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.     

Different effects of pentobarbital on two gamma-aminobutyrate receptors from rat brain: channel opening, desensitization, and an additional conformational change

The effect of pentobarbital on the responses of the gamma-aminobutyric acid (GABA) receptor from rat brain was studied in quantitative measurements of GABA-mediated chloride-exchange rates (reflecting channel-opening equilibrium) and receptor desensitization rates by using 36Cl- tracer ion with native membrane vesicles. Pentobarbital effected the two phases of 36Cl- influx in different ways, supporting previous evidence that these are mediated by two different receptors [Cash, D. J., & Subbarao, K. (1987) Biochemistry 26, 7556; Cash, D. J., & Subbarao, K. (1987) Biochemistry 26, 7562]. Both the chloride-exchange rate and the desensitization rate of the faster desensitizing receptor were increased by pentobarbital at concentrations above 20 microM by an allosteric effect shifting the response curve to lower GABA concentrations. A similar enhancement of the responses of the slower desensitizing receptor occurred up to 200 microM pentobarbital. Two pentobarbital effector sites were involved in the allosteric mechanism. Above 500 microM pentobarbital, both the initial chloride-exchange rate and the desensitization rate of the slower desensitizing receptor were decreased. This inhibition, which was immediate, occurred with saturating as well as low GABA concentrations and therefore was not attributed to decreased GABA binding but to inhibitory sites for pentobarbital, different from the allosteric activating sites and the GABA binding sites. The chloride ion exchange activity was seen to recover with time, at concentrations above 1000 microM pentobarbital, in a process with a very steep dependence on pentobarbital concentration. This reactivation was attributed to the conversion of an initial form of the receptor to a final form that was less inhibited by pentobarbital. The similarity of the effects of pentobarbital on the chloride ion exchange with its effects on electrophysiological measurements supports the fact that these different techniques study the same phenomena. Comparisons of the effects of pentobarbital on desensitization and on high-affinity ligand binding measurements suggest that increased GABA binding at equilibrium reflects an increased conversion to the desensitized state.     

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.     

Two desensitization processes of GABA receptor from rat brain. Rapid measurements of chloride ion flux using quench-flow techniques

Two rapid phases of GABA receptor desensitization, which proceeded with a 10-fold difference in rates, were detected in two types of experiment with membrane vesicle preparations from rat cerebral cortex. The time course of GABA-mediated 36Cl- influx progressed in two phases. The 36Cl- influx was decreased, by preincubation with GABA, in two phases. Measurements were made in the time range 10-1000 ms. The major loss of channel opening activity occurred in the faster phase, which was complete in 100 ms with saturating GABA concentrations. The remaining activity decreased in a slower phase in a few seconds with a 10-fold slower rate. The faster phase of desensitization was more than 10-fold faster than previously observed and the slower phase was slightly faster than previously reported measurements with GABA receptor. Both desensitization processes had a similar dependence on GABA concentration with a half response at approximately 100 microM GABA.     

gamma-Aminobutyric acid (GABA) mediated transmembrane chloride flux with membrane vesicles from rat brain measured by quench flow technique: kinetic homogeneity of ion flux and receptor desensitization

Transmembrane chloride flux mediated by the GABAA receptor and the desensitization of the receptor were followed using quench flow technique with 36Cl- and a membrane preparation from rat cerebral cortex. Measurements in short times allowed these two processes to be resolved. In general the ion-flux activity was desensitized in two phases. A fast phase took place in circa 200 ms (100 microM GABA) followed by a slower phase in several seconds. A minority (10%) of the membrane preparations did not display the fast phase. It is desirable to be able to separate these two phases of desensitization to facilitate analysis of the responses of the receptor. A short preincubation with GABA removed the fast phase from a subsequent measurement. In the absence of the fast phase the whole ion-flux equilibration was seen as a single phase. The measurements presented covering a time range of 0.01 seconds to 10 seconds show a single phase of ion flux which can be described by a first order ion influx process and a single first order desensitization process with a half time of circa 1 s (100 microM GABA). The results imply a single kinetically homogeneous population of vesicles containing a single population of GABA receptor (remaining active) with a single phase of desensitization. An understanding of this homogeneity, and how to ensure it, gives a basis for quantitatively testing the effects of drugs on these responses. Ion flux measurements with quench flow technique are a suitable tool for investigation of the mechanism of action of neurotransmitter receptors from 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.     

GABA-stimulated 36Cl- influx into reconstituted vesicles with purified GABAA/benzodiazepine receptor complex

Solubilized and Purified gamma-aminobutyric acid (GABA)A receptors from membrane vesicles of the bovine cerebral cortex were reconstituted into phospholipid vesicles and 36Cl- influx into the vesicles was examined. GABA induced a significant stimulation of the 36Cl- influx into reconstituted vesicles with 1.5% CHAPS/0.15% asolectin solubilized receptor and flunitrazepam further enhanced the GABA-stimulated influx. The purification of GABAA/benzodiazepine receptor complex and Cl- channel solubilized by 1.5% CHAPS/0.15% asolectin from membrane vesicles was achieved by 1012-S affinity column chromatography. The reconstituted vesicles with the purified receptor complex and Cl- channel also exhibited GABA-stimulated 36Cl- influx. This GABA-stimulated influx of 36Cl- was also enhanced by flunitrazepam, while suppressed by bicuculline, a GABAA receptor antagonist. These results strongly suggest that GABAA receptor is directly coupled with Cl- channel, whereas benzodiazepine receptor may be functionally coupled with GABAA receptor and modulates the GABA-stimulated Cl- influx through GABAA receptor. The present results also indicate that the purified GABAA receptor complex is coupled with Cl- channel and possesses functional characteristics as GABAA receptor.     

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.     

Transmembrane flux and receptor desensitization measured with membrane vesicles. Homogeneity of vesicles investigated by computer simulation.

The use of membrane vesicles to make quantitative studies of transmembrane transport and exchange processes involves an assumption of homogeneity of the membrane vesicles. In studies of 86Rb+ exchange mediated by acetylcholine receptor from the electric organ of Electrophorus electricus and of 36Cl- exchange mediated by GABA receptor from rat brain, measurements of ion exchange and receptor desensitization precisely followed first order kinetics in support of this assumption. In other measurements a biphasic decay of receptor activity was seen. To elucidate the molecular properties of receptors from such measurements it is important to appreciate what the requirements of vesicle monodispersity are for meaningful results and what the effect of vesicle heterogeneity would be. The experiments were simulated with single vesicle populations with variable defined size distributions as well as with mixtures of different populations of vesicles. The properties of the receptors and their density in the membrane could be varied. Different receptors could be present on the same or different membrane vesicles. The simulated measurements were not very sensitive to size dispersity. A very broad size distribution of a single vesicle population was necessary to give rise to detectable deviations from first order kinetics or errors in the determined kinetic constants. Errors could become significant with mixtures of different vesicle populations, where the dispersity in initial ion exchange rate constant, proportional to the receptor concentration per internal volume, became large. In this case the apparent rate of receptor desensitization would diverge in opposite directions from the input value when measured by two different methods, suggesting an experimental test for such kinetic heterogeneity. A biphasic decrease of receptor activity could not be attributed to vesicle heterogeneity and must be due to desensitization processes with different rates. Significant errors would not arise from the size dispersity apparent in subpopulations of vesicles seen by imaging techniques in membrane preparations.     

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.     

Prolonged exposure to GABA activates GABA-gated chloride channels in the presence of channel-blocking convulsants.

1. In assays of 36Cl- uptake into mouse brain vesicles, 100 microM GABA markedly increased both the initial rate of 36Cl- uptake and the total amount of chloride taken up over a 120-sec incubation period. Specific GABA-dependent 36Cl- uptake (the difference between total and background uptake) was essentially complete within 15 sec of incubation. 2. Incubation with GABA following preincubation with 10 microM endrin, a polychlorocycloalkane insecticide and established blocker of GABA-gated chloride channels, showed a stimulation of uptake over background levels that was much slower in onset than that observed with GABA alone but nevertheless achieved virtually the same level of stimulation above background levels after 90 sec of incubation with GABA. 3. In electrophysiological assays of GABA receptors expressed in Xenopus oocytes following injection with rat brain mRNA, endrin (20 microM) effectively blocked the transient currents elicited by brief exposure of oocytes to GABA (200 microM). However, prolonged exposure to GABA in the absence of perfusion produced a large, slowly-developing inward current. 4. The actions of several known GABA antagonists were also compared as inhibitors of GABA-dependent 36Cl- uptake into mouse brain vesicles at short (4 sec) and long (120 sec) incubation times using concentrations of inhibitors known to produce approximately 70-90% inhibition of GABA-dependent chloride uptake in 4-sec incubations. Picrotoxinin and TBPS, like endrin, were completely ineffective as inhibitors in 120-sec incubations. In contrast, bicuculline was almost as effective at 120 sec as at 4 sec, and avermectin Bla produced approximately 50% inhibition of the GABA response after 120 sec.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence measurements of anion transport by the GABAA receptor in reconstituted membrane preparations

A fluorescence assay for measuring the functional properties of the GABAA receptor in reconstituted membrane vesicles is described. This assay is based on a method previously described to measure monovalent cation transport mediated by the nicotinic acetylcholine receptor in membranes from Torpedo electric organ [Moore, H.-P.H., & Raftery, M. A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4509-4513]. The GABAA receptor has been solubilized from bovine brain membranes and reconstituted into phospholipid vesicles. Influx of chloride or iodide into the vesicles has been measured in stopped-flow experiments by monitoring the fluorescence quench of an anion-sensitive fluorophore trapped within the vesicles. Muscimol, a GABAA receptor agonist, stimulated a rapid uptake of either chloride or iodide. Stimulation of chloride influx was dependent on the concentration of muscimol, and the midpoint of the dose-response curve occurred at approximately 0.3 microM. Agonist-stimulated uptake was enhanced by diazepam and blocked by desensitization and by the antagonists bicuculline and picrotoxin. These receptor-mediated effects are shown to be qualitatively similar to measurements of 36Cl- and 125I- efflux using synaptoneurosomes prepared from rat cerebral cortex. The advantages of the fluorescence method in terms of its improved time resolution, sensitivity, and suitability for quantitating GABAA receptor function are discussed.     

A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO3- and SITS in rat intestinal epithelium

A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be measured 10-15-times more sensitive by determining the uptake of trace amounts of 125I- driven by a large Cl- gradient (in greater than out) generated by passing the vesicles through an anion-exchange column. Voltage clamping of the vesicle membrane with K+ and valinomycin did not effect the chloride driven 125I- uptake, showing that the 'overshooting' I- uptake was not mediated by an electrical diffusion potential, as might be generated by the Cl- gradient in the presence of a chloride channel. The Cl-/anion exchange was further characterized in brush-border membrane vesicles from both rat ileum and jejunum by studying the inhibitory action of various anions on the Cl- driven I- uptake. NO3-, Cl-, SCN- and formate at 2 mM could inhibit Cl-/I- exchange for more than 80%. The ileal brush-border membrane vesicles displayed a clear heterogeneity with respect to the inhibitory action of SO2-(4), SITS and HCO-3 on Cl-/I- exchange. Approximately 30% of the Cl-/I- exchange was insensitive to SO2-(4) and showed a relatively low sensitivity to SITS (IC50 = 1 mM) but could be inhibited for 80% by 2 mM HCO-3. Presumably this component represents Cl-/OH- or Cl-/HCO-3 exchange. The residual 70% showed a high sensitivity to SO2-(4) (IC50 = 0.5 mM) and SITS (IC50 = 2.5 microM) but was less sensitive to HCO-3. This part of the exchange activity showed inhibition characteristics very similar to the Cl-/I- exchange in the jejunal vesicles. The latter process was also inhibited for 80% by 2 mM oxalate. As discussed in this paper both exchangers may be involved in the electroneutral transport of NaCl across the apical membrane of the small intestinal villus cell.     

Responses of γ-aminobutyrate receptor from rat brain: Similarity of different preparation methods; muscimol induced desensitization and chloride exchange

Summary Chloride-36 exchange into three different membrane vesicle preparations from rat brain homogenate was followed. The different preparations all contained the same sealed vesicular components characterized by their rates of chloride exchange. The GABA-mediated³⁶Cl^– exchange in all the preparations occurred in two phases shown to be mediated by two distinguishable receptors present in the activity ratio of 51 as previously described (Cash, D.J., Subbarao, K. 1987.Biochemistry 26:7556, 7562). Reported differences do not result from differences in the membrane preparations used or from the use of a GABA-mimetic instead of GABA, but from experimental differences. The preparations compared were made with mild or vigorous homogenization and with different extents of purification from solutes or membrane components: (i) a synaptoneurosome preparation, (ii) a Ficoll gradient preparation, and (iii) a washed P₂ preparation. In each preparation the same four populations of membrane vesicles were characterized by their³⁶Cl^– influx rates: (i) a major population (40–50%) (t _1/2=1.4 min), (ii) a slower exchanging major population (40–55%) (t _1/2=24 min), (iii) a minor population (5–12%) containing active GABA receptor and having the GABA-independent permeability of the slower exchanging population, and (iv) a very small exchange (2%) (t _1/20.2 sec). The GABA-independent³⁶Cl^– exchange processes were kinetically first order. The relative quantities of the different vesicle populations varied slightly with the preparation and purification technique. The identity of these components, observed in the different preparations, was attributed to the vesicle formation being dependent on the morphology and properties of the membrane rather than the preparation method. The soluble brain extract was GABA-mimetic with the two observed receptors, causing channel opening and desensitization. But little washing of the membrane was required to observe the function of both receptors. Muscimol was GABA-mimetic with both receptors. With muscimol, channel opening occurred at 2.6-fold lower concentrations while desensitization was unaltered relative to GABA. This is additional evidence that these responses are mediated by different pairs of binding sites. The dependence of desensitization rate on muscimol concentration indicated that there are two binding sites mediating desensitization, as described with GABA.     

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.     

How fast does the gamma-aminobutyric acid receptor channel open? Kinetic investigations in the microsecond time region using a laser-pulse photolysis technique.

The gamma-aminobuytric acid(A) (GABA(A)) receptor is a membrane-bound protein that mediates signal transmission between neurons through formation of chloride ion channels. GABA is the activating ligand, which upon binding to the receptor triggers channel opening in the microsecond time domain and reversible desensitization of the receptor in the millisecond time region. We have investigated the channel-opening mechanism for this receptor in rat hippocampal neurons before the protein desensitizes by using a rapid flow method (cell-flow) with a 10 ms time resolution and a laser-pulse photolysis technique with a approximately 30 micros time resolution to determine the rate and equilibrium constants for channel opening and closing. Two different forms of the receptor, namely, a rapidly and a slowly desensitizing form, exist in the rat hippocampal cells and are characterized by their different rates for desensitization. At 250 microM GABA the rate constant for desensitization was 2.3 +/- 0.4 s(-)(1) for the rapidly desensitizing form and 0.4 +/- 0.1 s(-)(1) for the slowly desensitizing form. The dissociation constant of GABA from the site controlling channel opening was 100 +/- 40 microM for the rapidly desensitizing form and 120 +/- 60 microM for the slowly desensitizing form. The rate constants for channel closing did not differ significantly for the two forms, 85 +/- 20 s(-)(1) for the rapidly desensitizing and 100 +/- 60 s(-)(1) for the slowly desensitizing form. However, the channel-opening rate constant differed by a factor of 3, 1840 +/- 160 s(-)(1) for the rapidly desensitizing and 6700 +/- 330 s(-)(1) for the slowly desensitizing form. This difference in the rate constant for channel opening for the two forms, determined by the laser-pulse photolysis technique, is reflected as a shift in the channel-opening equilibrium constant, which is 7 +/- 5 and 20 +/- 15 for the rapidly and slowly desensitizing forms respectively, determined by the cell-flow method. These constants, together with the concentration of GABA and the concentration of receptor sites in the membrane, determine the number of channels that open as a function of GABA concentration, and the rate at which they open and close. These constants play an important role in determining the rate of the transmembrane ion flux and, therefore, the receptor-controlled changes in transmembrane voltage that trigger signal transmission.     

Trans-potassium effects on the chloride/proton symporter activity of guinea-pig ileal brush-border membrane vesicles.

To investigate the inhibitory effect of trans potassium on the Cl-/H+ symporter activity of brush-border membrane vesicles from guinea pig ileum, we measured both 36Cl uptake and, by the pyranine fluorescence method, proton fluxes, in the presence of appropriate H+ and K+ gradients. In the absence of valinomycin, a time-dependent inhibitory effect of chloride uptake by trans K+ was demonstrated. This inhibition was independent of the presence or absence of any K+ gradient. Electrical effects cannot be involved to explain these inhibitions because the intrinsic permeability of these vesicles to Cl- and K+ is negligibly small. Rather, our results show that, in the absence of valinomycin, the inhibitory effect of intravesicular K+ involves an acceleration of the rate of dissipation of the proton gradient through an electroneutral exchange of trans K+ for cis H+, catalyzed by the K+/H+ antiporter also present in these membranes. Valinomycin can further accelerate the rate of pH gradient dissipation by facilitating an electrically-coupled exchange between K+ and H+. To evaluate the apparent rate of pH-dissipating, downhill proton influx, we measured chloride uptake by vesicles preincubated in the presence of alkaline-inside pH gradients (pHout/pHin = 5.0/7.5), charged or not with K+. In the absence of intravesicular K+, proton influx exhibited monoexponential kinetics with a time constant k = 11 s-1. Presence of 100 mM K+ within the vesicles significantly increased the rate of pH gradient dissipation which, furthermore, became bi-exponential and revealed the appearance of an additional, faster proton influx component with k = 71 s-1. This new component we interpret as representing the sum of the electroneutral and the electrically-coupled exchange of trans K+ for cis H+, mentioned above. Finally, by using the pH-sensitive fluorophore, pyranine, we demonstrate that, independent of the absence or presence of a pH gradient, either vesicle acidification or alkalinisation can be generated by adding, respectively, Cl- or K+ to the extravesicular medium. Such results confirm the independent existence of both Cl-/H+ symporter and K+/H+ antiporter activities in our vesicle preparations, the relative activity of the former being larger under the conditions of the present experiments. The possible interplay of these two proton-transfer mechanisms in the regulation of the intracellular pH is discussed.     

Factors affecting actions of ethanol on GABA-activated chloride channels.

Effects of ethanol in vitro on membrane vesicles (microsacs prepared from mouse cerebral cortex) were evaluated by monitoring 36Cl- influx. Different assay parameters were tested to determine increased or decreased action of ethanol on GABA-activated chloride channels. The ability of 30 mM ethanol to augment 36Cl- flux was seen at 0 degrees C, in the absence of GABA ("direct" action of ethanol), and at 34 degrees C in the presence of GABA, using two different assay procedures. Picrotoxin blocked the direct effects of ethanol (at 0 degrees C) suggesting GABAa involvement. Endogenous GABA in the medium surrounding the microsacs was assayed at different temperatures both in the presence and absence of GABA and ethanol. The direct effect of ethanol did not appear to involve the action of endogenous GABA. In addition to temperature effects on the assay, time of membrane storage also influenced ethanol action. Microsacs stored on ice for 2 hours or more lost their ability to respond to ethanol but not to GABA, pentobarbital or flunitrazepam. When these drugs were tested on membranes from mice that had been sacrificed by cervical dislocation as opposed to decapitation, ethanol did not augment GABA-stimulated chloride flux. The method of sacrifice did not influence the response to GABA, pentobarbital or flunitrazepam.     

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.