larger GABA-activated currents did not. Immunocytochemical experiments confirmed the presence of glycine, GABAA, and GABAC receptor subunits on all physiologically identified ON-alpha ganglion cells in this study. In addition, the GABAB receptor immunolabeled puncta were present on the cells with larger glycine-activated currents, but not on the cells with the larger GABA-activated currents. In conclusion, the presence of different functional GABA and glycine receptors determined physiologically correlated well with the specific GABA and glycine receptor immunolabeling for two neuropharmacological types of rabbit ON-alpha ganglion cells.

RdlDv, a novel GABA-gated chloride channel gene from the American dog tick Dermacentor variabilis

The American dog tick Dermacentor variabilis is a major transmitter of bacterial and viral pathogens in human and animal populations, and compounds active against this species would benefit both human and animal health. Invertebrate GABA-gated chloride channels are validated targets of commonly used insecticides and acaricides. We cloned a novel member of the invertebrate GABA-gated chloride channel gene family from Dermacentor variabilis, Rdl Dv. The closest homologue of the predicted gene product of Rdl Dv is the RDL protein encoded by the GABA-gated chloride channel gene Drosophila Rdl (Resistance to Dieldrin), with which it shares 64% amino acid identity. When expressed in Xenopus oocytes, Rdl Dv produces GABA-activated currents blocked by the known insecticides and RDL antagonists fipronil and picrotoxinin. These results suggest that Rdl Dv encodes a GABA-gated chloride channel subunit, making it a potential target for compounds active against the tick D. variabilis.

Roles of protein kinase A and C in the opioid potentiation of the GABA A response in rat periaqueductal gray neuron

The periaqueductal gray (PAG) is the main target site of the opioid-induced analgesia. The present study was designed to examine the roles of protein kinase A (PKA) and C (PKC) in the opioid-induced modulation of the currents activated by an inhibitory neurotransmitter, γ-aminobutyric acid (GABA). The PAG neurons were acutely isolated and voltage-clamped under the nystatin-perforated patch-clamp mode. The GABA-activated current was sensitively blocked by a GABA A receptor antagonist, bicuculline, and selectively carried by chloride ions. The GABA A receptor-activated Cl − current was potentiated by a μ-opioid receptor agonist, [ d-Ala 2, N-MePhe 4,Gly 5-ol]-enkephalin acetate (DAMGO). The GABA response was also potentiated by phorbol-12-myristate-13-acetate (PMA). Pretreatment with PMA occluded the DAMGO potentiation. However, both chelerythrine and 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X) also potentiated the GABA response. Pretreatment with chelerythrine or GF109203X also occluded the DAMGO potentiation. Meanwhile, the GABA response was potentiated by N-(2-[ p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89), while not altered by forskolin. Pretreatment with H-89 occluded the potentiation effect of DAMGO on the GABA response. In addition, the DAMGO effect was completely blocked by pretreatment with forskolin. From the result, it can be suggested that activation of μ-opioid receptor potentiates the GABA A response through the mediation of PKA inhibition, and that PKC is not directly involved in the action mechanism of DAMGO.

Redox modulation of GABA A receptors obscured by Zn 2+ complexation

Redox reagents are thought to modulate γ-Aminobutyric acid type A (GABA A) receptors by regulating the redox state of the N-terminal disulphide bridge. Examining the redox sensitivity of recombinant GABA A receptors in human embryonic kidney cells, using whole-cell patch clamp techniques, revealed that α1β2 H267A and α1β2γ2 receptors, which are both less sensitive to Zn 2+ and H + modulation, ablated the potentiating effect of the reducing agent, dithiothreitol (DTT) seen for α1β2 receptors. This effect could result from disruption to the redox signal transduction pathway or be due to DTT chelating Zn 2+ from its H267 inhibitory binding site, consequently potentiating GABA-activated currents in α1β2 but not α1β2 H267A or α1β2γ2 receptors. A Zn 2+ chelating agent, tricine, potentiated GABA currents for the αβ constructs and vertically displaced GABA dose–response curves, suggesting that these receptors are subject to some inhibition by basal Zn 2+. Tricine, did not affect the GABA currents of either α1β2 H267A or α1β2γ2 receptors but did prevent the potentiation by 2 mM DTT and reduced the potentiation caused by 10 mM DTT on α1β2 receptors. Thus, at low concentrations of DTT, a substantial component of the potentiation probably occurs via Zn 2+ chelation from H267 in the ion channel. In contrast, at higher DTT concentrations, it is more likely to be acting as a redox agent, which modulates both αβ and αβγ subunit receptors.

Tryptophan scanning mutagenesis in TM4 of the GABA A receptor α1 subunit: implications for modulation by inhaled anesthetics and ion channel structure

Previous studies have shown that amino acid residues in trans-membrane (TM) segments 1, 2 and 3 of the α subunit are critical for the enhancement of GABA A receptor function by inhaled anesthetics. In this study we used tryptophan (Trp) scanning mutagenesis between Ile 406 and Asn 417 in the α1 subunit to determine the effects of Trp substitution in the fourth transmembrane segment (TM4) on receptor gating and anesthetic modulation. Wild-type and mutant α1 subunits were transiently expressed in HEK 293 cells with wild-type β2 and γ2s subunits and GABA-activated currents were recorded using whole-cell voltage clamp. The potentiation by three inhaled anesthetics (isoflurane, halothane and chloroform) of responses elicited by a submaximal concentration of GABA were also examined. EC 50 values for GABA at the mutant receptors were in the range 4–60 μM (wild-type=20 μM), indicating that Trp substitution can alter the apparent affinity of the receptor for GABA positively or negatively, dependent on position. The variation of the calculated EC 50 value for GABA exhibited an interesting periodicity, with the cycle length for each repeat corresponding to approximately 3.6 amino acids. These data are consistent with an α-helical structure for the TM4 segment of the α subunit. Several of these Trp point mutations altered the ability of one or more of the three inhaled anesthetics to modulate receptor function; four of the 12 mutations abolished receptor modulation by one or more of the anesthetics tested. These data are consistent with a role for these residues at the extracellular end of TM4 in anesthetic modulation of GABA A receptors.

Modulation of GABA A receptor-mediated currents by benzophenone derivatives in isolated rat Purkinje neurones

We investigated modulation of GABA A receptor-mediated whole-cell currents in cerebellar Purkinje neurones by several derivatives of benzophenone. A metabolite of phenazepam, 5-bromo-2’-chloro-2-aminobenzophenone ( I), caused dual modification of peak amplitudes of GABA-gated currents that depended upon the concentration of applied GABA and incubation time. Following short 10 s pre-incubations, 1–30 μM I facilitated activation and delayed deactivation of currents evoked by 500 ms pulses of 20 μM GABA. In addition, 10 μM I prominently enhanced desensitisation of currents during applications of 500 μM GABA mainly by decreasing the value of the fast time constant of the desensitisation. Continuous 6 min incubation with 10 μM I during GABA stimulation or its administration between but not during 1 s pulses of 500 μM GABA led to a gradual, partly reversible attenuation of GABA-activated currents. This inhibition was not observed when I was applied only during pulses of GABA, indicating that the blockade was not use-dependent. One of the possible mechanisms of this down-modulation could be an intracellular effect of I, because when applied intracellularly it caused slow inhibition of responses to consecutive GABA pulses. When 3–30 μM I was applied on the background of small ‘plateau’-like current 5–7 s after application of 500 μM GABA, it was able to block open channels with on and off rates similar to those observed with 30 μM picrotoxin but much slower than in the case of 500 μM benzylpenicillin. At a concentration of 10 μM, 5-substituted benzophenones, but not 2-aminobenzophenone or benzophenone itself, exhibited modulatory properties similar to I and distinct from those of picrotoxin and benzylpenicillin. Therefore, we conclude that derivatives of benzophenone are a novel class of GABA A receptor modulators with a unique pharmacological profile.

A Gene for JME at Last: The alpha1 GABA Receptor Subunit.

Mutation of GABRA1 in an Autosomal Dominant Form of Juvenile Myoclonic Epilepsy Cossette P, Liu L, Brisebois K, Dong H, Lortie A, Vanasse M, Saint-Hilaire JM, Carmant L, Verner A, Lu WY, Tian Wang Y, Rouleau GA Nat Genet 2002;31:184–189 Although many genes that predispose for epilepsy in humans have been determined, those that underlie the classical syndromes of idiopathic generalized epilepsy (IGE) have yet to be identified. We report that an Ala322Asp mutation in GABRA1, encoding the alpha1 subunit of the gamma-aminobutyric acid receptor subtype A (GABA(A)), is found in affected individuals of a large French Canadian family with juvenile myoclonic epilepsy. Compared with wildtype receptors, GABA(A) receptors that contain the mutant subunit show a lesser amplitude of GABA-activated currents in vitro, indicating that seizures may result from loss of function of this inhibitory ligand-gated channel. Our results confirm that mutation of GABRA1 predisposes towards a common idiopathic generalized epilepsy syndrome in humans.

Mutation of GABRA1 in an autosomal dominant form of juvenile myoclonic epilepsy.

Although many genes that predispose for epilepsy in humans have been determined, those that underlie the classical syndromes of idiopathic generalized epilepsy (IGE) have yet to be identified. We report that an Ala322Asp mutation in GABRA1, encoding the alpha1 subunit of the gamma-aminobutyric acid receptor subtype A (GABA(A)), is found in affected individuals of a large French Canadian family with juvenile myoclonic epilepsy. Compared with wildtype receptors, GABA(A) receptors that contain the mutant subunit show a lesser amplitude of GABA-activated currents in vitro, indicating that seizures may result from loss of function of this inhibitory ligand-gated channel. Our results confirm that mutation of GABRA1 predisposes towards a common idiopathic generalized epilepsy syndrome in humans.

Mechanisms of GABA A receptor blockade by millimolar concentrations of furosemide in isolated rat Purkinje cells

The action of diuretic furosemide on the GABA A receptor was studied in acutely isolated Purkinje cells using the whole-cell recording and fast application system. Furosemide blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC 50 value > 5 mM at −70 mV. The inhibition was rapid in the onset, fully reversible and did not require drug pre-perfusion. The termination of GABA and furosemide co-application was followed by transient increase in the inward current ‘tail’ current, which was not observed when furosemide was continuously present in the solution. The degree of furosemide block did not depend on GABA concentration. Furosemide block increased with membrane depolarization. Five millimolar furosemide depressed GABA currents by 32.4±1.3% at –70 mV and by 76.7±5.0% at +70 mV. Analysis of the voltage dependence of the block suggests that furosemide binds at the site located within GABA A channel pore with a dissociation constant of 5.3±0.5 mM at 0 mV and electric distance of 0.27. Our results provide evidence that furosemide interacts with Purkinje cell GABA A receptors (most probably composed of α1β2/3γ2 subunits) through a low affinity site located in channel pore and suggest that furosemide acts as a sequential open channel blocker, which prevents the dissociation of agonist while the channel is blocked.

Phosphorylation of the recombinant ρ1 GABA receptor

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. While a growing body of literature indicates that postsynaptic GABA receptors are regulated by phosphorylation, there is discrepancy as to the specific effects of phosphorylation on GABA receptor function. Here, we have identified phosphorylation sites on the human ρ1 GABA receptor for six protein kinases widely expressed in the brain: protein kinase C (PKC); cAMP-dependent protein kinase (PKA); calmodulin-dependent kinase (CaMKII); casein kinase (CKII); mitogen-activated protein kinase (MAPK); and cGMP-dependent protein kinase (PKG). We demonstrate that in nearly all cases, the consensus sites and actual phosphorylation sites do not agree supporting the risk of relying on a sequence analysis to identify potential phosphorylation sites. In addition, of the six kinases examined, only CKII phosphorylated the human ρ2 subunit. Site-directed mutagenesis of the phosphorylation sites, or activation/inhibition of select kinase pathways, did not alter the receptor sensitivity or maximal GABA-activated current of the ρ1 GABA receptor expressed in Xenopus laevis oocytes suggesting phosphorylation of ρ1 does not directly alter receptor properties. This study is a first and necessary step towards elucidating the regulation of ρ1 GABA receptors by phosphorylation.

Modulation of GABA-activated currents in rat isolated cerebellar neurons by lanthanum ions.

Lanthanum ions (10-1000 microM) potentiated GABA-activated currents and increased the sensitivity of GABA(A) receptors in isolated Purkinje cells from rat cerebellum. GABA receptors in Purkinje cells are comparable to receptors on other cells by their sensitivity lanthanum ions.

Evidence for the formation of functionally distinct alphabetagammaepsilon GABA(A) receptors.

1. We transiently introduced the human GABA(A) receptor epsilon subunit cDNA into a human embryonic kidney (HEK) cell line stably expressing alpha1beta3gamma2 receptors (WSS-1 cells) to establish whether the subunit competes with the gamma2 subunit for assembly into receptors. GABA-evoked currents were recorded using the patch-clamp technique from cells transfected with cDNA encoding green fluorescent protein (GFP) alone or in combination with the epsilon subunit cDNA. 2. The epsilon subunit did not change the potency of GABA: the GABA EC(50) was 34 +/- 6 microM in control WSS-1 cells and 37 +/- 6 microM in cells expressing the epsilon subunit. The introduction of the epsilon subunit reduced the peak current amplitude activated by GABA (1 mM) from 1.8 +/- 0.2 nA in control cells to 0.9 +/- 0.2 nA in cells expressing the epsilon subunit (P < 0.05). 3. The epsilon subunit caused the appearance of leak currents recorded in the absence of GABA. Outside-out patches excised from epsilon subunit-containing WSS-1 cells exhibited spontaneously opening GABA(A) channels not seen in patches excised from control GFP-expressing WSS-1 cells. Introduction of the epsilon subunit did not alter the GABA-evoked single-channel cord conductance. 4. The anaesthetic 2,6-diisopropylphenol (propofol, 3 microM) and the benzodiazepine flunitrazepam (1 microM) potentiated GABA-evoked currents recorded from control cells labelled with GFP. The epsilon subunit reduced potentiation by both agents 48-96 h after transfection. 5. The introduction of the epsilon subunit had no effect on the ability of propofol (3-30 microM) relative to GABA (1 mM) to activate GABA(A) receptors in WSS-1 cells. High concentrations of propofol (> or = 100 microM) produced a more marked desensitization of GABA(A) receptor activity in WSS-1 cells transfected with cDNA for the epsilon subunit than in control cells. 6. There was no difference in the potency of Zn(2+) as an inhibitor of currents recorded from control cells (IC(50) = 165 +/- 34 microM) or cells expressing the epsilon subunit (IC(50) = 179 +/- 11 microM). 7. GABA-activated currents recorded both from control cells and cells expressing the epsilon subunit reversed in sign at the Cl- equilibrium potential and exhibited outward rectification. 8. The introduction of the epsilon subunit changes the functional properties of GABA(A) receptors in WSS-1 cells. The resulting receptors have a unique combination of properties indicative of the co-assembly of alpha, beta, gamma and epsilon subunits.

Functional analysis of GABA A receptors in nucleus tractus solitarius neurons from neonatal rats

To gain insight into specific GABA A receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1–14 day old rats using whole-cell patch clamp recordings. GABA A− receptor-mediated currents were detected in all neurons tested, with an average EC 50 of 22.2 μM. GABA currents were consistently stimulated by diazepam (EC 50=63 nM), zolpidem (EC 50=85 nM), loreclezole (EC 50=10.1 μM) and the neurosteroid 5α-pregnan-3α-hydroxy-20-one (3α-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn 2+ (IC 50=33.6 μM) picrotoxin (IC 50=2.4 μM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10–1000 μM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant α subunit isoform present in GABA A receptors of the NTS appears to be the α1 and/or α2 subunit. β2 and/or β3 subunits are the major β isoform, while the predominant γ subunit is likely γ2. Our data suggest the contribution to NTS GABA currents by α3–α6, β1, γ1 and δ subunits, if present, is minor by comparison.

First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2-subunit gene.

Major advances in the identification of genes implicated in idiopathic epilepsy have been made. Generalized epilepsy with febrile seizures plus (GEFS+), benign familial neonatal convulsions and nocturnal frontal lobe epilepsy, three autosomal dominant idiopathic epilepsies, result from mutations affecting voltage-gated sodium and potassium channels, and nicotinic acetylcholine receptors, respectively. Disruption of GABAergic neurotransmission mediated by gamma-aminobutyric acid (GABA) has been implicated in epilepsy for many decades. We now report a K289M mutation in the GABA(A) receptor gamma2-subunit gene (GABRG2) that segregates in a family with a phenotype closely related to GEFS+ (ref. 8), an autosomal dominant disorder associating febrile seizures and generalized epilepsy previously linked to mutations in sodium channel genes. The K289M mutation affects a highly conserved residue located in the extracellular loop between transmembrane segments M2 and M3. Analysis of the mutated and wild-type alleles in Xenopus laevis oocytes confirmed the predicted effect of the mutation, a decrease in the amplitude of GABA-activated currents. We thus provide the first genetic evidence that a GABA(A) receptor is directly involved in human idiopathic epilepsy.

Modulation of GABA A receptors by benzodiazepines and barbiturates is autonomous of PKC activation

Previous studies have suggested that activation of calcium-phospholipid-dependent protein kinase (PKC) enhances benzodiazepine (BZD)- and pentobarbital (PB)- mediated potentiation of α 1β 1γ 2 GABA A receptors (GABA A-Rs). To delineate the underlying mechanism(s), voltage-clamp recordings were performed on recombinant α 1β 1γ 2 GABA A receptors functionally expressed in Xenopus laevis oocytes. GABA A-Rs were tested for their sensitivity to diazepam and PB before and after incubation in phorbol 12-myristate 13-acetate (PMA). PMA (25 nM) significantly attenuated the GABA A current ( p<0.05, n=12–19) up to 90%. PMA treatment, however, did not alter the sensitivity to diazepam or pentobarbital. Similar results were obatined with recombinant α 1β 2γ 2 GABA receptors. These data suggest that PKC activation does not alter the allosteric modulation of GABA A-Rs by benzodiazepines and barbiturates and is consistent with the observation from other studies in oocytes that PMA decreases the amplitude of the GABA-activated currents via receptor internalization rather than modification of receptor kinetics.

Proton sensitivity of rat cerebellar granule cell GABAA receptors: dependence on neuronal development.

The effect of GABAA receptor development in culture on the modulation of GABA-induced currents by external H+ was examined in cerebellar granule cells using whole-cell and single-channel recording. Equilibrium concentration-response curves revealed a lower potency for GABA between 11 and 12 days in vitro (DIV) resulting in a shift of the EC50 from 10.7 to 2.4 uM. For granule cells before 11 DIV, the peak GABA-activated current was inhibited at low external pH and enhanced at high pH with a pKa of 6.6. For the steady-state response, low pH was inhibitory with a pKa of 5.56. After 11 DIV, the peak GABA-activated current was largely pH insensitive; however, the steady-state current was potentiated at low pH with a pKa of 6.84. Single GABA-activated ion channels were recorded from outside-out patches of granule cell bodies. At pH 5.4-9.4, single GABA channels exhibited multiple conductance states occurring at 22-26, 16-17 and 12-14 pS. The conductance levels were not significantly altered over the time period of study, nor by changing the external H+ concentration. Two exponential functions were required to fit the open-time frequency histograms at both early (< 11 DIV) and late (> 11 DIV) development times at each H+ concentration. The short and long open time constants were unaffected either by the extracellular H+ concentration or by neuronal development. The distribution of all shut times was fitted by the sum of three exponentials designated as short, intermediate and long. At acidic pH, the long shut time constant decreased with development as did the relative contribution of these components to the overall distribution. This was concurrent with an increase in the mean probability of channel opening. In conclusion, this study demonstrates in cerebellar granule cells that external pH can either reduce, have no effect on, or enhance GABA-activated responses depending on the stage of development, possibly related to the subunit composition of the GABAA receptors. The mode of interaction of H+ at the single-channel level and implications of such interactions at cerebellar granule cell GABAA receptors are discussed.

Heterogeneity of GABA(A) receptor-mediated responses in the human IMR-32 neuroblastoma cell line.

The gamma-aminobutyric acid (GABA) response profiles of IMR-32 human neuroblastoma cells were examined using whole-cell patch clamp and RT-PCR techniques. GABA activated a concentration-dependent and bicuculline-sensitive current, and RT-PCR revealed the expression of multiple GABA(A) receptor subunit mRNAs (alpha(1), alpha(3), alpha(4), beta(1), beta(3), gamma(2), and delta). A pharmacological profile of the GABA-induced current was derived using several subunit-selective agents. Diazepam, which requires the presence of a gamma subunit in order to modulate GABA(A) receptor-mediated responses, potentiated GABA-induced currents in a subset of IMR-32 cells. Two populations of GABA-activated currents were also evident based on sensitivity to modulation by zinc. Comparison of zinc- and diazepam-induced modulation of GABA-induced current responses in the same cells revealed an inverse correlation between these two modulators. No differences, however, were observed with the GABA(A) receptor modulators loreclezole, allopregnanolone, and pentobarbital. Thus, IMR-32 cells maintained in culture are heterogeneous in terms of expression of GABA(A) receptor isoforms.