Effect of adsorbed cations on phosphorus uptake by excised roots

Pretreatment of excised roots of Hordeum vulgare, Zea mays, and Glycine max with various salt solutions affected their subsequent rate of phosphorus absorption from 2 × 10⁻⁵m KH₂PO₄. The rate of absorption was greatest for roots pretreated with trivalent cations, intermediate with divalent cations and lowest with monovalent cations. It appeared that the pretreatment involved a rapid exchange reaction at the root surface which was reversible. A 1 min pretreatment was effective for more than 20 min. The acceleration of phosphorus uptake by roots produced by polyvalent cations may be due partly or entirely to a greater reduction in the electrical potential at the root surface or within the pores of the negatively charged cell wall by polyvalent cations than by monovalent cations.     

Ion association reactions with biological membranes,studied with the fluorescent dye 1-anilino-8-naphthalenesulfonate

Summary (1) When salts are added to buffered suspensions of membrane fragments containing the fluorochrome 1-anilino-8-naphthalenesulfonate (ANS), there is an increased fluorescence. This is caused by increased binding of the fluorochrome; the intrinsic fluorescence characteristics of the bound dye remain unaltered. These properties make ANS a sensitive and versatile indicator of ion association equilibria with membranes. (2) Alkali metal and alkylammonium cations bind to membranes in a unique manner. Cs⁺ binds most strongly to rat brain microsomal material, with the other alkali metals in the order Cs⁺>Rb⁺>K⁺>Na⁺>Li⁺. The reaction is endothermic and entropy driven. Monovalent cations are displaced by other monovalent cations. Divalent cations and some drugs (e. g., cocaine) displace monovalent cations more strongly. (3) Divalent cations bind to membranes (and to lecithin micelles) at four distinct sites, having apparent association constants between 50 and 0.2mm ^–1. The characteristics of the titration suggest that only one species of binding site is present at any one time, and open the possibility that structural transitions of the unassociated coordination sites may be induced by divalent cation binding. Divalent cation binding at the weakest site (like monovalent cation binding) is endothermic and entropy driven. At the next stronger site, the reaction is exothermic. Monovalent cations affect divalent cation binding by reducing the activity coefficient: they do not appear to displace divalent cations from their binding sites.     

Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons

Summary A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na⁺, K⁺ and Cs⁺, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage-and concentration-dependent block of Na⁺ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg²⁺ was >1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg²⁺ block of Na^– permeation, and demonstrating the channel's presence in arthropods.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH,cations and phosphate

The uptake of Ca²⁺ and Sr²⁺ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics.The rate of Ca²⁺ and Sr²⁺ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca²⁺ and Sr²⁺ at low pH may be explained by a decrease of the surface potential.The inhibition of Ca²⁺ and Sr²⁺ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane.Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Extracellular Ca2+ controls outward rectification by apical cation channels in toad urinary bladder: Patch-clamp and whole-bladder studies

Summary Outward rectifying. cation channels were observed in the epithelial cells of the urinary bladder of the toad.Bufo marinus. As studied in isolated cells using the patch-clamp technique, the channel has an average conductance of 24 and 157 pS for pipette potentials between 0 and +60 mV and –60 to –100 mV, respectively, when the major cation in both bath and pipette solutions is K⁺. The conductance of the cannel decreasen with increasing dehydration energy of the permeant monovalent cation in the oder Rb⁺=K⁺>Na⁺>Li⁺. Reversal potentials near zero under biionic conditions imply that the permeabilities for all four of these cations are smiliar. The channel is sensitive to quinidine sulfate but not to amiloride. It shares several pharmacological and biophysical properties with an outwardly-rectifying, vasopressin-sensitive pical K⁺ conductive pathway described previously for the toad urinary bladder. We demonstrate, in both single-channel and whole-bladder studies, that the outward rectification is a consequence of interaction of the chanel with extracellular divalent cations, particularly Ca²⁺, which blocks inward but not outward current. Various divalent cations impart different degrees of outward rectification to the conductive pathway. Concentrations of Mg²⁺ and Ca²⁺ required for halfmaximal effect are 3×10^–4 and 10^–4 m, resopectively. For Co²⁺ the values are 10^–6 m at +50 mV and a 10^–4 m at +200 mV. The mechanism of blockade by divalent cations is not established, but does not seem to involve a voltage-dependent interaction in which the blocker penetrates the transmembrane electric field. In the absence of divalent cations in the mucosal solution, the magnitudes of inward current carried by Rb⁺, K⁺, Na⁺ and Li⁺ through the apical K⁺ pathway at any transepithelial voltage, are in the same order as in the single-channel studies. We propose that the cation channel observed by us in isolated epithelial cells is the single-channel correlate of the vasopressin-sensitive apical K⁺ conductive pathway in the toad urinary bladder and is also related to the oxytocin- and divalent cation-sensitive apical condictivity observed in frog skin and urinary bladder.     

An in situ approach for measuring root-associated respiration and nitrate uptake of forest trees

The ecophysiological characteristics of fine roots of mature forest plants are poorly understood because of difficulties of measurement. We explored a root in-growth approach to measure respiration and nitrate uptake of woody plant roots in situ. Roots of seven species were grown into sand-filled chambers. Root-associated respiration was measured as CO ₂ emission on four dates and nitrate uptake was quantified using ¹⁵N. All the roots were younger than 3 months at the time of measurement. Fine root respiration measured over the temperature range of 14.5–15.5 °C averaged 18.9–36.5 nmol gDM ^–1 s ^–1 across species. Nitrate uptake rates by these fine roots (1.3–6.8 nmol gDM ^–1 s ^–1) were comparable to other studies of forest trees. The root respiration rates were several times higher than measurements on detached roots of mature trees, concurring with literature observations that young roots respire much more rapidly than older roots. The root in-growth approach appears promising for providing information on the metabolic activity of fine roots of mature forest trees growing in soil.     

Pyruvate kinase: Activation by and catalytic role of the monovalent and divalent cations

Summary This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K⁺ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact,E. coli synthesizes depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4–8 ? of the divalent cation activat or and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg⁺² is optimal for the physiological reaction, however, Co⁺², Mn⁺², and Ni⁺² also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.     

Activation of a nonselective cation channel by swelling in atrial cells

Cell swelling has been shown to increase the permeability of the plasma membrane to ions such as K⁺, Na⁺, Ca²⁺ or Cl^– in many types of cells. In cardiac cells, swelling has been reported to increase Cl^– conductance, but whether cation-selective currents are activated by swelling is not known. Low Cl^– or Cl^–-free solutions were used to study the presence of such currents. Lowering the osmolarity of the extracellular medium from 299 to 219 mOsm resulted in cell swelling and concurrent activation of a cation-selective whole-cell current. When cell-attached patches were formed on swollen cells, opening of bursting single channel currents were observed in 18% of the patches studied. Ion substitution experiments indicated that the channel discriminated poorly among monovalent cations, and was impermeable to Cl^–. The channel was permeable to Ca²⁺. In symmetrical 140 mM K⁺, the current-voltage relation was linear with a single channel conductance of 36 ± 3 pS. Depolarization increased channel open probability. Interestingly, depending on the membrane patch studied, application of negative pressure to the pipette caused either an increase or a decrease in the open probability of the channel already activated by swelling. Thus, the sensitivity to tension of the swelling-activated channel was different from those of previously reported stretch-activated channels. These findings suggest that nonselective cation channels exist in rat atrial cells and may be involved in swelling-induced changes in cell function.Dr. Kim is an Established Investigator of the American Heart Association.     

Relative importance of Na+, Cl−, and abscisic acid in NaCl induced inhibition of root growth of rice seedlings

The relative importance of endogenous abscisic acid (ABA), as well as Na⁺ and Cl^– in NaCl-induced responses related to growth in roots of rice seedlings were investigated. The increase in ammonium, proline and H₂O₂ levels, and cell wall peroxidase (POD) activity has been shown to be related to NaCl-inhibited root growth of rice seedlings. Increasing concentrations of NaCl from 50 to 150 mM progressively decreased root growth and increased both Na⁺ and Cl^–. Treatment with NaCl in the presence of 4,4-diisothiocyano-2,2-disulfonic acid (DIDS, a nonpermeating amino-reactive disulfonic acid known to inhibit the uptake of Cl^–) had less Cl^– level in roots than that in the absence of DIDS, but did not affect the levels of Na⁺, and responses related to growth in roots. Treatment with 50 mM Na-gluconate (the anion of which is not permeable to membrane) had similar Na⁺ level in roots as that with 100 mM NaCl. It was found that treatment with 50 mM Na-gluconate effected growth reduction and growth-related responses in roots in the same way as 100 mM NaCl. All these results suggest that Cl^– is not required for NaCl-induced responses in root of rice seedlings. Endogenous ABA level showed no increase in roots of rice seedlings exposed to 150 mM NaCl. It is unlikely that ABA is associated with NaCl-inhibited root growth of rice seedlings.     

The uptake and extrusion of monovalent cations by isolated heart mitochondria

Summary The factors involved in the movement of monovalent cations across the inner membrane of the isolated heart mitochondrion are reviewed. The evidence suggests that the energy-dependent uptake of K⁺ and Na⁺ which results in swelling of the matrix is an electrophoretic response to a negative internal potential. There are no clear cut indications that this electrophoretic cation movement is carrier-mediated and possible modes of entry which do not require a carrier are examined. The evidence also suggests that the monovalent cation for proton exchanger (Na⁺ > K⁺) present in the membrane may participate in the energy-dependent extrusion of accumulated ions. The two processes, electrophoretic cation uptake (swelling) and exchange-dependent cation extrusion (contraction) may represent a means of controlling the volume of the mitochondrion within the functioning cell. A number of indications point to the possibility that the volume control process may be mediated by the divalent cations Ca⁺² and Mg⁺². Studies with mercurial reagents also implicate certain membrane thiol groups in the postulated volume control process.An invited article.     

Triton-stimulated nucleoside diphosphatase: Characterization

Summary Our previous report indicated a Triton-stimulated NDPase was specifically associated with Golgi membranes isolated from corn roots. Characterization of the enzyme indicates that UDP is the slightly preferred substrate with Mn²⁺ the preferred divalent cation. Monovalent cations do not further activate the Triton-stimulated UDPase activity. The enzyme has a pH optimum at 6.5 and a temperature optimum between 38–40°C. Kinetic analyses indicate that UDP-Mn²⁺ is the substrate for the enzyme.Properties of the Triton-stimulated NDPase are compared to other membrane associated NDPases isolated from plants, animals and fungi. Characteristics and subcellular location of NDPase activity are discussed in relation to the possible biochemical role of the enzyme.This research was supported in part by National Science Foundation grant CDP 79-7927121 and funds provided by Bronfman Science Center, Williams College.     

Cyclic AMP-dependent regulation of activities of synthetase and phosphodiesterase of 2′,5′-oligoadenylate in NIH 3T3 cells

Summary Dihydrofolate synthetase (EC 6.3.2.12) from N. gonorrhoeae was isolated and enzyme characteristics were determined. The purified enzyme was found quite stable when stored at –60 °C. About 50% of the enzyme activity wag destroyed within 6 weeks when kept at 4 °C. Maximum velocity was observed at pH 9.3. The enzyme required a monovalent cation, K⁺ or NH₄ ⁺ , and divalent cation, Mg²⁺ or Mn²⁺ for its function. ATP at 5 mM concentration gave maximum activity. K_m values for dihydropteroate and L-glutamate at pH 9.3 were 3.5 × 10^–5 M and 6.5 × 10^–4 M, respectively. Patterns of product inhibition by dihydrofolate were found to be non-competitive with respect to dihydropteroate, having a K_i value of 5.1 ± 0.8 × 10^–4 M, and competitive with respect to L-glutamate, having a K_i value of 6.2 × 10^–4 M.     

Hydrogel modified uptake of salt ions and calcium in<Emphasis Type="Italic"> Populus euphratica</Emphasis> under saline conditions

The effects of hydrogel on growth and ion relationships of a salt resistant woody species, Populus euphratica , were investigated under saline conditions. The hydrogel used was Stockosorb K410, a highly cross-linked polyacrylamide with about 40% of the amide group hydrolysed to carboxylic groups. Amendment of saline soil (potassium mine refuse) with 0.6% hydrogel improved seedling growth (2.7-fold higher biomass) over a period of 2 years, even though plant growth was reduced by salinity. Hydrogel-treated plants had approximately 3.5-fold higher root length and root surface area than those grown in unamended saline soil. In addition, over 6% of total roots were aggregated in gel fragments. Tissue and cellular ion analysis showed that growth improvement appeared to be the result of increased capacity for salt exclusion and enhancement of Ca²⁺ uptake. X-ray microanalysis of root compartments indicated that the presence of polymer restricted apoplastic Na⁺ in both young and old roots, and limited apoplastic and cytoplastic Cl^– in old roots while increasing Cl^– compartmentation in cortical vacuoles of both young and old roots. Collectively, radical transport of salt ions (Na⁺ and Cl^–) through the cortex into the xylem was lowered and subsequent axial transport was limited. Hydrogel treatment enhanced uptake of Ca²⁺ and microanalysis showed that enrichment of Ca²⁺ in root tissue mainly occurred in the apoplast. In conclusion, enhanced Ca²⁺ uptake and the increased capacity of P. euphratica to exclude salt were the result of improved Ca²⁺/Na⁺ concentration of soil solution available to the plant. Hydrogel amendment improves the quality of soil solutions by lowering salt level as a result of its salt-buffering capacity and enriching Ca²⁺ uptake, because of the polymers cation-exchange character. Accordingly, root aggregation allows good contact of roots with a Ca²⁺ source and reduces contact with Na⁺ and Cl^–, which presumably plays a major role in enhancing salt tolerance of P. euphratica.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning α-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of “the cation” in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca²⁺, Mg²⁺ and Mn²⁺) and monovalent metal cations (Li⁺, Na⁺, K⁺ and Cs⁺), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.     

Structural Effects of Cl and Other Anions on the Water Oxidizing Complex of Chloroplast Photosystem II

To further our understanding of the role of Cl⁻ and certain other monovalent anions in the oxygen evolving photosystem II of chloroplasts, dissociating and stabilizing anion effects on the extrinsic 17 and 23 kilodalton polypeptides of the photosynthetic water oxidizing complex were investigated. It was found that (a) the dissociation of the two polypeptides in Cl⁻ free media of pH ≈ 7 was enhanced by millimolar concentrations of the divalent anion SO₄²⁻ and also by divalent cations like Mg²⁺ and Ca²⁺; (b) the dissociation was opposed by relatively low concentrations of monovalent anions with an order of effectiveness Cl⁻ = Br⁻ > NO₃⁻ > F⁻ > ClO₄⁻; (c) at molar concentrations, SO₄²⁻ stabilized the binding of the 23 kilodalton polypeptide, while Cl⁻ and Br⁻ became dissociating agents, in agreement with studies by Blough and Sauer (1984 Biochim Biophys Acta 767: 377-381); (d) the binding of the polypeptides was strengthened at room temperature relative to 0°C, indicating an involvement of hydrophobic forces. It is suggested that a specific binding of Cl⁻, or certain substitutes, organizes the protein surfaces and/or the adjacent water layers in the water oxidizing complex in a way that not only stabilizes its assembly, but is essential for the catalytic mechanism as well. Binding of, or charge screening by, divalent ions interferes with this process. At high salt concentrations, all these effects are overridden by “lyotropic” actions of the solutes that affect the integrity of the water oxidizing protein complex by stabilizing or disrupting critical hydrophobic domains.     

Discrimination between monovalent and divalent cations by hydrophobic solvent-saturated membranes containing fixed negative charges

Summary Cellulose acetate-nitrate filters were saturated with hydrophobic solvent and interposed between various aqueous solutions. The membranes thus formed are cation permselective. The discrimination between a monovalent cation such as K⁺ and the alkaline earth group divalent cations is very sharp. The discrimination ratio is at least a few thousand times in favor of the monovalent cation. A major part of this discrimination is caused by the very low mobility of the divalent cation within the membrane compared with that of the monovalent cation. The remainder of the discrimination is caused by the selectivity of the membranes which prefer monovalent to divalent cations. There is a clear discrepancy between Ba⁺⁺ diffusibility and mobility within, the membrane. This implies that Ba⁺⁺ may move within the hydrophobic membrane as a neutral complex. Some similarity with natural biological membranes is indicated.     

The effect of cations on reassociation of the components of the cellulosome cellulase complex synthesized by the bacterium Clostridium thermocellum

The cellulosome multienzyme complex was dissociated into 12–14 components when incubated at 30° C in a reaction mixture that was buffered at pH 5.0 and was 50 mm with respect to sodium dodecyl sulphate and 10 mm with respect to both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT). The dissociated components reassociated into a complex when dialysed against 20 mm TRIS/HCl buffer, pH 7.7, containing 2.5 mm DTT. When incubated in the presence of Ca²⁺ and DTT the reassociated complex had the same activity to hydrogen-bond-ordered cellulose as the undissociated cellulosome. However, when Ca²⁺ ions were incorporated into the TRIS/HCl-DTT dialysis medium the reconstituted complex had very little activity towards cellulose. Other divalent cations such as Mg²⁺ and Ba²⁺ had the same effect, but the monovalent cation Na⁺ resulted in a complex that was very active on crystalline cellulose. The results are interpreted as indicating that the divalent cations bind to one or more of the dissociated polypeptide components and induce changes in conformation that prevent their reassociation into a complex with activity towards crystalline cellulose. Correspondence to: T. M. Wood     

Evidence for a Na+/K+/Cl− cotransport system in basolateral membrane vesicles from the rabbit parotid

Summary Sodium (²²Na) transport was studied in a basolateral membrane vesicle preparation from rabbit parotid. Sodium uptake was markedly dependent on the presence of both K⁺ and Cl^– in the extravesicular medium, being reduced 5 times when K⁺ was replaced by a nonphysiologic cation and 10 times when Cl^– was replaced by a nonphysiologic anion. Sodium uptake was stimulated by gradients of either K⁺ or Cl^– (relative to nongradient conditions) and could be driven against a sodium concentration gradient by a KCl gradient. No effect of membrane potentials on KCl-dependent sodium flux could be detected, indicating that this is an electroneutral process. A KCl-dependent component of sodium flux could also be demonstrated under equuilibrium exchange conditions, indicating a direct effect of K⁺ and Cl^– on the sodium transport pathway. KCl-dependent sodium uptake exhibited a hyperbolic dependence on sodium concentration consistent with the existence of a single-transport system withK _m =3.2mm at 80mm KCl and 23°C. Furosemide inhibited this transporter withK _0.5=2×10^–4 m (23°C). When sodium uptake was measured as a function of potassium and chloride concentrations a hyperbolic dependence on [K] (Hill coefficient =1.31±0.07) were observed, consistent with a Na/K/Cl stoichiometry of 112. Taken together these data provide strong evidence for the electroneutral coupling of sodium and KCl movements in this preparation and strongly support the hypothesis that a Na⁺/K⁺/Cl^– cotransport system thought to be associated with transepithelial chloride and water movements in many exocrine glands is present in the parotid acinar basolateral membrane.     

Relationship of Cell Sap pH to Organic Acid Change During Ion Uptake

Excised roots of barley (Hordeum vulgare, var. Campana) were incubated in KCl, K₂SO₄, CaCl₂, and NaCl solutions at concentrations of 10⁻⁵ to 10⁻² n. Changes in substrate solution pH, cell sap pH, and organic acid content of the roots were related to differences in cation and anion absorption. The pH of expressed sap of roots increased when cations were absorbed in excess of anions and decreased when anions were absorbed in excess of cations. The pH of the cell sap shifted in response to imbalances in cation and anion uptake in salt solutions as dilute as 10⁻⁵ n. Changes in cell sap pH were detectable within 15 minutes after the roots were placed in 10⁻³ n K₂SO₄. Organic acid changes in the roots were proportional to expressed sap pH changes induced by unbalanced ion uptake. Changes in organic acid content in response to differential cation and anion uptake appear to be associated with the low-salt component of ion uptake.     

The Ca2+-induced leak current in Xenopus oocytes is indeed mediated through a Cl− channel

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl^– channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately –15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl^–. Readdition of Ca²⁺ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca²⁺ inactivated Cl^– channel (CaIC). The inhibitory potency of Ca²⁺ was a function of the external Ca²⁺ concentration with a half maximal blocker concentration of about 20 m.These channels were inhibited by the Cl^– channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4-acetamido-4-isothiocyanatostilbene-2,2-disulfonicacid (SITS), another Cl^– channel blocker, led to activation of this Cl^– channel. Like other Cl^– channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl^– current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton.The results indicate that removal of divalent cations activates Cl^– channels in Xenopus oocytes which share several features with Cl^– channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl^– channels.The microelectrode measurements are part of the PhD thesis of K. Liebold; the patch clamp contributions are part of the PhD thesis of F.W. Reifarth. This study was supported by the Deutsche Forschungsgemeinschaft (We1858/2-l) and by Sonderforschungsbereich 249.