Derek J. Cash

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. (i) The time course of GABA‐mediated 36Cl− influx progressed in two phases. (ii) 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 ∼ 100 μM GABA.

Transmembrane 36CI− Flux Measurements and Desensitization of the γ‐Aminobutyric AcidA Receptor

Abstract: Some data on the concentration range of response and the concentration for half‐response (EC50) of γ‐aminobutyric acid (GABA) for the GABAA receptor are reviewed and compared. An analysis of the 36CI− flux assay demonstrates that both the EC50 and the slope of a Hill plot depend on the ion influx or efflux assay time. The effects of depletion of the 36CI− concentration gradient during the assay and of receptor desensitization on the result for a range of assay times are considered. The EC50 can be decreased by orders of magnitude by increasing the assay time. The EC50 measured in a finite time is less than the half‐response concentration for the response(s) of the receptor. The extent of this difference depends on the receptor concentration per internal volume. The maximal decrease of EC50 depends on the rate of receptor desensitization. The computer simulations showed that a GABAA receptor with a half‐response concentration of 100 μM GABA can give 36CI− flux measurements with an EC50 value 100‐fold lower.

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 μM, sufficient to give maximum enhancement of chloride exchange rate) but sigmoid in its absence. Enhancement of channel opening (10‐fold at 0.3 μM GABA) decreased with increasing GABA concentration. The maximal response, above 1,000 μM GABA, was unaltered. The half‐response concentration was reduced from 80 μM to 50 μM. 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 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.

Modulation of GABA flux across rat brain membranes resolved by a rapid quenched incubation technique

The progress and inhibition of [3H]GABA influx in native plasma membrane vesicles from the rat cerebral cortex was studied on a subsecond to minute time scale under different conditions by applying a rapid quenched incubation technique. In the absence of Ca2+ ([Ca2+]free = 10(-8) M), the progress of influx followed by the addition of 10 nM [3H]GABA to the membrane vesicle suspension with time (500 ms to 15 min) can be described by a first-order rate equation giving an overall rate constant, k, of 3.93 +/- 0.48 x 10(-3) s-1 and equilibrium influx value, INFe, of 8.84 +/- 0.41 pmol [3H]GABA/mg protein. In the presence of Ca2+ ([Ca2+]free = 2.4 x 10(-3) M) a significant increase in the INFe value was observed (k = 4.64 +/- 0.41 x 10(-3) s-1 and INFe = 13.9 +/- 0.40 pmol [3H]GABA/mg protein). Multiplicity of GABA transporters was indicated in the time-dependent inhibition of [3H]GABA influx by different uptake blockers. In the absence of Ca2+, depolarization (75 mM KCl) inhibited the influx of [3H]GABA into the vesicles by approximately 70% and initiated the efflux from vesicles loaded with [3H]GABA. Different uptake blockers inhibited the Ca(2+)-independent translocation of [3H]GABA in both directions with similar specificities.

γ-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.

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

SummaryChloride-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 5∶1 as previously described (Cash, D.J., Subbarao, K. 1987.Biochemistry26:7556, 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%) (t1/2=1.4 min), (ii) a slower exchanging major population (40–55%) (t1/2=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%) (t1/2∼0.2 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.

Filter assay technique and quench-flow experiments: examples of receptor-mediated transmembrane ion-exchange measured with membrane vesicles.

Modifications to a quench-flow apparatus are described which allow a rapid, in-line filter assay with immediate washing, in conditions to give minimum background. A design for an effluent spout is presented, which decelerates the liquid by a large factor, prevents splashes, limits the area of the filter exposed to the sample and allows an immediate wash over a larger area. A design for a filter assay funnel for general use is also presented. These devices feature minimal contact of the funnels with the filter disc. Examples are given in which in-line filtration was used to follow transmembrane ion flux in membrane vesicle preparations. In measurements of transmembrane flux with membrane vesicles and radioisotope the filter assay background can be resolved into three components. These are, (1) the uptake of radioactivity by the filter, (2) the radioactivity inside the vesicles not taking part in the specific measurement and (3) the occlusion of radioactivity in aggregated membrane particles on the filter. These different components depend on the conditions in different ways. Techniques for minimizing the background in filter assays are discussed. The importance of rapid filtration and immediate washing is demonstrated. The examples given illustrate that the function of the acetylcholine receptor from E. electricus is not affected by diisopropylfluorophosphate in the conditions used, and that added GABA is not removed from solution in a brain membrane preparation by the GABA uptake mechanisms in the short times of the experiments.

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.

Inhibition of γ-aminobutyric acid uptake by bicuculline analogues

Abstract Enantiomers of norbicuculline, (+)[1S,9R] and (−)[1R,9S]erythro-1-[1′-(4′,5′-methylenedioxyphthalidyl)]-6,7-methylenedioxy-1,2,3,4-tetrahydroisoquinoline and of the N-methyl derivatives {(+)[1S,9R] and (−)[1R,9S]bicuculline} were found to inhibit the progress of the γ-aminobutyric acid transporter-mediated uptake of 40 μM [ 14 C ]γ-aminobutyric acid into native plasma membrane vesicles from the rat cerebral cortex at 30°C. The values for the dissociation constants of the reversible inhibition, relative to (+)[1S,9R]bicuculline, in order of increasing inhibition, were: (−)[1R,9S]bicuculline, 3.3; (+)[1S,9R]-bicuculline, 1.0; (−)[1R,9S]norbicuculline, 0.4≈(+)[1S,9R]norbicuculline; guvacine, 0.02. The norbicucullines have higher potencies than (+)[1S,9R]bicuculline for the γ-aminobutyric acid transporter, in contrast to the relative potencies of these inhibitors for the inhibition of function and γ-aminobutyric acid binding of the γ-aminobutyric acid type A receptor.

Use of 82Br− radiotracer to study transmembrane halide flux: The effect of a tranquilizing drug, chlordiazepoxide on channel opening of a GABAA receptor

We used the short-lived radionuclide, 82Br− to follow γ-aminobutyrate (GABA) receptor-mediated halide exchange into membrane vesicles from rat cerebral cortex in millisecond and second time regions using quench-flow technique. The radioisotope was prepared by neutron capture [81Br−(n,γ)82Br−] on irradiation of a natural isotope of bromine, 81Br− in a neutron flux. 82Br− decays by β-emission with secondary γ-emission. Possible advantages of 82Br− over 36Cl− in anion tracer measurements include, (a) a short lifetime (t1/2 = 35.3 hr), which alleviates contamination and disposal problems, (b) high counting efficiency (1.54) due to the secondary radiation, (c) measurement with a γ-counter as well as a β-counter, (d) a simple preparation not requiring subsequent purification steps giving a specific activity depending on the irradiation time. With 6 hr irradiation time the specific activity was sufficient to make measurements with <1 mm Br−, which is less than the bromide concentration known to affect the properties of GABAA receptor. The radiotracers, 82Br− and 36Cl− could be compared with the same solution composition. In conditions where a direct effect of binding of halide to receptor does not contribute to a difference in measured ion-flux, 82Br− was translocated only marginally faster than 36Cl−. The effect of chlordiazepoxide (CDPX) (2–250 μm) on the progress of GABA (10 μm)-mediated 82Br− uptake was measured in a time range of 200 msec to 20 sec using quench-flow technique. The two phases of anion exchange previously reported in this experimental model with GABA alone were observed. The rate of 82Br− exchange was increased 2.3-fold at 30–60 μm CDPX and was not further increased with increasing [CDPX]. The rate of halide exchange is a measure of open channel concentration. The isotope exchange rate constant, J, in a membrane vesicle preparation, is a measure of the membrane permeability per internal volume/surface area, J = PmA/V. Receptor desensitization rate was also increased by CDPX, but unlike the isotope exchange rate, it continued to increase up to at least 250 μm CDPX.