Measurement of chloride movement in neuronal preparations.

In this unit, protocols are described for biochemical and optical techniques that have been used by investigators to measure ligand-gated chloride movement in vesicular structures called synaptoneurosomes (also referred to as microsacs), in cultured neurons, and in the acute brain slice. These techniques can be applied to other ions as well. The measurement of uptake and efflux of radioisotopic chloride in synaptoneurosomes is used to study the responses of gamma-aminobutyric acid (GABA) receptors, which are coupled to chloride channels. Similar chloride flux assays for primary neuronal cultures are also presented. Alternatively, the efflux of chloride from synaptoneurosomes and primary neuronal cultures can be studied using fluorescent dyes and photometry. Finally, the measurement of chloride uptake can be studied in individual neurons in brain slices using fluorescent dyes and optical imaging by nonconfocal and confocal microscopy. Several support protocols are provided as well, outlining the preparation of synaptoneurosomes from specific brain regions, and the preparation, loading, and calibration of chloride-sensitive fluorescent dyes.

A single glycine residue at the entrance to the first membrane-spanning domain of the gamma-aminobutyric acid type A receptor beta(2) subunit affects allosteric sensitivity to GABA and anesthetics.

Site-directed mutagenesis of the gamma-aminobutyric acid type A (GABA(A)) receptor beta(2) subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous rho(1) residue phenylalanine alters the modulating effects of four different i.v. anesthetics: pentobarbital, alphaxalone, etomidate, and propofol. Using the baculovirus expression system in Spodoptera frugiperda 9 cells, anesthetic-induced enhancement of [(3)H]muscimol and [(3)H]flunitrazepam binding in receptors containing the beta(2)(G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA(A) receptors containing the alpha(1)(G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. Conversely, the homologous point mutation in rho(1) receptors (F261G) changed the i.v. anesthetic-insensitive receptor to confer anesthetic modulation of [(3)H]muscimol binding. Consistent with the binding, functional analysis of pentobarbital-enhanced GABA currents recorded with whole-cell patch clamp demonstrated the beta(2)(G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly, propofol-enhanced GABA currents were potentiated less in alpha(1)beta(2)(G219F)gamma(2) receptors than in alpha(1)beta(2)gamma(2) receptors. Although ligand binding displayed comparable K(D) values for muscimol among wild-type, alpha(1)beta(2)gamma(2), and mutant receptors, patch-clamp recordings showed that alpha(1)beta(2)(G219F)gamma(2) receptors had a significantly more potent response to GABA than did alpha(1)beta(2)gamma(2) or alpha(1)(G223F)beta(2)gamma(2). The alpha(1)beta(2)(G219F)gamma(2) receptors also were more sensitive to direct channel activation by pentobarbital and propofol in the absence of GABA. These results suggest that the first transmembrane glycine residue on the beta(2) subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.

Ethanol specifically inhibits NMDA receptors with affinity for ifenprodil in the low micromolar range in cultured cerebellar granule cells.

The effect of ethanol on the intracellular Ca2+ concentration response to NMDA in rat cerebellar granule cells grown in low or high KCl concentrations has been studied using image analysis. The cells grown in low KCl displayed high sensitivity for glycine. The subtype-selective antagonist ifenprodil inhibited the response with high (in the low micromolar range) and low (in the high micromolar range) potency. Ethanol affected the high-potency component in these cultures. In cells grown in high KCl the glycine sensitivity was lower, and a low potency for ifenprodil (high micromolar) dominated. These cells were not significantly sensitive to ethanol. The results indicate that the component displaying potency for ifenprodil in the low micromolar range with properties of the NR2B subunit is the target for ethanol action on the NMDA receptor.

Glycine and GABAA receptor-mediated chloride fluxes in synaptoneurosomes from different parts of the rat brain.

Strychnine-sensitive, inhibitory glycine receptors have not until lately been considered to play a significant role in neurotransmission in mammalian forebrain regions. In order to investigate the role of glycine as a neurotransmitter in brain we have measured glycine induced chloride fluxes in different adult rat forebrain areas using synaptoneurosomes and a chloride-sensitive fluorescent indicator. The results have been compared to those obtained with GABA. The synaptoneurosomes from every brain area investigated responded to both glycine and GABA with chloride fluxes in a picrotoxin sensitive manner. The effect of glycine was inhibited by strychnine, which had no effect on the GABA-induced Cl-flux. Bicuculline inhibited the effect of GABA, but had no effect on the glycine-induced Cl-flux. Addition of GABA did not affect the response to glycine and vice versa. The endogenous content of glycine and GABA in the synaptoneurosome preparations was about the same and synaptoneurosomes from every brain area investigated released both glycine and GABA upon depolarisation with KCl. The depolarisation induced release of both GABA and glycine was partly Ca(2+)-dependent and partly Ca(2+)-independent. These results indicate that glycine can induce inhibitory Cl- fluxes distinct from GABA induced fluxes in every investigated brain area and that glycine can be released upon depolarisation.

Determination of the intracellular free chloride concentration in rat brain synaptoneurosomes using a chloride-sensitive fluorescent indicator.

The chloride-sensitive fluorescent indicator MQAE (N-(6-methoxyquinolyl) acetoacetyl ester) has been used for determination of the intracellular free chloride concentration in rat brain synaptoneurosomes. Loading of the synaptoneurosomes with MQAE occurs by transmembrane diffusion. Calibration of the intracellular MQAE was done by determining the correlation between fluorescence intensity and intrasynaptoneurosomal Cl- concentration in the presence of the Cl-/OH- exchanger tributyltin and the K+/H+ exchanger nigericin, starting from zero Cl- concentration. The total quenchable signal of MQAE was determined by adding KSCN in the presence of the K+ ionophore valinomycin. The correlation between the reciprocal of the fluorescence intensity and the chloride concentration was linear at least up to 50 mM Cl-. The fluorescence of freshly prepared synaptoneurosomes was then measured and the obtained value was plotted into the calibration curve and the corresponding Cl- was read. The mean intrasynaptoneurosomal chloride concentration was 14 +/- 4 mM. We also quantitatively estimated the Cl- flux after addition of the barbiturate, pentobarbitone that opens GABAA receptor-Cl(-)-channels, to the synaptoneurosomes. An addition of 1 mM pentobarbitone corresponded to an approx. 0.59 mM change in the intrasynaptoneurosomal free chloride concentration. The results show that the chloride-sensitive fluorescent indicator MQAE is a useful tool when determining intracellular chloride activity, and in quantitative determination of chloride fluxes in living cells and subcellular preparations.

Ethanol-induced Cl- flux in rat cerebellar granule cells as measured by a fluorescent probe.

Cl- fluxes through the GABAA receptor gated ion channels in cultured rat cerebellar granule cells were measured using the chloride-sensitive fluorescent probe SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium) incorporated into the cells. The fluorescence of SPQ is quenched by Cl- ions. The cells were bathed in a low Cl- medium so that the Cl- gradient was directed outward. Ethanol increased the SPQ fluorescence indicating a decrease in intracellular Cl- due to Cl- efflux. Picrotoxin inhibited the effect at low concentrations of ethanol (less than 50 mM) in a concentration dependent manner. The effects of ethanol were potentiated at low concentrations (less than 10 microM) of gamma-aminobutyric acid (GABA), but inhibited at higher concentrations (0.3-2.0 mM). The results support the hypothesis that ethanol may act via the GABAA receptor gated ion channel. The results also suggest that SPQ is a suitable probe for measuring GABAA receptor-coupled Cl- fluxes through the GABAA receptor-gated channels in living cells.

Effect of ethanol on gamma-aminobutyric acid and glycine receptor-coupled Cl- fluxes in rat brain synaptoneurosomes.

Chloride fluxes in synaptoneurosomes in response to additions of gamma-aminobutyric acid, glycine, and ethanol were measured using a chloride-sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). The Cl- gradient was directed outward by bathing cells in a medium low in Cl- concentration. The synaptoneurosomes responded to both gamma-aminobutyric acid and glycine by outflow of Cl- ions, as judged from an increase in SPQ fluorescence. These effects were inhibited by picrotoxin and strychnine, respectively. Ethanol also produced an outflow of Cl- ions from the synaptoneurosomes. Both picrotoxin and strychnine inhibited this effect. When the antagonists were used together, the inhibiting effect was additive. These results indicate that ethanol affects both gamma-aminobutyric acid and glycine receptor-linked chloride fluxes in the rat brain.

Determination of GABA receptor-linked Cl- fluxes in rat cerebellar granule cells using a fluorescent probe SPQ.

Gamma-Aminobutyric acid (GABA)-induced Cl- fluxes in cultured rat cerebellar granule cells were measured using the chloride-sensitive fluorescent probe SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium) incorporated into the cells. The fluorescence of SPQ is quenched by Cl-ions. GABA and pentobarbitone increased the fluorescence of the probe when the Cl- gradient was directed outward by bathing cells, grown in the presence of GABA, in a low Cl- medium. Picrotoxin and bicuculline inhibited the response to GABA. The results suggest that SPQ is a suitable probe for measuring GABA-induced Cl- fluxes in living cells.

Is calmodulin inhibition involved in shape transformations induced by amphiphiles in erythrocytes?

Potent antihaemolytic and shape transforming amphiphilic compounds were studied for their ability to inhibit calmodulin-activated phosphodiesterase activity. Some echinocytogenic and stomatocytogenic amphiphiles were potent calmodulin inhibitors. The most potent echinocytogenic and stomatocytogenic amphiphiles, however, had no or only weak inhibitory effect. Our results show that there is no causal relationship between the ability of amphiphiles to induce antihaemolysis or shape transformations in erythrocytes and their ability to inhibit calmodulin-activated phosphodiesterase activity, and it is suggested that calmodulin is not involved in shape transformations induced by amphiphiles.

On the time-dependence of amphiphile-induced haemolysis.

The time-course of haemolysis induced by alkyltrimethylammonium bromides, alkyl sulphates, octaethyleneglycol alkyl ethers, and ZWITTERGENT detergents (C10-C16) was studied. A considerable lag in the development of haemolysis was observed for the C10 and C12 derivatives. The lag exceeded 30 min at low or moderate concentrations of the surfactants. Most of the C14 and C16 derivatives showed no lag but the time-course of the haemolytic reaction of these derivatives indicated biphasic kinetics of haemolysis. With those surfactants showing a lag the critical micelle concentration (cmc) of the surfactants was above the concentrations used in the experiments, whereas cmc in the case of those surfactants showing no lag was close to or slightly below. It is concluded that the differences in the time-course of the haemolytic reaction shown by different derivatives are not connected with the occurrence of micelles in the bulk solution, but that the differences in time-course of the haemolytic reaction reflect differences in the rate of those changes in the membrane molecular organization which precede haemolysis.

Permeability alterations and antihaemolysis induced by amphiphiles in human erythrocytes.

In an attempt to define the parameters in amphiphilic molecules important for their interaction with the erythrocyte membrane, the effects of cationic, anionic, zwitterionic and nonionic amphiphilic agents (C10-C16) on osmotic fragility and transport of potassium and phosphate in human erythrocytes were studied. All the amphiphiles protected the erythrocytes against hypotonic haemolysis. Half-maximum protection occurred at a concentration which was about 15% of that inducing 50% haemolysis. The concentrations of amphiphiles required to induce protection or haemolysis were related to the length of the alkyl chain in a way indicating that a membrane/aqueous phase partition is the mechanism whereby the amphiphile monomers intercalate into the membrane. At antihaemolytic concentrations all the amphiphiles increased potassium efflux and passive potassium influx. The increase in the fluxes was about the same in both directions through the membrane and there were no clear differences in the effects of the different amphiphilic derivatives at equi-protecting concentrations. Active potassium influx was decreased by cationic, zwitterionic and non-ionic amphiphiles. The ability of the amphiphiles to inhibit the influx was not related to the length of the alkyl chain. Anionic amphiphiles had no or only a weak stimulatory effect on the influx. Phosphate efflux was reduced by all the amphiphiles. The inhibitory potency of the different amphiphiles decreased in the following order; anionic greater than zwitterionic, non-ionic greater than cationic. Short-chained amphiphiles were more potent inhibitors than long-chained. The possible participation of non-bilayer phases (mixed inverted micelles) in the intercalation of amphiphiles into the membrane is discussed.