Channel opening of gamma-aminobutyric acid receptor from rat brain: molecular mechanisms of the receptor responses

The function of gamma-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of 36Cl- isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific 36Cl- influx was completed in three phases of ca. 3% (t 1/2 = 0.6 s), 56% (t 1/2 = 82 s), and 41% (t 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent 36Cl- influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles with initial first-order rate constants of 9.5 s-1 and 2.3 s-1 and desensitizing with first-order rate constants of 21 s-1 and 1.4 s-1, respectively, at saturation. The half-response concentrations were similar for both receptors, 150 microM and 114 microM GABA for desensitization and 105 microM and 82 microM for chloride exchange, for the faster and slower desensitizing receptors, respectively. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of "low-affinity" to "high-affinity" GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative efficacies of 1,4-diazepines on GABA-stimulated chloride influx in rat brain vesicles

The effects of 1,4-diazepines with two annelated heterocycles [brotizolam (WE 941), ciclotizolam (WE 973) and WE 1008] on gamma-aminobutyric acid (GABA)-stimulated chloride influx into rat brain membrane vesicles were examined. Brotizolam enhanced GABA (30 microM)-stimulated 36Cl- influx (146.1% of control), while ciclotizolam and WE 1008 showed only a small enhancement (119.3% and 119.1%, respectively) of GABA-stimulated 36Cl- uptake. Brotizolam resulted in a left shift of the GABA dose response curve at lower concentrations of GABA (10 microM), while at higher concentrations of GABA (1 mM), brotizolam caused a reduction of the maximal response. The enhancement of GABA-stimulated 36Cl- uptake by brotizolam (0.1 microM) was antagonized by Ro 15-1788. At higher concentration of GABA (300 microM), brotizolam inhibited GABA-stimulated 36Cl- uptake in a dose dependent manner and Ro15-1788 failed to antagonize this effect. These results suggest that 1) brotizolam produces an enhancement of GABA (30 microM)-stimulated chloride influx through the benzodiazepine receptor. 2) brotizolam inhibition of GABA (300 microM)-stimulated chloride influx involves an additional mechanism, and 3) the sedative-hypnotic action of brotizolam may be related to its high efficacy at the benzodiazepine/GABA-gated chloride channel.     

γ-Aminobutyric Acid-Activated 36Cl- Influx: A Functional In Vitro Assay for CNS γ-Aminobutyric Acid Receptors of Insects

The effects of gamma-aminobutyric acid (GABA) on the uptake of 36Cl- into a membrane microsac preparation from isolated nerve cords of the cockroach Periplaneta americana was studied. On addition of 1 microM GABA (after 4-s incubation, then rapid quenching) the influx of 36Cl- was stimulated to a level 75% above that of the control value. This stimulation was reduced by picrotoxin (100 microM), but was not significantly affected by bicuculline (100 microM). Results of 36Cl- influx experiments are in agreement with data obtained from radiolabelled ligand binding assays and electrophysiological investigations on the same tissue. The method described represents a functional in vitro assay for CNS GABA receptors of insects.     

The effect of benzodiazepines and beta-carbolines on GABA-stimulated chloride influx by membrane vesicles from the rat cerebral cortex

Benzodiazepine agonists such as diazepam, flunitrazepam and clonazepam enhanced GABA (30 microM)-stimulated 36Cl- uptake in membrane vesicles from the rat cerebral cortex. The rank order of potencies was flunitrazepam greater than diazepam = clonazepam. beta-Carboline-3-carboxylate esters beta-CCM, beta-CCE and DMCM inhibited GABA-stimulated 36Cl- uptake. The rank order of inhibitory potencies was DMCM greater than beta-CCM greater than beta-CCE. The benzodiazepine antagonist Ro15-1788 antagonized the enhancement of flunitrazepam and the inhibition of DMCM on GABA-stimulated 36Cl- uptake in a competitive inhibitory manner. These results suggest that benzodiazepine receptors regulate GABA-stimulated 36Cl- uptake and there is a functional coupling between the GABA and benzodiazepine receptors, and chloride channels in membrane vesicles from the rat cerebral cortex.     

Aging reduces the GABA-dependent 36Cl- flux in rat brain membrane vesicles

The function of the chloride channel associated to GABAA receptor complex was analyzed in the brain of aged rats by measuring the chloride flux across the neuronal membrane and its modulation by drugs acting at the level of the GABA receptor complex and 35S-TBPS binding. The basal 36Cl- uptake by brain membrane vesicles of aged rats was higher (22%) than that observed in those of adult rats. The higher 36Cl- uptake found in cortical membrane vesicles of senescent rats was not sensitive to the action of bicuculline indicating that it was not the consequence of a tonic GABAergic modulation. Moreover, the stimulation of 36Cl- uptake induced by GABA was markedly lower in membrane vesicles of aged rats than that observed in those of adult rats. Accordingly, the stimulation of 36Cl- efflux elicited by GABA (18%) and pentobarbital (26%) was higher in membrane vesicles of adult rats with respect to that (8 and 16%, respectively) of old rats. Finally, a significant decrease of 35S-TBPS binding was observed in membrane preparation from the cerebral cortex, cerebellum and hippocampus of aged-rats. Scatchard plot analysis indicated that the decrease was entirely due to a reduction in the total number of binding sites with no change in their affinity. All together the results indicate that in the rat brain the function of the chloride channel coupled to the GABA/benzodiazepine/barbiturate receptor complex is reduced by aging.     

Activation of gamma-aminobutyric acid insensitive chloride channels in mouse brain synaptic vesicles by avermectin B1a.

The interaction of avermectin B1a (AVMB1a) with mouse brain chloride channels was characterized using a radiochloride efflux assay. The loss of intravesicular chloride from synaptoneurosomes preloaded with 36Cl involved an initial rapid phase followed by a slower phase that approached equilibrium within 10 min. AVMB1a stimulated a 30% loss of intravesicular chloride within the first 2 s of exposure; however, AVMB1a had no effect on the rate of the slower phase of chloride loss. Experiments with lysed synaptoneurosomes showed that both chloride loading and basal and AVMB1a-stimulated chloride release required the presence of intact vesicles. The efflux of 36Cl from mouse brain synaptosomes and the stimulation of efflux by AVMB1a were qualitatively similar to the results obtained with synaptoneurosomes but involved much lower overall levels of chloride loading and release. AVMB1a produced half-maximal stimulation of chloride efflux from synaptoneurosomes at a concentration of 2.1 +/- 0.3 microM and a 35.4 +/- 1.4% maximal loss of intravesicular chloride at saturating concentrations. gamma-Aminobutyric acid (GABA), bicuculline, or the chloride channel blockers picrotoxinin, t-butylbicyclophosphorothionate (TBPS) 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene 9-carboxylic acid (9-CA) had little or no effect on the loss of chloride from synaptoneurosomes either in the presence or the absence of AVMB1a. However, the chlorinated cycloalkane insecticides dieldrin and lindane were equally effective as inhibitors of GABA-dependent chloride uptake and AVMB1a-stimulated chloride efflux. These data demonstrate that AVMB1a-stimulated chloride efflux from mouse brain synaptic vesicles results from the activation of GABA-insensitive chloride channels and that this action is distinct from their previously documented effects on GABA-gated chloride channels in mouse brain preparations. Our findings imply that both GABA-gated and GABA-insensitive chloride channels may be toxicologically significant targets for the action of avermectins.     

Characterization of GABAA receptor-mediated 36chloride uptake in rat brain synaptoneurosomes

gamma-Aminobutyric acid (GABA) receptor-mediated 36chloride (36Cl-) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated 36Cl- uptake in a concentration-dependent manner with the following order of potency: Muscimol greater than GABA greater than piperidine-4-sulfonic acid (P4S)greater than 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP) = 3-aminopropanesulfonic acid (3APS) much greater than taurine. Both P4S and 3APS behaved as partial agonists, while the GABAB agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated 36Cl- uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated 36Cl- uptake was also dependent on the anion present in the media. The muscimol response varied in media containing the following anions: Br- greater than Cl- greater than or equal to NO3- greater than I- greater than or equal to SCN- much greater than C3H5OO- greater than or equal to ClO4- greater than F-, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl- channel.     

Interaction of t-Butylbicyclophosphorothionate with γ-Aminobutyric Acid-Gated Chloride Channels in Cultured Cerebral Neurons

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of gamma-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 microM for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 microM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 microM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 microM) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- greater than or equal to SCN- greater than Cl- greater than or equal to NO3- greater than I- greater than F- greater than gluconate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of the effects of anthranilic and (indanyloxy) acetic acid derivatives on chloride transport in membrane vesicles.

The effects of the Cl- channel blockers, NPPB, IAA94/95 and a number of related compounds on 36Cl- transport in membrane vesicles from bovine kidney cortex and rabbit ileum mucosa brush borders have been studied. These vesicles have been previously shown to be enriched in Cl- channel and Cl-/anion cotransport activity, respectively. Chloride transport was assayed in both types of vesicles by measuring the uptake of 36Cl- in response to an outwardly-directed Cl- concentration gradient. In kidney microsomes, a large proportion of the observed 36Cl- uptake was mediated by an electrogenic uniport and could be substantially reduced by clamping the membrane potential at zero mV using K+ and valinomycin. Chloride uptake was inhibited by both NPPB and IAA94/95 with apparent IC50 values of around 10 microM under optimal conditions (i.e., 4 min uptake at 4 degrees C). Under other conditions (e.g., 10 min uptake at 25 degrees C), where uptake had reached a steady-state level, much higher concentrations of inhibitor were required to cause inhibition. Therefore, previous differences in the reported potency of these compounds may, in part, have been due to the conditions under which Cl- uptake was measured. In addition, both NPPB and, to a lesser extent, IAA94/95 were found to have other effects on the vesicles, in that, when added at a concentration of 100 microM, they induced a leakage of pre-accumulated 36Cl-. This was probably caused by either dissipation of membrane potential or damage to the vesicle membranes. The sulphonic acid derivatives of NPPB and IAA94/95 (NPPB-S and ISA94/95, respectively) blocked 36Cl- uptake with around the same potency as NPPB and IAA94/95, but did not cause any non-specific Cl- leakage, when added at concentrations up to 100 microM. Inhibition of 36Cl- uptake by all four compounds was almost completely reversible. However, when vesicles were incubated with the inhibitors in the presence of an outward Cl- concentration gradient, or if vesicles were freeze/thawed in the presence of the compounds, inhibition could be only partially reversed. In rabbit brush border membrane vesicles, 36Cl- uptake was not reduced when the vesicles were voltage clamped using valinomycin and K+, and was therefore probably mediated by Cl-/Cl- exchange. However, despite the lack of effect of valinomycin, 36Cl- uptake was inhibited by both NPPB (approx. 80% inhibition at 100 microM) and, to a lesser extent, by IAA94/95 (approx. 30% inhibition at 100 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of GABA-stimulated chloride influx into membrane vesicles from rat cerebral cortex by triazolobenzodiazepines

The effects of triazolobenzodiazepines on GABA-stimulated 36Cl- uptake by membrane vesicles from rat cerebral cortex were examined. Triazolam and alprazolam showed a significant enhancement of GABA-stimulated 36Cl- uptake at 0.01-10 microM. On the other hand, adinazolam showed a small enhancement at 0.1-1 microM followed by a significant inhibition of GABA-stimulated 36Cl- uptake at 100 microM. The enhancement of GABA-stimulated 36Cl- uptake by 1 microM alprazolam was antagonized by Ro15-1788, a benzodiazepine antagonist, but the inhibition of this response by 30 microM adinazolam was not antagonized by Ro15-1788. These results indicate that triazolobenzodiazepines enhanced GABA-stimulated 36Cl- uptake through benzodiazepine receptors. High concentrations of adinazolam inhibit GABA-stimulated 36Cl- uptake which may be due to the direct blockade of GABA-gated chloride channel.     

GABAA receptor-controlled 36Cl- influx in cultured rat cerebellar granule cells

gamma-Aminobutyric acid (GABA)-mediated and bicuculline-sensitive 36Cl- influx and bicuculline-sensitive [3H] GABA binding were demonstrated in cultures of rat cerebellar granule cells. The addition of 10(-5) M GABA produced a two-fold increase in 36Cl-influx over the basal level and the maximal increase was observed after approximately 20 sec. Progressive occupation of GABAA receptor by [3H]-(1S-9R)-bicuculline methiodide decreased 36Cl- influx activated by 10 microM GABA. The above results suggest that primary cultures of rat cerebellar granule cells provide a new and reliable model for studying the GABA activated chloride fluxes.     

Potentiation of γ-Aminobutyric Acid-Mediated Chloride Flux by Pentobarbital and Diazepam but Not Ethanol

The influx of 36Cl- into cerebral cortical and cerebellar microsacs from ICR mice and Sprague-Dawley rats was studied in incubations lasting 3 s, 500 ms, or 21 ms. In the 3-s assay, 10-40 mM ethanol did not affect either basal or gamma-aminobutyric acid (GABA)-mediated Cl- flux, at any GABA concentration tested. Only at a concentration of 600 mM did ethanol potentiate Cl- flux in both mouse and rat preparations. Ethanol (20 mM) also did not affect the significant potentiation of GABA-mediated flux produced by 50 microM pentobarbital or 2 microM diazepam in ICR mouse microsacs. In 21- and 500-ms incubations (quench-flow method), 50 microM pentobarbital significantly potentiated GABA-mediated Cl- flux in rat cortical microsacs, but 10-50 mM ethanol did not. These studies suggest that some as yet unrecognized factor is essential for ethanol enhancement of GABA-mediated Cl- flux, as reported by others in brain homogenates and in tissue culture.     

Block of an insect central nervous system GABA receptor by cyclodiene and cyclohexane insecticides

The effects of a cyclodiene (endrin) and a cyclohexane (lindane) insecticide have been tested on gamma-aminobutyric acid (GABA) receptors in the central nervous system of the cockroach (Periplaneta americana), by using electrophysiological methods and an in vitro functional receptor assay. In electrophysiological experiments on an identified motor neuron (Df), endrin blocked the GABA response with a 50% inhibition concentration of 5.0 x 10(-7) M in a non-competitive manner. The actions of endrin were irreversible under the experimental conditions adopted. Increasing the intracellular chloride concentration reduced the effectiveness of endrin, whereas a change in the potassium concentration failed to influence the block by endrin of GABA responses. Lindane exhibited similar actions to endrin on insect GABA receptors, but was approximately an order of magnitude less effective. In a microsac preparation from cockroach nerve cords, endrin, at a concentration of 1.0 x 10(-5) M, completely blocked GABA-stimulated 36Cl- uptake, whereas the same concentration of lindane was less potent, only blocking about 40% of uptake under similar conditions. Neither insecticide had any effect on L-glutamate-activated chloride channels. The results demonstrate that endrin and lindane block functional insect neuronal GABA receptors.     

Distribution and Pharmacological Properties of the GABAA/ Benzodiazepine/Chloride Ionophore Receptor Complex in the Brain of the Fish Anguilla anguilla

In the present study, we characterized the distribution and the pharmacological properties of the different components of the GABAA receptor complex in the brain of the eel (Anguilla anguilla). Benzodiazepine recognition sites labeled "in vitro" with [3H]flunitrazepam ([3H]FNT) were present in highest concentration in the optic lobe and in lowest concentration in the medulla oblongata and spinal cord. A similar distribution was observed in the density of gamma-[3H]aminobutyric acid ([3H]GABA) binding sites. GABA increased the binding of [3H]FNT in a concentration-dependent manner, with a maximal enhancement of 45% above the control value, and, vice versa, diazepam stimulated the binding of [3H]GABA to eel brain membrane preparations. The density of benzodiazepine and GABA recognition sites and their reciprocal regulation were similar to those observed in the rat brain. In contrast, the binding of the specific ligand for the Cl- ionophore, t-[35S]butylbicyclophosphorothionate ([35S]TBPS), to eel brain membranes was lower than that found in the rat brain. In addition, [35S]TBPS binding in eel brain was less sensitive to the inhibitory effects of GABA and muscimol and much more sensitive to the stimulatory effect of bicuculline, when compared with [35S]TBPS binding in the rat brain. Moreover, the uptake of 36Cl- into eel brain membrane vesicles was only marginally stimulated by concentrations of GABA or muscimol that significantly enhanced the 36Cl- uptake into rat brain membrane vesicles. Finally, intravenous administration of the beta-carboline inverse agonist 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methyl ester (20 mg/kg) and of the chloride channel blocker pentylenetetrazole (80 mg/kg) produced convulsions in eels that were antagonized by diazepam at doses five to 20 times higher than those required to produce similar effects in rats. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.     

Effect of a benzodiazepine (chlordiazepoxide) on a GABAA receptor from rat brain. Requirement of only one bound GABA molecule for channel opening.

Chlordiazepoxide (CDPX) enhanced the rate of chloride exchange mediated by the major GABAA receptor found on sealed native membrane vesicles from rat cerebral cortex. The initial rate constant for chloride exchange for this receptor, (JA), a measure of open channel, was determined from the progress of GABA-mediated influx of 36Cl-. The dependence of JA on GABA concentration was hyperbolic in the presence of CDPX (150 microM, sufficient to give maximum enhancement of chloride exchange rate) but sigmoid in its absence. Enhancement of channel opening (10-fold at 0.3 microM GABA) decreased with increasing GABA concentration. The maximal response, above 1,000 microM GABA, was unaltered. The half-response concentration was reduced from 80 microM to 50 microM. CDPX alone caused no measurable 36Cl- exchange. In the presence of CDPX, channel opening occurred with only one bound GABA molecule, whereas in its absence, channel opening with two bound GABA molecules was much more favorable. This could not be direct allosteric modulation of the channel opening conformational change by binding of CDPX at effector sites, but could be explained by an additional change of the receptor on binding CDPX to give a closed state which gave channel opening mediated by a single GABA binding site. Another possibility is that CDPX could act at one of the channel opening binding sites without a postulated, second closed conformational state.     

Regulation of ion uptake in membrane vesicles from rat brain by thiamine compounds

We examined the effects of thiamine derivatives on ion uptake in rat brain membrane vesicles. Thiamine triphosphate (1 mM) and pyrithiamine (0.1 mM) increase chloride uptake. Preincubation of crude homogenate with thiamine or pyrithiamine increases chloride uptake while oxythiamine has the reverse effect. Thiamine and oxythiamine also affect 22Na+ and 86Rb+ uptake in the same way as for 36Cl- but to a lesser extent. Thiamine-dependent 36Cl- uptake is activated by sodium bicarbonate (10 mM) and partially inhibited by bumetanide (0.1 mM) and 2,4-dinitrophenol (0.1 mM). Preincubation with thiamine increases the thiamine triphosphate content of the vesicles. The hypothesis that TTP is the activator of a particular chloride uptake mechanism is discussed.     

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.     

γ-Aminobutyric AcidA Receptor Pharmacology in Rat Cerebral Cortical Synaptoneurosomes

Equilibrium binding interactions at the gamma-aminobutyric acid (GABA) and benzodiazepine recognition sites on the GABAA receptor-Cl- ionophore complex were studied using a vesicular synaptoneurosome (microsacs) preparation of rat brain in a physiological HEPES buffer similar to that applied successfully in recent GABAergic 36Cl- flux measurements. NO 328, a GABA reuptake inhibitor, was included in the binding assays to prevent the uptake of [3H]muscimol. Under these conditions, the equilibrium dissociation constant (KD) values for [3H]muscimol and [3H]diazepam bindings are 1.9 microM and 40 nM, respectively. Binding affinities for these and other GABA and benzodiazepine agonists and antagonists correlate well with the known physiological doses required to elicit functional activity. This new in vitro binding protocol coupled with 36Cl- flux studies should prove to be of value in reassessing the pharmacology of the GABAA receptor complex in a more physiological environment.     

ATP-dependent Cl- uptake by plasma membrane vesicles from the rat brain

Uptake of Cl- by plasma membrane vesicles from the rat brain was stimulated by ATP at 37 degrees C, but not by beta, gamma-methylene ATP or at 0 degrees C. The addition of Triton X-100 or sucrose to the incubation medium diminished the ATP-stimulated Cl- uptake, suggesting that Cl- was transported across the membranes into the intravesicular space. This ATP-stimulated Cl- uptake was not affected by 1 mM ouabain. 1 microM oligomycin, 0.1 mM gamma-aminobutyric acid or 0.1 mM picrotoxin. Thus, non-mitochondrial ATP-driven Cl- transport through a system other than Na, K-ATPase or Cl- channels occurs in neuronal plasma membrane vesicles.     

Desensitization of gamma-aminobutyric acid receptor from rat brain: two distinguishable receptors on the same membrane

Transmembrane chloride flux mediated by gamma-aminobutyric acid (GABA) receptor can be measured with a mammalian brain homogenate preparation containing sealed membrane vesicles. The preparation can be mixed rapidly with solutions of defined composition. Influx of 36Cl- tracer initiated by mixing with GABA was rapidly terminated by mixing with bicuculline methiodide. The decrease in the isotope influx measurement due to prior incubation of the vesicle preparation with GABA, which increased with preincubation time and GABA concentration, was attributed to desensitization of the GABA receptor. By varying the time of preincubation with GABA between 10 ms and 50 s with quench-flow technique, the desensitization rates could be measured over their whole time course independently of the chloride ion flux rate. Most of the receptor activity decreased in a fast phase of desensitization complete in 200 ms (t 1/2 = 32 ms) at saturation with GABA. Remaining activity was desensitized in a few seconds (t 1/2 = 533 ms). These two phases of desensitization were each kinetically first order and were shown to correspond with two distinguishable GABA receptors on the same membrane. The receptor activities could be estimated, and the faster desensitizing receptor was the predominant one, giving on average ca. 80% of the total activity. The half-response concentrations were similar, 150 and 114 microM for the major and minor receptors, respectively. The dependence on GABA concentration indicated that desensitization is mediated by two GABA binding sites. The fast desensitization rate was approximately 20-fold faster than previously reported rates while the slower desensitization rate was slightly faster than previously reported rates.