Loading of nitrate into the xylem: apoplastic nitrate controls the voltage dependence of X-QUAC,the main anion conductance in xylem-parenchyma cells of barley roots

We report here that NO(3)(-) in the xylem exerts positive feedback on its loading into the xylem through a change in the voltage dependence of the Quickly Activating Anion Conductance, X-QUAC. Properties of this conductance were investigated on xylem-parenchyma protoplasts prepared from roots of Hordeum vulgare by applying the patch-clamp technique. Chord conductances were minimal around -40 mV and increased with plasma membrane depolarisation as well as with hyperpolarisation. Two gates with opposite voltage dependences were postulated. When 30 mM Cl- in the bath was replaced by NO(3)(-), a shift in the midpoint potential of the depolarisation-activated gate by about -60 mV from 43 to -16 mV occurred (K(m) = 3.4 mM). No such effect was seen when chloride was replaced by malate. Addition of 10 mM NO(3)(-)to the pipette solution and reduction of [Cl-] from 124 to 4 mM (to simulate cytoplasmic concentrations) did not interfere with the voltage dependence of X-QUAC activation, nor was it affected by changes in external [K+]. If only the NO(3)(-) effect on gating was considered, an increase of the NO(3)(-) concentration in the xylem sap to 5 mM would result in an enhancement of NO(3)(-) efflux by about 30%. Although the driving force for NO(3)(-) efflux would be reduced simultaneously, NO(3)(-) efflux into the xylem through X-QUAC would be maintained with high NO(3)(-) concentrations in the xylem sap; a situation which occurs for instance during the night.     

The regulation of anion loading to the maize root xylem

The regulation of anion loading to the shoot in maize (Zea mays) was investigated via an electrophysiological characterization of ion conductances in protoplasts isolated from the root stele. Two distinct anion conductances were identified. In protoplasts from well-watered plants, Z. mays xylem-parenchyma quickly-activating anion conductance (Zm-X-QUAC) was the most prevalent conductance and is likely to load the majority of NO(3)(-) and Cl(-) ions to the xylem in nonstressed conditions. Z. mays xylem-parenchyma inwardly-rectifying anion conductance was found at a lower frequency in protoplasts from well-watered plants than Zm-X-QUAC, was much smaller in magnitude in all observed conditions, and is unlikely to be such a major pathway for anion loading into the xylem. Activity of Z. mays xylem-parenchyma inwardly-rectifying anion conductance increased following a water stress prior to protoplast isolation, but the activity of the putative major anion-loading pathway, Zm-X-QUAC, decreased. Addition of abscisic acid (ABA) to protoplasts from well-watered plants also inhibited Zm-X-QUAC activity within minutes, as did a high free Ca(2+)concentration in the pipette. ABA was also seen to activate a Ca(2+)-permeable conductance (Z. mays xylem-parenchyma hyperpolarization activated cation conductance) in protoplasts from well-watered plants. It is postulated that the inhibition of anion loading into the xylem (an important response to a water stress) due to down-regulation of Zm-X-QUAC activity is mediated by an ABA-mediated rise in free cytosolic Ca(2+).     

Sulfate Is Both a Substrate and an Activator of the Voltage-Dependent Anion Channel of Arabidopsis Hypocotyl Cells

On the basis of the anion content of in vitro-cultured Arabidopsis plantlets, we explored the selectivity of the voltage-dependent anion channel of the plasma membrane of hypocotyl cells. In the whole-cell configuration, substitution of cytosolic Cl(-) by different anions led to the following sequence of relative permeabilities: NO(3)(-) (2.6) >/= SO(4)(2-) (2.0) > Cl(-) (1.0) > HCO(3)(-) (0.8) > malate(2-) (0.03). Large whole-cell currents were measured for NO(3)(-) and SO(4)(2-), about five to six times higher than the equivalent Cl(-) currents. Since SO(4)(2-) is usually considered to be a weakly permeant or non-permeant ion, the components of the large whole-cell current were explored in more detail. Aside from its permeation through the channel with a unitary conductance, about two-thirds that of Cl(-), SO(4)(2-) had a regulatory effect on channel activity by preventing the run-down of the anion current both in the whole-cell and the outside-out configuration, increasing markedly the whole-cell current. The fact that the voltage-dependent plasma membrane anion channel of hypocotyl cells can mediate large NO(3)(-) and SO(4)(2-) currents and is regulated by nucleotides favors the idea that this anion channel can contribute to the cellular homeostasis of important metabolized anions.     

Characteristics of anion channels in the tonoplast of the liverwort Conocephalum conicum

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca(2+) activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg(2+) and low Ca(2+) concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg(2+) with K(+) in the bath. Sr(2+) and Ba(2+) were also effective activators of the currents. With a Cl(-) gradient, 10 mM in the bath and 100 mM in the lumen, currents were significantly reduced and the current-voltage characteristics shifted towards the reversal potential of Cl(-), indicating Cl(-) selectivity. Currents almost vanished after substituting Cl(-) with gluconate. They were strongly reduced by anion channel inhibitors 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (A9C; 2 mM) and ethacrinic acid (0.5 mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conducting anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physiological calcium concentration. The channels were almost equally permeable to Cl(-), NO3(-) and SO4(2-), and much less permeable to malate(2-). Anion channels did not respond to ATP addition. cAMP (10 microM) had a weak effect on anion channels. Protein kinase A (0.4 U) added to the medium caused no significant effect on anion channels.     

Anion Selectivity of Slow Anion Channels in the Plasma Membrane of Guard Cells (Large Nitrate Permeability)

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to Cl- of NO3- > Br- > F- ~ Cl- ~ I- > malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple conductance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of Cl- and initial malate efflux required for mediation of stomatal closure. The large NO3- permeability as well as the significant permeabilities of all anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels.     

Tefluthrin modulates a novel anionic background conductance (I(AB)) in guinea-pig ventricular myocytes

This report describes for the first time a novel anionic background current (I(AB)) identified in guinea-pig isolated ventricular myocytes. It also shows that I(AB) has both novel and differential pharmacology from other (cardiac) chloride currents. Using the whole-cell patch-clamp technique and external anion substitution, I(AB) was found to be outwardly rectifying and highly permeable to NO(-)(3), with a relative permeability sequence of NO(-)(3) > I(-) > Cl(-). I(AB) was not blocked by 50 microM DIDS, by hypertonic external solution, or by the nonselective protein kinase inhibitor H7-DHC. Exposure to the pyrethroid agent tefluthrin (10 microM) increased the current density of I(AB) significantly at positive voltages (P < 0.05), but had no significant effect on other cardiac chloride currents. We conclude that I(AB) possesses a distinct pharmacology and does not fall into the three major classes of cardiac chloride conductance commonly reported.     

Anion permeation in an apical membrane chloride channel of a secretory epithelial cell

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.     

Anion permeation in the proximal tubule of Necturus kidney: the shunt pathway.

The effect of foreign anions on transepithelial potential difference and transepithelial input conductance was studied in the isolated perfused Necturus kidney. Two microelectrodes (recording and current-injecting) were inserted into the lumen of single proximal tubules and the peritubular perfusate was shifted reversibly for 30-60 sec from a physiologic Ringer's solution to a test solution in which chloride was replaced isosmotically by a foreign anion. The permeability sequence, obtained by potential measurements, was: lactate less than glutamate less than gluconate less than pyruvate less than benzene sulfonate less than or equal to acetate less than or equal to F less than propionate less than BrO3 less than formate less than ClO3 less than Cl than ClO4 less than I less than or equal to Br less than NO3 less than SCN. Transepithelial conductance decreased when the tissue was perfused with anions less permeable than chloride but the conductance sequence was different from the permeability sequence. Such discrepancies were more pronounced during perfusion with hyperpolarizing anions; ClO4 and I- (both more permeable than chloride) produced an important decrease in transepithelial conductance, followed by incomplete reversibility when the perfusion was shifted again to chloride Ringer's. The results are best explained by the presence of weak positive fixed charges, governing anion permeation, at the shunt pathway of the proximal tubule. An analysis of the data allows tentative estimates of shape and size of the sites.     

Characterization of anion channels in the plasma membrane of Arabidopsis epidermal root cells and the identification of a citrate-permeable channel induced by phosphate starvation

Organic-acid secretion from higher plant roots into the rhizosphere plays an important role in nutrient acquisition and metal detoxification. In this study we report the electrophysiological characterization of anion channels in Arabidopsis (Arabidopsis thaliana) root epidermal cells and show that anion channels represent a pathway for citrate efflux to the soil solution. Plants were grown in nutrient-replete conditions and the patch clamp technique was applied to protoplasts isolated from the root epidermal cells of the elongation zone and young root hairs. Using SO4(2-) as the dominant anion in the pipette, voltage-dependent whole-cell inward currents were activated at membrane potentials positive of -180 mV exhibiting a maximum peak inward current (I(peak)) at approximately -130 mV. These currents reversed at potentials close to the equilibrium potential for SO4(2-), indicating that the inward currents represented SO4(2-) efflux. Replacing intracellular SO4(2-) with Cl- or NO3(-) resulted in inward currents exhibiting similar properties to the SO4(2-) efflux currents, suggesting that these channels were also permeable to a range of inorganic anions; however when intracellular SO4(2-) was replaced with citrate or malate, no inward currents were ever observed. Outside-out patches were used to characterize a 12.4-picoSiemens channel responsible for these whole-cell currents. Citrate efflux from Arabidopsis roots is induced by phosphate starvation. Thus, we investigated anion channel activity from root epidermal protoplasts isolated from Arabidopsis plants deprived of phosphate for up to 7 d after being grown for 10 d on phosphate-replete media (1.25 mm). In contrast to phosphate-replete plants, protoplasts from phosphate-starved roots exhibited depolarization-activated voltage-dependent citrate and malate efflux currents. Furthermore, phosphate starvation did not regulate inorganic anion efflux, suggesting that citrate efflux is probably mediated by novel anion channel activity, which could have a role in phosphate acquisition.     

Biophysical characterization of a large conductance anion channel in hypodermal membranes of the gastrointestinal nematode,Ascaris suum

Patch-clamp recordings from muscle- and cuticle-facing hypodermal membranes of the gastrointestinal nematode Ascaris suum reveal a high-conductance, voltage- sensitive Ca(2+) -dependent Cl(-) channel. The hypodermal channel has a conductance of 195 pS in symmetrical 160 mM NaCl. The open probability of the channel is highly voltage-sensitive, and channel activity is not observed when Ca(2+) is reduced to <100 microM. The channel is permeable to organic anions that are major end-products of carbohydrate metabolism in A. suum, including acetate, butyrate and 2-methylvalerate. The conductances and relative permeabilities of these organic anions are inversely related to size, with 2-methylvalerate being only approximately 3% as permeable as Cl(-). The diameter of the channel pore was 12.3+/-0.2 A, calculated from the relative permeability coefficients of Cl(-) and the organic anions. Results of this study are consistent with the hypothesis that the large conductance anion channel in A. suum hypodermal membranes provides a low energy pathway for organic anion excretion from the hypodermal compartment, followed by diffusion across the aqueous channels of the cuticle matrix.     

Single chloride channels in cultured rat neurones

Single-channel currents through chloride channels were recorded in cultured hippocampal neurones from rats using the patch-clamp method. The channel is active at voltages between -80 and +80 mV, and the time spent in the open state increases with depolarization (almost fourfold for 120 mV). The channel conductance is 62 pS in symmetrical 150 mM NaCl saline. In salt gradient conditions the channel was measurably permeable to Na+. Substitution of NO3- and Br- for Cl- gave higher single-channel currents, meaning a higher permeability of the channel toward the two anions than to Cl-. SO4(2-) ions were poorer charge carriers, yet contributed measurable inward current at negative voltages. Channel activity appeared independent of intracellular Ca2+ concentration. Taken together, these features would suggest for this channel a role in stabilizing resting membrane potential and in maintaining normal cell excitability.     

Calcium-regulated anion channels in the plasma membrane of Lilium longiflorum pollen protoplasts

? Currents through anion channels in the plasma membrane of Lilium longiflorum pollen grain protoplasts were studied under conditions of symmetrical anionic concentrations by means of patch-clamp whole-cell configuration. ? With Cl(-) -based intra- and extracellular solutions, three outward-rectifying anion conductances, I(Cl1) , I(Cl2) and I(Cl3) , were identified. These three activities were discriminated by differential rundown behaviour and sensitivity to 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), which could not be attributed to one or more channel types. All shared strong outward rectification, activated instantaneously and displayed a slow time-dependent activation for positive potentials. All showed modulation by intracellular calcium ([Ca(2+) ](in) ), increasing intensity from 6.04 nM up to 0.5 mM (I(Cl1) ), or reaching a maximum value with 8.50 μM (I(Cl2) and I(Cl3) ). ? After rundown, the anionic currents measured using NO(3) (-) -based solutions were indistinguishable, indicating that the permeabilities of the channels for Cl(-) and NO(3) (-) are similar. Additionally, unitary anionic currents were measured from outside-out excised patches, confirming the presence of individual anionic channels. ? This study shows for the first time the presence of a large anionic conductance across the membrane of pollen protoplasts, resulting from the presence of Ca(2+) -regulated channels. A similar conductance was also found in germinated pollen. We hypothesize that these putative channels may be responsible for the large anionic fluxes previously detected by means of self-referencing vibrating probes.     

Anion-cation interactions in the pore of neuronal background chloride channels

Background Cl channels in neurons and skeletal muscle are significantly permeable for alkali cations when tested with asymmetrical concentrations of the same salt. Both anion and cation permeation were proposed to require binding of an alkali cation with the pore (Franciolini, F., and W. Nonner. 1987. Journal of General Physiology. 90:453-478). We tested this hypothesis by bilaterally substituting large alkali cations for Na and found no significant changes of unitary conductance at 300 mM symmetrical concentrations. In addition, all organic cations examined were permeant in a salt gradient test (1,000 mM internal@300 mM external), including triethanolamine, benzyltrimethylamine, and bis-tris-propane (BTP, which is divalent at the tested pH of 6.2). Inward currents were detected following substitution of internal NaCl by the Na salts of the divalent anions of phosphoric, fumaric, and malic acid. Zero-current potentials in gradients of the Na and BTP salts of varied anions (propionate, F, Br, nitrate) that have different permeabilities under bi-ionic conditions, were approximately constant, as if the permeation of either cation were coupled to the permeation of the anion. These results rule out our earlier hypothesis of anion permeation dependent on a bound alkali cation, but they are consistent with the idea that the tested anions and cations form mixed complexes while traversing the Cl channel.     

Functional and structural analysis of ClC-K chloride channels involved in renal disease

ClC-K channels belong to the CLC family of chloride channels and are predominantly expressed in the kidney. Genetic evidence suggests their involvement in transepithelial transport of chloride in distal nephron segments; ClC-K1 gene deletion leads to nephrogenic diabetes insipidus in mice, and mutations of the hClC-Kb gene cause Bartter's syndrome type III in humans. Expression of rClC-K1 in Xenopus oocytes yielded voltage-independent currents that were pH-sensitive, had a Br(-) > NO(3)(-) = Cl(-) > I(-) conductance sequence, and were activated by extracellular calcium. A glutamate for valine exchange at amino acid position 166 induced strong voltage dependence and altered the conductance sequence of ClC-K1. This demonstrates that rClC-K1 indeed functions as an anion channel. By contrast, we did not detect currents upon hClC-Kb expression in Xenopus oocytes. Using a chimeric approach, we defined a protein domain that, when replaced by that of rClC-K1, allowed the functional expression of a chimera consisting predominantly of hClC-Kb. Its currents were linear and were inhibited by extracellular acidification. Contrasting with rClC-K1, they displayed a Cl(-) > Br(-)> I(-) > NO(3)(-) conductance sequence and were not augmented by extracellular calcium. Insertion of point mutations associated with Bartter's syndrome type III destroyed channel activity. We conclude that ClC-K proteins form constitutively open chloride channels with distinct physiological characteristics.     

An electrogenic pump in the xylem parenchyma of barley roots

Analysis of an electrogenic pump in the plasma membrane of xylem-parenchyma protoplasts from barley roots was performed using the patch-clamp technique in the whole-cell configuration. Particularly with regard to understanding xylem loading and unloading, the study of the electrogenic pump from this cell type is important; its functional confirmation was lacking to date. About one-half of the investigated protoplasts displayed current responses with reversal potentials between −80 and −200 mV. The application of fusicoccin, an H⁺-pump stimulator, caused an increase in currents recorded at a membrane potential of 0 mV and a shift of the reversal potential by about −50 mV. Treatment with dicylohexylcarbodiimid, an H⁺-pump inhibitor, resulted in the reduction of the current at 0 mV. The Ca²⁺-pump inhibitor, erythrosin B, showed no effect on current density at 0 mV and on the polarisation of the membrane potential. Enlarging the transmembrane pH gradient by raising the pH of the extracellular solution from 5.8 to 8.8 stimulated the currents. These are strong indications that the electrogenic pump was an H⁺-pump. Neither intracellular pH nor the intracellular Ca²⁺ concentration affected its activity. Simultaneous activity of the electrogenic pump and anion conductances could produce states in which protoplasts exhibited 'intermediate' reversal potentials. It was concluded that the electrogenic pump was not directly involved in the loading of KCl and KNO₃ into the xylem but, in combination with anion channel activities, contributed to the establishment of membrane potentials at which electroneutral salt transport and acid release can proceed.     

Chloride conductance of CFTR facilitates basal Cl-/HCO3- exchange in the villous epithelium of intact murine duodenum

Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl(-)/HCO(3)(-) exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl(-)/HCO(3)(-) exchange rate was reduced by approximately 35% in CF compared with WT villous epithelium. Cl(-)/HCO(3)(-) exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl(-)(out)/HCO(3)(-)(in) exchange in both epithelia. In anion substitution studies, anion(in)/HCO(3)(-)(out) exchange rates were greater in WT epithelium using Cl(-) or NO(3)(-), but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO(4)(2-). Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl(-)/HCO(3)(-) exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl(-)/HCO(3)(-) exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl(-) channel activity of CFTR facilitates apical membrane Cl(-)(in)/HCO(3)(-)(out) exchange by providing a Cl(-) "leak" under basal conditions.     

Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent

ClC-3 is a member of the ClC family of anion channels/transporters. Recently, the closely related proteins ClC-4 and ClC-5 were shown to be Cl(-)/H(+) antiporters (39, 44). The function of ClC-3 has been controversial. We studied anion currents in HEK293T cells expressing wild-type or mutant ClC-3. The basic biophysical properties of ClC-3 currents were very similar to those of ClC-4 and ClC-5, and distinct from those of the swelling-activated anion channel. ClC-3 expression induced currents with time-dependent activation that rectified sharply in the outward direction. The reversal potential of the current shifted by -48.3 +/- 2.5 mV per 10-fold (decade) change in extracellular Cl(-) concentration, which did not conform to the behavior of an anion-selective channel based upon the Nernst equation, which predicts a -58.4 mV/decade shift at 22 degrees C. Manipulation of extracellular pH (6.35-8.2) altered reversal potential by 10.2 +/- 3.0 mV/decade, suggesting that ClC-3 currents were coupled to proton movement. Mutation of a specific glutamate residue (E224A) changed voltage dependence in a manner similar to that observed in other ClC Cl(-)/H(+) antiporters. Mutant currents exhibited Nernstian changes in reversal potential in response to altered extracellular Cl(-) concentration that averaged -60 +/- 3.4 mV/decade and were pH independent. Thus ClC-3 overexpression induced a pH-sensitive conductance in HEK293T cells that is biophysically similar to ClC-4 and ClC-5.     

Novel properties of the wheat aluminum tolerance organic acid transporter (TaALMT1) revealed by electrophysiological characterization in Xenopus Oocytes: functional and structural implications

Many plant species avoid the phytotoxic effects of aluminum (Al) by exuding dicarboxylic and tricarboxylic acids that chelate and immobilize Al(3+) at the root surface, thus preventing it from entering root cells. Several novel genes that encode membrane transporters from the ALMT and MATE families recently were cloned and implicated in mediating the organic acid transport underlying this Al tolerance response. Given our limited understanding of the functional properties of ALMTs, in this study a detailed characterization of the transport properties of TaALMT1 (formerly named ALMT1) from wheat (Triticum aestivum) expressed in Xenopus laevis oocytes was conducted. The electrophysiological findings are as follows. Although the activity of TaALMT1 is highly dependent on the presence of extracellular Al(3+) (K(m1/2) of approximately 5 microm Al(3+) activity), TaALMT1 is functionally active and can mediate ion transport in the absence of extracellular Al(3+). The lack of change in the reversal potential (E(rev)) upon exposure to Al(3+) suggests that the "enhancement" of TaALMT1 malate transport by Al is not due to alteration in the transporter's selectivity properties but is solely due to increases in its anion permeability. The consistent shift in the direction of the E(rev) as the intracellular malate activity increases indicates that TaALMT1 is selective for the transport of malate over other anions. The estimated permeability ratio between malate and chloride varied between 1 and 30. However, the complex behavior of the E(rev) as the extracellular Cl(-) activity was varied indicates that this estimate can only be used as a general guide to understanding the relative affinity of TaALMT1 for malate, representing only an approximation of those expected under physiologically relevant ionic conditions. TaALMT1 can also mediate a large anion influx (i.e. outward currents). TaALMT1 is permeable not only to malate but also to other physiologically relevant anions such as Cl(-), NO(3)(-), and SO(4)(2-) (to a lesser degree).     

Amino acid-permeable anion channels in early mouse embryos and their possible effects on cleavage

Effects of several Cl(-) channel blockers on ionic currents in mouse embryos were studied using whole-cell patch-clamp and microelectrode methods. Microelectrode measurements showed that the resting membrane potential of early embryonic cells (1-cell stage) was -23 mV and that reduction of extracellular Cl(-) concentration depolarized the membrane, suggesting that Cl(-) conductance is a major contributor for establishing the resting membrane potential. Membrane currents recorded by whole-cell voltage clamp showed outward rectification and confirmed that a major component of these embryonic currents are carried by Cl(-) ions. A Cl(-) channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suppressed the outward rectifier current in a voltage- and concentration-dependent manner. Other Cl(-) channel blockers (5-nitro-2-[3-phenylpropyl-amino] benzoic acid and 2-[3-(trifluoromethyl)-anilino] nicotinic acid [niflumic acid]) similarly inhibited this current. Simultaneous application of niflumic acid with DIDS further suppressed the outward rectifier current. Under high osmotic condition, niflumic acid, but not DIDS, inhibited the Cl(-)current, suggesting the presence of two types of Cl(-) channels: a DIDS-sensitive (swelling-activated) channel, and a DIDS-insensitive (niflumic acid-sensitive) Cl(-) channel. Anion permeability of the DIDS-insensitive Cl(-) current differed from that of the compound Cl(-) current: Rank order of anion permeability of the DIDS-sensitive Cl(-) channels was I(-) = Br(-) > Cl(-) > gluconate(-), whereas that of the DIDS-insensitive Cl(-) channel was I(-) = Br(-) > Cl(-) > gluconate(-). These results indicate that early mouse embryos have a Cl(-) channel that is highly permeable to amino acids, which may regulate intracellular amino acid concentration.     

Identification of a region of strong discrimination in the pore of CFTR

The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.