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.     

Chloride channels as tools for developing selective insecticides

Ligand-gated chloride channels underlie inhibition in excitable membranes and are proven target sites for insecticides. The gamma-aminobutyric acid (GABA(1)) receptor/chloride ionophore complex is the primary site of action for a number of currently used insecticides, such as lindane, endosulfan, and fipronil. These compounds act as antagonists by stabilizing nonconducting conformations of the chloride channel. Blockage of the GABA-gated chloride channel reduces neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions, and death. We recently investigated the mode of action of the silphinenes, plant-derived natural compounds that structurally resemble picrotoxinin. These materials antagonize the action of GABA on insect neurons and block GABA-mediated chloride uptake into mouse brain synaptoneurosomes in a noncompetitive manner. In mammals, avermectins have a blocking action on the GABA-gated chloride channel consistent with a coarse tremor, whereas at longer times and higher concentrations, activation of the channel suppresses neuronal activity. Invertebrates display ataxia, paralysis, and death as the predominant signs of poisoning, with a glutamate-gated chloride channel playing a major role. Additional target sites for the avermectins or other chloride channel-directed compounds might include receptors gated by histamine, serotonin, or acetylcholine.The voltage-sensitive chloride channels form another large gene family of chloride channels. Voltage-dependent chloride channels are involved in a number of physiological processes including: maintenance of electrical excitability, chloride ion secretion and resorption, intravesicular acidification, and cell volume regulation. A subset of these channels is affected by convulsants and insecticides in mammals, although the role they play in acute lethality in insects is unclear. Given the wide range of functions that they mediate, these channels are also potential targets for insecticide development.     

Actions of avermectin B1a on the γ-aminobutyric AcidA receptor and chloride channels in rat brain

The interaction of avermectin B_1a (AVM) with the γ-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [³H]flunitrazepam and inhibited the binding of both [³H]muscimol and [³⁵S]t-butylbicyclo-phosphorothionate to the GABA_A receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of ³⁶chloride (Cl) flux were studied. The ³⁶Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, ³⁶Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced ³⁶Cl influx into these microsacs in a dose–dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced ³⁶Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABA_A-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage–dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

Two types of chloride transporters are required for GABA(A) receptor-mediated inhibition in C. elegans

Chloride influx through GABA-gated Cl(-) channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl(-) gradient established in part by K(+)-Cl(-) cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na(+)-driven Cl(-)-HCO(3)(-) exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K(+)-Cl(-) cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl(-) extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl(-) gradient such that Cl(-) flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl(-) gradient. Our results show that Na(+)-driven Cl(-)-HCO(3)(-) exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.     

Chloride and potassium movements from frog's sartorius muscle in the presence of aromatic anions

Summary The efflux of³⁶Cl and⁴²K from frog's sartorius muscles equilibrated in Ringer's fluid with added KCl were measured in the absence and presence of salicylate, benzoate, and acetylsalicylate. The transmembrane potential and resistance were also measured in sartorii under similar conditions. Although the rate coefficient for loss of⁴²K remained reasonably constant over extended experimental periods for untreated muscles, the rate coefficient for loss of³⁶Cl fluctuated in many muscles giving rise to minima and maxima. The aromatic anions mentioned increased the efflux of chloride while having no detectable effect on the potassium efflux. The aromatic anion-stimulated chloride efflux was insensitive to alterations of external pH and was markedly reduced when nitrate replaced external chloride. No detectable changes in transmembrane potential or resistance were produced by salicylate, the most extensively studied aromatic anion. The results suggest that salicylate and the other aromatic anions stimulate an exchange diffusion mechanism for chloride.     

Ion transport and permeability in the mouse egg

Sodium, potassium, and chloride unidirectional fluxes have been studied in the mature mouse egg. Their relationship to cell membrane potential and conductance has been investigated. Unidirectional Na efflux is composed of a ouabain sensitive component, presumably representing an active Na efflux, an external Na-dependent component and a diffusional component. The data indicate that the external Na-dependent component represents a Na:Na exchange mechanism. There also exists an ouabain-sensitive component of K influx. The stoichiometry of the ouabain-sensitive fluxes is approx. 2.7:1 (Na to K). From the diffusional components of Na and K flux, the membrane permeability to these cations has been estimated. P_Na and P_K are 1.2 × 10⁻⁷ cm sec⁻¹ and 0.8 × 10⁻⁷ cm sec⁻¹ respectively. These permeabilities, in conjunction with the internal exchangeable fractions of Na and K and the external concentrations, predict an egg membrane potential of −11 mV (inside negative). Microelectrode measurements yield an egg membrane potential of −14 ± 0.4 mV, indicating that the cell membrane potential is predominantly a result of the Na and K permeabilities and distributions. Internal exchangeable Cl is 67 ± 3 mM in standard medium, as determined from ³⁶Cl distribution. The chloride equilibrium potential is therefore −15 mV, which is not significantly different from the egg membrane potential. This suggests that Cl distributes passively across the egg membrane, reflecting the egg membrane potential. Hyperpolarization of the egg membrane potential to −27 ± 1.5 mV by reduction of external Na results in an exchangeable internal Cl of 49 ± 8 mM. This yields a Cl equilibrium potential of −24 mV, indicating that the Cl distribution shifts in the predicted manner upon a change in cell membrane potential. Tracer flux data indicate that Cl conductance comprises the bulk of the total membrane conductance with Na and K sharing the remainder in approximately equal amounts.     

Effect of furosemide on GABA<Subscript>A</Subscript>-induced <Superscript>36</Superscript>Cl transport and Cl--ATPase activity in synaptic membranes of carp brain (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

We studied the effect of furosemide on GABA_A-induced ³⁶Cl transport and GABA_A-induced Cl^--ATPase activity in synaptic membranes of fish brain. At physiological pH 7.4, GABA (0.1–100 µM) stimulated ³⁶Cl influx in synaptoneurosomes and Cl^--ATPase activity in synaptic membranes. Furosemide (0.1–0.5 mM) removed the activating effect of the mediator on chloride transport and enzyme activity (I₅₀ equaled 0.16 and 0.12 mM, respectively). In the absence of the mediator, picrotoxin (50 µM) activated the basal ³⁶Cl influx in synaptoneurosomes and the basal Mg²⁺-ATPase activity of synaptic membranes. Furosemide (1 mM) removed the activating effect of picrotoxin on both biochemical processes. The obtained data demonstrated similar sensitivities of GABA_A-induced transport of ³⁶Cl in synaptoneurosomes and of GABA_A-induced Cl^--ATPase activity in the synaptic membranes to furosemide and indicated the involvement of the ATPase in GABA_A-induced processes. The soluble ATPase, recovered by sodium deoxycholate solubilization of the membranes, remained sensitive to GABA_A-ergic ligands, which suggested proximity of their binding sites with ATP hydrolysis sites in the protein molecule and their structural coupling.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 18–22.Original Russian Text Copyright © 2005 by Menzikov, Menzikova.     

Comparative aspects of nitrobenzylthioinosine and dipyridamole inhibition of adenosine accumulation in rat and guinea pig synaptoneurosomes

Dipyridamole (DPR) and nitrobenzylthioinosine (NBI) inhibition of adenosine accumulation in synaptoneurosomes derived from rat cerebral cortex, rat cerebellum, guinea pig cerebral cortex and guinea pig cerebellum was investigated. The inhibition of adenosine accumulation by NBI was observed to be distinctly biphasic in both guinea pig and rat synaptoneurosomes. Such biphasic inhibition consisted of a nM potency component to inhibition, accounting for 20–30% of the maximum inhibition, and a μM potency component, accounting for the remaining 70–80% maximum inhibition. Such an inhibitory profile contrasts sharply with that of DPR which appears monophasic, with a mean IC₅₀ of between 10⁻⁷ M and 10⁻⁶ M, in all rat and guinea pig synaptoneurosomes preparations studied.Further differences between the potency of NBI and DPR in inhibiting [³H]adenosine accumulation were also noted. DPR was more potent in inhibiting [³H]adenosine accumulation in guinea pig cerebellar synaptoneurosomes than in cerebral cortex synaptoneurosomes. In rat synaptoneurosomes, the reverse selectivity was observed. DPR was also 2–6 fold (depending on brain region of comparison) more potent in inhibiting adenosine accumulation in guinea pig synaptoneurosomes than in inhibiting such accumulation in rat synaptoneurosomes. In contrast, NBI was approximately equipotent in inhibiting adenosine accumulation in rat and guinea pig synaptoneurosomes. Additional binding studies using [³H]NBI are also reported. The data presented are entirely consistent with the hypotheses that (1) NBI and DPR bind to functionally relevant sites and (2) there are different populations of nucleoside transporters in mammalian brain.     

Effects of divalent ions on vesicle-vesicle fusion studied by a new luminescence assay for fusion

Summary A new assay has been developed for vesicle-vesicle fusion based upon the mixing of intravesicular contents of two sets of vesicles. Purified firefly luciferase and MgCl₂ were incorporated into one set of vesicles (LV) and ATP into the other (AV). Vesicles were prepared from soybean phospholipids. The luminescence that resulted from hydrolysis of ATP by luciferase was measured to determine the extent of mixing of the intravesicular contents. In the absence of divalent ions, incubation of a mixture of LV and AV did not produce luminescence. However, if Ca⁺⁺ or other divalent ions were present at millimolar concentrations, luminescence occurred. The luminescence did not result from extravesicular reaction of vesicle contents that had leaked into the medium. Instead, luminescence resulted from the mixing of intravesicular spaces of AV and LV in fused vesicles. Optical density changes and negative stain electron microscopy indicated that Ca⁺⁺ induced extensive aggregation of vesicles. However, quantitation of the maximum possible luminescence indicates that only a small percentage (less than 1%) of the vesicles actually fused in a fusion experiment.Addition of EDTA to chelate Ca⁺⁺ after luminescence had been induced resulted in a two-to threefoldincrease in light emission which then rapidly decayed. These results suggest that the sudden removal of Ca⁺⁺ caused a transient increase in fusion after which subsequent fusion was inhibited. It was also found that the vesicles were relatively stable to hypotonic solutions.     

The existence of a group translocation transport mechanism in animal cells: Uptake of the ribose moiety of inosine

After exposure to inosine, transport-competent plasma membrane vesicles isolated from SV -40-transformed Balb/c 3T3 cells accumulate intravesicular ribose 1-PO₄ at a concentration 200-fold greater than the extravesicular concentration. An analysis of the purine nucleoside phosphorylase activity distribution in various subcellular fractions, relative to other enzyme activities, indicated the presence of plasma membrane-associated purine nucleoside phosphorylase activity. The plasma membrane vesicles appear relatively impermeable to hypoxanthine. However, hypoxanthine, which is a competitive inhibitor of the transport reaction, is the only compound tested capable of mediating efflux of already accumulated ribose 1-PO₄. In addition, hypoxanthine does not result in the efflux of transported uridine which is accumulated in these membrane vesicles as uridine. Exogenous ribose 1-PO₄ neither results in counterflow nor does it inhibit the original uptake reaction. The following transport reaction is proposed: uptake occurs by group translocation, mediated by membrane-localized purine nuceloside phosphorylase. The data are consistent with sites for inosine and hypoxanthine being on the outer membrane surface whereas the ribose 1-PO₄ site is only on the inner surface.     

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

Summary In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a _i Cl^–), sodium (a _i Na⁺) and potassium (a _i K⁺), pH_i, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a _i Cl^–=35 meq/liter, a _i Na⁺=11 meq/liter and a _i K⁺=95 meq/liter. During bilateral substitution the latter two did not change while a _i Cl^– dropped to virtually zero.Mucosal membrane potentials (_ms) were: control,-53 mV; serosal substitution,-51 mV; bilateral substitution,-66 mV; while during mucosal substitution a transient depolarization occurred and the final steady state _ms was-66 mV.During control and bilateral substitution the transepithelial potentials (_ms) did not differ from zero. During unilateral substitutions _ms was small, in the order of magnitude of the errors in the liquid junction potentials near the measuring salt bridges.During bilateral substitution pH _i increased 0.4 pH units. Cellular volume decreased during mucosal substitution to 88% in 40 min; after serosal substitution it transiently increased, but the new steady-state value was not significantly above its control value.Three minutes after mucosal substitution ana _i Cl^– of approx. 10 meq/liter was measured.Chemical concentrations of Na, K and Cl were determined under control conditions and bilateral substitution. Cl concentrations were also measured as a function of time after unilateral substitutions.The data indicate an electrically silent chloride influx mechanism in the brush border membrane and an electrodiffusional chloride efflux in the basolateral membrane. A substantial bicarbonate permeability is present in the basolateral membrane. The results are in agreement with the observed changes in membrane resistances, volume changes and pH changes.     

Effects of mutations in cAMP-dependent protein kinase on chloride efflux in Caco-2 human colonic carcinoma cells

In order to evaluate the importance of cAMP and cAMP-dependent protein kinase (cAMPdPK) in the regulation of chloride efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, Caco-2, human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of regulator subunit of cAMPdPK under control of the mouse metallothionein 1 promoter. Four stable transformants were isolated that expressed the mutant subunit in a Zn²⁺-inducible manner and exhibited Zn²⁺-inducible inhibition of cAMPdPK activity. The parental and transformed Caco-2 cells were examined for their abilities to regulate chloride efflux in response to various secretagogues using a radioactive iodide-efflux assay. In the transformants, induction of the protein kinase mutation with ZnSo₄ markedly decreased chloride efflux in response to forskolin, the 8-(4-chlorophenylthio) analog of cAMP, vasoactive intestial polypeptide, prostaglandin E2 and isoproterenol, whereas Zn²⁺-treated parental cells remained responsive to these secretagogues. Treatment with carbachol, calcium ionophores or phorbol ester did not acutely affect chloride efflux. Together, these studies indicate that cAMP and cAMPdPK are essential components of secretagogue-regulated chloride channel activity in the Caco-2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK mutation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intracellularly, reversed the inhibition. These latter results demonstrate that the CFTR chloride channels in the protein kinase-defective transformants are normal and that the protein kinase mutation specifically affects their regulation, presumably by direct phosphorylation. © 1995 Wiley-Liss, Inc.     

Modes of Cl− transport across the mucosal and serosal membranes of urodele intestinal cells

Summary The characteristics of Cl^– movement across luminal and basolateral membranes ofAmphiuma intestinal absorptive cells were studied using Cl^–-sensitive microelectrodes and tracer³⁶Cl techniques. Intracellular Cl^– activity (a _Cl ⁱ ) was unchanged when serosal Cl^– was replaced; when luminal Cl^– was replaced cell Cl^– was rapidly lost. Accordingly, the steady statea _Cl ⁱ could be varied by changing the luminal [Cl]. As luminal [Cl] was raised from 1 to 86mM,a _Cl ⁱ rose in a linear manner, the mucosal membrane hyperpolarized, and the transepithelial voltage became serosa negative. In contrast, the rate of Cl^– transport from the cell into the serosal medium, measured as the SITS-inhibitable portion of the Cl^– absorptive flux, attained a maximum whena _Cl ⁱ reached an apparent value of 17mm, indicating the presence of a saturable, serosal transport step. The stilbeneinsensitive absorptive flux was linear with luminal [Cl], suggestive of a paracellular route of movement. Intracellulara _Cl was near electrochemical equilibrium at all but the lowest values of luminal [Cl] after interference produced by other anions was taken into account.a _Cl ⁱ was unaffected by Na replacement, removal of medium K, or elevation of medium HCO ₃ ^– . Mucosae labeled with³⁶Cl lost isotope into both luminal and serosal media at the same rate and from compartments of equal capacity. Lowering luminal [Cl] or addition of theophylline enhanced luminal Cl^– efflux. It is concluded that a conductive Cl leak pathway is present in the luminal membrane. Serosal transfer is by a saturable, stilbene-inhibitable pathway. Luminal Cl^– entry appears to be passive, but an electrogenic uptake cannot be discounted.     

Effect of FeCl3 on ion transport in isolated frog skin

Summary The effect of addition of FeCl₃ to the media bathing the isolated skin ofRana pipiens was studied by measuring short-circuit current, transepithelial potential, and resistance, and by determining the influx and efflux of sodium (J ₁₃ ^Na andJ ₃₁ ^Na , respectively) and the influx and efflux of chloride (J ₁₃ ^Cl andJ ₃₁ ^Cl , respectively) across the epithelium. With normal Ringer's solution on both sides of the skin, addition of 10^–3 m FeCl₃ to the external medium resulted in nearly complete inhibition of active Na transport (J ₁₃ ^Na decreased from 1.30±0.14 to 0.10±0.04 eq/cm² hr (N=8)) and in appearance of active chloride transport in outward direction due to an 80% increase inJ ₃₁ ^Cl . Average (J ₃₁ ^Cl –J ₁₃ ^Cl ) obtained from means of 8 skins in 6 consecutive control and last 3 experimental periods was –0.17±0.04 and 0.38±0.05 eq/cm² hr, respectively. FeCl₃ added to external medium also induced substantial net chloride movement in outward direction when external medium contained Na-free choline chloride Ringer's or low ionic strength solution. Under the latter condition net Na movement was virtually eliminated by external FeCl₃. After addition of FeCl₃ to serosal medium there was delayed inhibition ofJ ₁₃ ^Na but no change in chloride fluxes. Immediate and profound changes in Na and Cl transport systems seen after external application of FeCl₃ indicate charge effects of Fe³⁺ on surface of apical cell membranes, possibly close to or in ion channels.     

Voltage-dependent,monomeric channel activity of colicin E1 in artificial membrane vesicles

Summary The dependence of colicin channel activity on membrane potential and peptide concentration was studied in large unilamellar vesicles using colicin E1, its COOH-terminal thermolytic peptide and other channel-forming colicins. Channel activity was assayed by release of vesicle-entrapped chloride, and could be detected at a peptide: lipid molar ratio as low as 10^–7. The channel activity was dependent on the magnitude of atrans-negative potassium diffusion potential, with larger potentials yielding faster rates of solute efflux. For membrane potentials greater than –60mV (K _in ⁺ /K _out ⁺ 10), addition of valinomycin resulted in a 10-fold increase in the rate of Cl^– efflux. A delay in Cl^– efflux observed when the peptide was added to vesicles in the presence of a membrane potential implied a potential-independent binding-insertion mechanism. The initial rate of Cl^– efflux was about 1% of the single-channel conductance, implying that only a small fraction of channels were initially open, due to the delay or latency of channel formation known to occur in planar bilayers.The amount of Cl^– released as a function of added peptide increased monotonically to a concentration of 0.7 ng peptide/ml, corresponding to release of 75% of the entrapped chloride. It was estimated from this high activity and consideration of vesicle number that 50–100% of the peptide molecules were active. The dependence of the initial rate of Cl^– efflux on peptide concentration was linear to approximately the same concentration, implying that the active channel consists of a monomeric unit.     

Characteristics of anion transport in cat and dog red blood cells

Summary Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rates of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of³⁵SO₄ efflux on the intracellular SO₄ concentration, the pH dependence and the inhibition of³⁵SO₄ efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of³⁶Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higherV _max andK _1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H₂DIDS, where DIDS is 4,4-diisothiocyano-2,2 stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H₂DIDS.     

A dominant‐negative form of Arabidopsis AP‐3 β‐adaptin improves intracellular pH homeostasis

Intracellular pH (pH_i) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pH_i regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid‐tolerant 1‐1D (wat1‐1D) are due to the expression of a truncated form of AP‐3 β‐adaptin (encoded by the PAT2 gene) that behaves as a as dominant‐negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pH_i and a more depolarized plasma membrane electrical potential than wild‐type cells. Additional phenotypes of wat1‐1D roots include increased rates of acetate efflux, K⁺ uptake and H⁺ efflux, the latter reflecting the in vivo activity of the plasma membrane H⁺‐ATPase. The in vitro activity of the enzyme was not increased but, as the H⁺‐ATPase is electrogenic, the increased ion permeability would allow a higher rate of H⁺ efflux. The AP‐3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1‐1D mutant can be explained if loss of function of the AP‐3 β‐adaptin causes activation of channels or transporters for organic anions (acetate) and for K⁺ at the plasma membrane, perhaps through miss‐localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone

The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D_f) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D_f motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.